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ANMJAL  REPORT 

of 

PROGRAM  ACTIVITIES 

MTIOKAL  HEART  AND  LUNG  INSTITUTE 
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Fiscal  Year  1971 

PART  II 


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INTRAMURAL  RESEARCH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1970  -  June  30,  1971 


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INTRAMURAL  RESEARCH 

Project  Reports 
Cardiology  Branch 
Summary  1 

1.  Potentiation  of  the  Inotropic  Effects  of  Norepinephrine, 
Glucagon,  and  Dibutyryl  Cyclic  AMP  by  Theophylline  17 

2.  Comparison  of  Left  Ventricular  and  Pulmonary  Arterial 
Injection  Sites  in  the  Determination  of  Cardiac  Output  by 

the  Indicator  Dilution  Technique  18 

3.  Acquired  Right  Ventricular  Infundibular  Stenosis  in  Patients 

.  with  Ventricular  Septal  Defect  20 

4.  Hemodynamic  Assessment  of  Kay  Shiley  Prosthetic  Valves  22 

5.  Reversibility  of  Impaired  Heart  Rate  Response  to  Sympathetic 
Stimulation  in  Patients  with  Cardiac  Decompensation  24 

6.  Effects  of  Aging,  Type  IV  Hyperlipoproteinemia,  and  Coronary 
Artery  Disease  on  the  Diurnal  Pattern  of  Fibrinolytic 

Activity 25 

7.'   Propranolol  in  the  Treatment  of  Arrhythmias  Occurring  During 

Acute  Myocardial  Ischemia  in  Conscious  Dog  27 

8.  The  Effects  of  Training  in  Patients  with  Coronary  Artery 
Disease  and  Angina  Pectoris  29 

9.  The  Effects  of  Atropine  on  the  Degree  of  Myocardial  Ischemia 
During  Coronary  Occlusion  in  the  Conscious  Dog  30 

10.  The  Role  of  Cyclic  AMP  in  Myocardial  Contractility 32 

11.  A  Completely  Automated  Video-Tracking  Technique  for  the 
Determination  of  Dynamic  Changes  in  Ventricular  Volume  34 

12.  Mechanism  of  Action  of  the  Inotropic  Effects  of  Theophylline-   35 

13.  Enzyme  and  Receptor  Activities  of  Subcellular  Systems  Iso- 
lated from  Ischemic  Rabbit  Hearts  37 

14.  Physiological  and  Biochemical  Characteristics  of  a  New  Group 

of  Inotropic  Agents:   Analogues  of  Angiotensin  II  38 

15.  Supersensitivity  of  Hyperthyroid  Myocardium  to  Decreased 
Extracellular  Calcium  40 

16.  Control  of  Heart  Rate  in  Hyperthyroid  Cats 41 

17.  Effects  of  Hypoxia  on  the  Cardiac  Response  to  Inotropic 
Interventions  42 

18.  The  Natural  History  of  the  Floppy  Mitral  Valve  Syndrome  44 

19.  Disseminated  Intravascular  Coagulation  (DIVC)  Complicating 
Severe  Congestive  Heart  Failure  (CHF)  46 

20    A  Radioisotope  Imaging  Technique  for  the  Identification  of 

Myocardial  Scar 47 

21.  Correlation  of  Antiarrhythmic  Effects  of  Diphenylhydantoin 
with  Digoxin-induced  Changes  in  Contractility,  Na-K  ATPase 

and  K+  Efflux 49 

22.  Hemodynamic  Changes  During  Exertional  Syncope  in  Dogs  with 
Pulmonary  Artery  Constriction  50 

23.  Comparison  of  Simultaneously  Determined  Inotropic  and 
Chronotropic  Effects  of  Practolol  and  Propranolol  51 

24.  Effects  of  Chronic  Heart  Failure  on  the  Capacity  of  Glucagon 
to  Enhance  Contractility  and  Adenyl  Cyclase  Activity  of 

Human  Papillary  Muscles  52 


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25.  Comparison  of  the  Inotropic  and  Chronotropic  Activity  of 
Glucagon  and  Isoproterenol  in  the  Intact  Canine  Heart  53 

26.  The  Dynamic  Nature  of  Left  Ventricular  Outflow  Obstruction 

in  Idiopathic  Hypertrophic  Subaortic  Stenosis  54 

27.  Dissociation  of  Cardiac  Inotropic  and  Membrane  Effects  of 
Ouab  ain 

28.  Calcium  Ion  Movement  in  Skeletal  Muscle 56 

29.  The  Treatment  of  Ventricular  Arrhythmias  Occurring  During 
Acute  Coronary  Artery  Occlusion  in  Conscious  Dogs:   Efficacy 

of  Atropine  and  Lidocaine 58 

30.  The  Effects  of  Operative  Correction  of  Congenital  Heart 

Disease  on  the  Functional  Capacity  of  the  Heart 60 

31.  Ventricular  Tachycardia  Following  Release  of  Coronary  Artery 
Occlusion  in  Conscious  Dogs  and  the  Antiarrhythmic  Effects 

of  Atropine  "^ 

32.  Mechanical  Behavior  of  Large  Coronary  Arteries  64 

33.  Blood  Velocity  Profiles  and  Wall  Shear  in  the  Aorta  and  its 
Major  Branches  66 

3A.    Vascular  Mechanics:   Arterial  Wall  Properties  68 

35.  In  vitro  Studies  of  the  Influence  of  Mechanical  Factors  on 
Transvascular  Albumin  Flux  70 

36.  In  vitro  Study  of  the  Influence  of  Temperature  on  Trans- 
vascular  Albumin  Flux 72 

37.  In  vivo  Studies  of  Aortic  Intimal  Histologic  and  Chemical 
Response  to  Acutely  Induced  Mechanical  Stress  74 

38.  The  Development  of  Methods  for  the  in  vitro  Study  of  Vascular 
Interfacial  Transport  Mechanics  76 

39 .  Development  of  Canine  and  Miniature  Swine  Experimental 
Atherosclerotic  Animal  Colonies  77 

40.  The  Topography  of  Experimental  Atherosclerotic  Lesions  in  the 
Dog 79 

41.  Endothelial  Nuclear  Orientation  and  Morphology  and  its 
Relation  to  Hemodynamic  Factors  81 

42.  Healed  Valvular  Infective  Endocarditis  83 

43.  Active  Non-infective  Thrombotic  Endocarditis  84 

44.  Active  Valvular  Infective  Endocarditis  85 

45.  Active  Infective  Endocarditis  Confined  to  Mural  Endocardium  -   86 

46.  The  Spleen  in  Type  I  Hyperlipoproteinemia:   Histochemical, 
Biochemical,  Microfluorimetric  and  Electron  Microscopic 
Observations  87 

47.  Ultrastructural  Studies  of  Cardiac  Biopsies  in  Primary  Myo- 
cardial Disease 88 

48.  Electron  Microscopic  and  Histochemical  Studies  of  the  Liver 

in  GMx  Gangliosidosis  89 

49.  A  Histochemical  and  Electron  Microscopic  Study  of  Cardiac 
Myxomas  90 

50.  Effects  of  Hyperosmotic  Perfusate  on  Ultras tructure  and 
Function  of  the  Isolated  Canine  Heart  91 

51.  Papillary  Muscle  Dysfunction  Before  and  After  Auscultatory 
Mitral  Regurgitation.   Hemodynamic  and  Morphologic 
Documentation  92 


52.  Aneurysm  of  the  Non-patent  Ductus  Arteriosus  93 

53.  Congenital  Aortic  Stenosis  Resulting  from  a  Unicommissural 
Valve.   Clinical  and  Anatomic  Features  in  21  Adult  Patients  -   94 

54.  Acute  and  Chronic  Effects  of  Normothermic  Anoxia  on  Canine 
Hearts:   Light  and  Electron  Microscopic  Evaluation  95 

55.  Causes  of  Death  and  Other  Anatomic  Observations  after  Cardiac 
Valve  Replacement  96 

Surgery  Branch 

Summary 99 

56.  Development  of  a  Percutaneous  Transthoracic  Pacing  Wire  for 

Both  Routine  and  Emergency  Use 103 

57.  Preoperative  Assessment  of  Aortic  and  Mitral  Valve  Sizes  to 
Facilitate  Valve  Replacement  with  Autogenous  Tissue  Valves  —  105 

58.  A  New  Cause  for  Diastolic  Murmur  Following  Caged-ball  Re- 
placement of  the  Aortic  Valve  106 

59.  Four  Years'  Experience  with  Fabric-covered  Starr-Edwards 

Ball  Valves 107 

60.  An  Exploration  of  the  Usefulness  of  Secondary  Parameters  in 
Improving  Accuracy  of  Stroke  Volume  Estimation  by  Computer 
Analysis  of  Aortic  Pulse  Contour  108 

61.  The  Direct  and  Reflex  Effects  of  Isolated  Changes  in  pH,  p02 , 
and  pC02  upon  Systemic  and  Pulmonary  Vascular  Resistance  109 

62.  Measurement  of  Left  Ventricular  Diameter  and  Contraction 
Velocity  by  Ultrasound  111 

6  3.    Effects  of  Chronic  Cardiac  Denervation  on  the  Cardiac 

Response  to  Exercise  112 

64.  The  Effects  of  Chronic  Right  Heart  Failure  on  Left 
Ventricular  Function  113 

65.  Evaluation  of  Glutaraldehyde-fixed  Fascia  Lata  as  a  Material 

for  Tissue  Valve  Leaflets  114 

66.  Effect  of  Positive  Pressure  Ventilation  on  Pulmonary 

Vascular  Resistance  115 

67.  Evaluation  of  the  Operative  Treatment  of  Mitral  Stenosis  116 

68.  Closure  of  Atrial  Septal  Defect  with  a  Perforated  Patch  117 

69.  Augmentation  of  Myocardial  Contractility  with  Coronary  Bypass 
Grafts  118 

70.  The  Effect  of  Protamine  Sulfate  on  Myocardial  Contractility 

in  the  Intact  Dog 119 

71.  Evaluation  of  the  Effects  of  Corticosteroids  on  Myocardial 
Contractility  120 

72.  Mechanisms  of  the  Pulmonary  Vascular  Response  to  Hypovolemic 
Shock 121 

73.  Hemodynamic  Effects  of  Bretylium  Tosylate  in  the  Intact  Dog  -  122 

74.  The  Use  of  Isobutyl  Cyanoacrylate  in  Microvascular 
Anastomosis  123 

75.  Prolongation  of  Cardiac  Allografts  in  the  Rat  by  Alloanti- 

sera 124 

76.  Mixed  Leucocyte  Reaction  (MLR)  as  an  Assay  for  the  Presence 

of  Enhancing  Alloantiserum  125 

77.  Immediate  in  vitro  Leukocyte  DNA  Synthesis:   An  Early 
Indicator  of  Heart  Allograft  Rejection  126 


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78.  Creation   of  Aorto-pulmonary   Shunts   with   Cyanoacrylate 

Tissue   Adhesive   ^^' 

Experimental  Therapeutics   Branch 

Summary  ^ 

79.  Urinary  Excretion  of  Kallikrein  137 

80.  Studies  of  the  Biological  Role  of  Histamine:   Histaminase 
Activity  in  Various  Physiological  and  Pathological  States  139 

81.  Studies  on  6-Azuridine  Triacetate  (Azaribine)  in  Animals  and 

Man 1^  3 

82.  Metabolism  of  Hydroxyproline  and  Collagen  146 

83.  Effects  of  Pharmacological  Agents  on  Central  Neurotrans- 
mission Processes  using  the  Spinal  Cord  as  a  Simple  Model 

System ^^^ 

84.  Clinical  Investigation  of  Cardiovascular  Drugs  152 

85.  Studies  on  the  Biological  Role  of  Histamine:   Examination  of 
Histaminase  Activity  in  Rat  Tissues  and  Its  Release  by 

Heparin l^" 

86.  Studies   on  Catecholamines   in  Human  Disease   160 

87.  Studies    on   the   Biological   Role  of  Histamine:      Metabolism  of 
S-3H-histamine   in  Man 162 

88.  Studies    of   the   Biological  Role   of  Histamine:      Application  of 
the  Enzymatic  Assay   of  Histamine    to    the  Measurement   of  Hista- 
mine in  Urine    164 

89.  Isolation   and  Characterization  of  Hydroxyindole   0-methyl 

Trans  ferase 167 

90.  A  Sensitive   and  Automated  Method    for  Recording  Gastric  pH 
Changes   and  H^  Ion  Secretion   in  Experimental  Animals    and    the 
Use  of   this  Method   for   Studying   the  Pharmacology  of   Gastric 
Secretion 169 

91.  Tryptophan   Hydroxylase   and   Serotonin   in  Spinal   Cord  and 
Brainstem  Before   and  After  Chronic  Transection   171 

92.  Studies   on   the   Biological   Role   of  Histamine:      Participation 

of  Histamine  in  Inflammation  Following  Heat    Injury    173 

9  3.        Studies    on    the  Mechanisms    of   Binding  and  Release   of    Biogenic 

Amines    175 

94.  Studies  on  the  Biosynthesis  and  Metabolism  of  Physiologically 
Active  Amines  178 

95.  Shock-induced  Aggression  in  Hypertensive  Rats  181 

96.  Renin-angiotensin  System  of  the  Spontaneously  Hypertensive 

Rat  183 

97.  Tryptophan  Hydroxylase  and  Phenylalanine  Hydroxylase: 
Comparative  Properties,  Purification  and  Interactions  185 

98.  Studies  on  the  Isolation  and  Characterization  of  Clostridial 
Electron  Transfer  Proteins  and  Other  Iron-Sulfur  Proteins  188 

99.  Catecholamine  Metabolism  and  Blood  Pressure  of  Spontaneously 
Hypertensive  Rats  192 

100.  Studies  on  a  Pineal  Gland  Protein  Kinase  195 

101.  Studies  on  Naturally  Occurring  Vasoactive  Substances  197 

102.  A  Sensitive  Assay  for  Esterase  Activity  Employing  Radioactive 
Substrates:   Application  to  Plasma  Kallikrein  201 


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103.  Studies  on  the  Enzymes  Involved  in  the  Activation  of  Human 
Plasma  Kallikrein  204 

104.  Analysis  of  Amino  Acid  Phenylthiohydantoin  by  Gas 
Chromatography  210 

105.  Application  of  Counter current  Chromatography  to  the  Iso- 
lation and  Characterization  of  Substances  of  Biochemical 
Interests  213 

106.  Studies  on  the  Structure  of  Villikinin 216 

10  7.    Unusual  Linkages  in  Peptides  218 

108.  Preparation  of  Affinity  Adsorbents  220 

109.  Biochemistry  of  the  Kallikrein-kininogen-kinin  System  222 

110.  Effects  of  Steroid  Hormones,  Protein  Hormones  and 
Electrolytes  on  Neural  Function  227 

111.  Trace  Metal  Metabolism  237 

112.  Taste  and  Olfaction 248 

Laboratory  of  Kidney  and  Electrolyte  Metabolism 

Summary  273 

113.  Mechanism  of  Salt  and  Water  Transport  by  Proximal  Renal 

Tubules _ 279 

114.  Study  of  Ion  Transport  in  Renal  Cortical  Collecting  Tubules-   285 

115.  A  Study  of  the  Mechanism  of  Stimulation  of  Sodium  Transport 
by  Vasopressin  and  by  Aldosterone  by  Determining  their 
Effect  on  the  Electrolyte  Content  and  Energy  Metabolism  of 

the  Epithelial  Cells  of  the  Toad  Urinary  Bladder 288 

116.  A  Study  of  the  Effect  of  Vasopressin  and  Certain  other 
Hormones  and  Drugs  on  the  Concentration  of  Adenosine  3',5'- 
Monophosphate  in  the  Epithelial  Cells  of  the  Toad  Urinary 
Bladder 290 

117.  A  Study  of  Cyclic-AMP  Dependent  Protein  Kinase  in  the  Toad 
Urinary  Bladder  292 

118.  A  Study  of  the  Effect  of  Prostaglandin  E^  and  7-oxa-13- 
Prostynoic  Acid  on  the  Sodium  Transport  and  Permeability 
Properties  of  the  Toad  Urinary  Bladder 294 

119 .  Volume  Regulation  in  Duck  Erythrocytes  296 

120.  Immunochemical  Comparison  of  Cardioglobulin  and  Rat  Muscle 

E-C  Coupling  Complex  301 

121.  Mechanism  of  Cardiac  Failure  in  Hamsters  with  Hereditary 
Cardiomyopathy  303 

'-.ab  oratory  of  Biochemical  Genetics 

Summary  307 

122.  The  Initiation  of  Mammalian  Protein  Synthesis  311 

123.  Studies  on  Aminoacyl-tRNA  Synthetases 312 

124.  The  Neuroblastoma  System  as  a  Model  for  Neuron  Dif- 
ferentiation    313 

125.  Mammalian  Peptide  Chain  Termination  315 

126.  Biochemistry  of  Rare  Constituents  of  tRNA 317 

127.  Genes  for  Neuronal  Properties  Expressed  in  Neuroblastoma  x  L 

Cell  Hybrids  320 

128.  Acetylcholinesterase  in  Neuroblastoma  Cells  322 


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129.  Cyclic  AMP  Metabolism  in  Neuronal  and  Glial  Clonal  Cell 
Lines  

Laboratory  of  Biochemistry 

Summary   

130.  Regulation  of   Bacterial   Purine   and  Pyrimidine    Base   and 
Nucleoside   Utilization   

131.  Studies    on   the   a-Lysine  Mutase   Complex  

132.  Genetics    of   E_.    coli   Glutamine   Synthetase 

133.  A  Dithiol   Dehydrogenase   of   Clostridium  sticklandii   

134.  1.    Methionine   Biosynthesis   and   its    Regulation   in   Fungi   and 

Bacteria 

2.  Mechanisms  of  Pyridoxal  Phosphate  Enzyme  Catalyzed 
Elimination  and  Replacement  Reactions 

3.  Microbial  Genetics  of  Microtubular  Proteins  and  their 
Assembly  

135.  Enzyme  Structure  and  Mechanisms  of  Action  and  Control  

136.  Zinc  Induced  Paracrystalline  Aggregation  of  Glutamine 
Synthetase  

137.  Studies  on  L-asparaginase  in  Anaerobic  Bacteria  

138.  1.  Radioisotopic  Assays  for  Glutamine  Synthetase  and 

Glutaminase . 
2.  Regulation  of  Glutaminase  Activity  in  E^.  coli 

139.  Studies  on  Lysine  Fermentation  in  Clostridia.   The  D-a- 
Lysine  Pathway  

140.  1.  Anaerobic  Metabolism  of  Certain  Amino  Acids  and  other 

Nitrogen  Compounds  with  Especial  Reference  to  the  Role 
of  Bi2  Coenzyme  and  to  Electron  Transfer  and  Phos- 
phorylation Reactions  Involved. 
2.  Methane  Biosynthesis  and  One-carbon  Compound  Metabolism  - 

141.  1.  Effects  of  Divalent  Cations  and  Energy  Charge  Upon  the 

Activity  of  Glutamine  Synthetase  

142.  Antibodies  to  E^.  coli  Glutamine  Synthetase 

143.  1.  Application  of  Physical  Methods  in  the  Determination  of 

Structures  of  Organic  Compounds . 
2.  Synthetic  Studies  of  Organic  Compounds  of  Biological 

Interest  

144.  Enzymatic  Fragmetation  of  Fibrinogen  

145.  Isolation  and  Characterization  of  Cellular  Growth  Factors 
from  Calf  Serum  

146.  Effects  of  Conformation  on  the  Free  Radical  Districutions  of 
Irradiated  Proteins  

147.  Studies  on  the  Tritiation  of  a  Peptide  Derived  from  Nisin  — 

148.  Biological  Oxidation  Capabilities  of  Membrane  Fragments 
Obtained  from  E^.  coli 

149.  Location  of  Tritium  in  Tritiated  Methionine  

150.  Biosynthetic  Capabilities  of  Membrane  Fragments  Obtained 
from  E^.  coli 

151.  The  Interaction  of  Actin  and  Myosin  

152.  Structure  of  the  Muscle  Protein,  Myosin  

15  3.    Plasmin  Digestion  of  Fibrinogen  


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340 
342 
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344 
348 

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366 


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154.  The  Structure  and  Biochemical  Activity  of  Actin  and  Myosin  -  396 

155.  The  Isolation  and  Purification  of  Human  Platelet  Myosin  398 

156.  Membrane  Biochemistry  400 

157.  Cytology  of  Acanth amoeba 402 

158.  Study  of  Motility  in  Ameba 404 

Laboratory  of  Chemical  Pharmacology 

Summary 409 

159.  An  Investigation  of  the  Mechanisms  by  which  Various  Steroids 
Alter  the  Hepatic  Microsomal  Mixed  Function  Oxidase  System 

to  Alter  Drug  Metabolism '■ 417 

160.  Studies  on  the  Oxidation  of  NADH  Stimulated  by  NADPH  and 

Drugs  in  Liver  Microsomes  421 

161.  An  Approach  to  Measure  the  Stoichiometric  Relationship 
between  Hepatic  Microsomal  Drug  Metabolism  and  NADPH 

Oxidation 423 

162.  A  Comparison  of  the  Effects  of  Halothane  and  CCI4  on  the 
Hepatic  Drug  Metabolizing  System  426 

163.  Studies  on  the  Resolution  of  Components  of  Mixed  Function 
Oxidases  Concerned  with  Drug  Metabolism  428 

164.  Autoradiographic  Studies  on  the  Localization  of  -"-^C-Labeled 

Hep ato toxins  430 

165.  Effect  of  3-Methylcholanthrene  Hepatotoxic  Effects  Caused 

by  Various  Toxicants  in  Rats  and  Mice 432 

166.  The  Relationship  between  Bromobenzene  Metabolism  and  Hepatic 
Necrosis  434 

167.  Studies  on  the  Covalent  Binding  of  -'-^C-bromobenzene  in  vivo-   436 

168.  Possible  Explanation  for  the  Zonal  Distribution  of 
Chemically-induced  Hepatic  Necrosis  438 

169.  Binding  and  Metabolism  of  Aromatic  Hydrocarbons  in  the  Lung-   440 

170.  Accumulation  of  Radioactivity  After  Incubation  of  Rat  Brain 
Slices  with  ^H-choline  442 

171.  Studies  on  the  Possible  Role  of  Choline  in  the  Regulation 

of  Acetylcholine  Levels  in  Rat  Brain 444 

172.  The  Effect  of  Prostaglandin  E^  on  the  EEC  and  the  Levels  of 
Brain  Acetylcholine  in  Rats  447 

173.  Effect  of  Pilocarpine  on  the  Metabolism  of  Serotonin  and 
Acetylcholine  in  the  Brain 449 

174.  Correlation  between  Beta  Adrenergic  Receptor  Activity  and 

Enzyme  Activity  of  Arterial  and  Cardiac  Muscle  451 

175.  Interaction  of  Microtubule  Protein  with  Colchicine  and  other 
Antimitotic  Drugs  453 

176.  The  Prevention  of  Isoproterenol  Tachyphylaxis  and  the 

Reversal  of  Contractile  Effects  in  Vascular  Smooth  Muscle  —   457 

177.  The  Influence  of  Age  on  the  Response  of  Smooth  Muscle  to 
Pharmacologic  Agents  459 

178.  Characterization  of  the  Enzymatic  Ion  Transport  System  461 

179.  Correlation  of  Antiarrhythmic  Effect  of  Diphenylhydantoin 
with  Digoxin-induced  Changes  in  Contractility,  NaK  ATPase 

and  K+  Efflux 464 

180.  Synthesis  of  Spin  Labeled  Cardiac  Aglycones  for  Electron 

Spin  Resonance  Studies  of  Membrane  Function  466 


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181.  The  Mechanism  of  Drug  Phototoxicity  and  Photosensitivity  —    469 

182.  Effects  of  Bromobenzene  and  CCl^  Administration  on  Plasma 

and  Hepatic  Protein  Synthesis  by  Perfused  Rat  Liver  471 

183.  The  Effect  of  Cedrene  on  Drug  Metabolism  by  Hepatic  Micro- 
somes     473 

184.  The  Binding  of  Sulfaphenazole  to  Fetal  Plasma  Albumin  and 

to  Albumin  Isolated  from  the  Plasma  of  Infants  and  Adults  475 

185.  Desoxyisoproterenol:  An  Alpha  and  Beta  Adrenergic  Receptor 
Stimulating  and  Blocking  Agent  479 

186.  Effects  of  Chronic  Exposure  to  Atmospheric  Ozone  and  of 

Drugs  on  Enzymes  481 

187.  The  Detection  of  Conformation  Shifts  in  Cell  Membranes  during 
Normal  and  Altered  Function  by  the  Use  of  Electron  Spin 
Resonance  483 

188.  Physicochemical  Studies  of  Complexes  Betvjeen  Drugs  and  Bio- 
molecules.   V.   Interaction  of  Anionic  Drugs  with  Acetyl- 
salicylic  Acid  Treated  Human  Serum  Albumin  485 

189.  Physicochemical  Studies  of  Complexes  Between  Drugs  and  Bio- 
molecules.  VIII.  The  Interaction  of  Spin-labelled  Sulfon- 
amides with  Bovine  Erythrocyte  Carbonic  Anhydrase  B  489 

190.  Physicochemical  Studies  of  Complexes  Between  Drugs  and  Bio- 
molecules.   VII.   The  Use  of  Fluorescent  Probes  to  Monitor 

Drug  Interactions  with  Red  Cell  Ghost  Membranes 492 

191.  Studies  on  the  Hepatotoxicity  of  Halogenated  Aryl  Hydro- 
carbons.  3.   Pulse  Label  Studies  on  Bromobenzene  Metabolism-  496 

192.  Studies  on  Drug-induced  Hemolytic  Anemia  498 

193.  Inhibition  by  Colchicine  Derivatives  and  other  Antimitotic 
Agents  of  Rat  Hindpaw  Edema  Induced  by  Sodium  Urate  Crystals-  500 

194.  The  Effects  of  Colchicine  and  Antagonists  of  Histamine  and 
Serotonin  on  Inflammation  Induced  by  Sodium  Urate  Crystals  —  504 

195.  Correlations  Between  the  Effects  of  Hepatotoxic  Compounds 
on  Plasma  and  Liver  Triglyceride  Levels  and  the  Development 

of  Hepatic  Necrosis  508 

196.  Antihistamines  and  Inflammation  Induced  by  Histamine, 
Serotonin,  Bradykinin  and  Compound  48/80  511 

197.  Salivation  in  Mice  as  an  Index  of  Adrenergic  Activity  516 

198.  Effects  of  Sodium  and  Potassium  on  the  Kinetics  of  Serotonin 

and  Norepinephrine  Transport  by  Rabbit  Synaptosomes  518 

199.  The  Effect  of  Divalent  Ions  on  the  Kinetics  of  Release  of 

h3-NE  from  Rat  Heart  Slices  521 

200.  The  Effect  of  Enzyme  Induction  of  Synthesis  and  Catabolism 
of  Corticosterone  in  Rats.   Analysis  by  Means  of  Steady- 
State  Kinetics  524 

201.  Comparison  of  the  Effects  of  K^-free  Media  and  Ouabain  on 

the  Transport  of  5-HT  and  Norepinephrine  by  Synaptosomes  526 

202.  Effect  of  Electrolytes  on  the  Accumulation  and  Metabolism 

of  Biogenic  Amines  by  Subcellular  Fractions  of  Rat  Brain  529 

203.  Relationship  between  Potassium  and  the  Sodium  Requirement 
for  Transport  and  Storage  of  5-Hydroxytryptamine  and  Norepi- 
nephrine by  Synaptosomes  531 

204.  Enhancement  of  Motor  Activity  in  Rats  Following  Withdrawal 

of  Chronic  Treatment  with  Alpha-Methyltyrosine  534 


205.  Evidence  for  a  Balance  in  the  Basal  Ganglia  between 
Cholinergic  and  Dopaminergic  Activity  536 

206.  Cerebral  Serotonin  Metabolism  and  Tolerance  to  Selective 
REM-Sleep  Deprivation  in  Normal  and  Hypophysectomized  Rats  -   538 

20  7.    Alterations  in  Brain  Monoamines  and  their  Amino  Acid  Pre- 
cursors in  Chronically  Jaundiced  (Gunn)  Rats  540 

208.  Dynamic  Aspects  of  Hormonal  Control  of  the  Formation  of 

Cyclic  AMP  in  Human  and  Rabbit  Platelets  542 

209.  Studies  on  the  Biliary  Excretion  of  Bromobenzene 

Metabolites  545 

210.  Studies  on  the  Effects  of  Phenobarbital  and  3-Methyl- 
cholanthrene  Pretreatment  on  the  in  vitro  Metabolism  of 
Bromobenzene  in  3  Species 547 

211.  Studies  on  the  Requirements  for  the  Formation  of  Glutathione 
Conjugate  of  Bromobenzene  in  vitro  549 

212.  Studies  on  the  Metabolism  of  -'-^C -Bromobenzene  Metabolism  by 
Rabbit  Kidney  Microsomes  552 

213.  Differentiation  of  APT  and  Cyclic  AMP  Pools  in  the  Fat  Cell-   554 

214.  Mechanism  of  Bromobenzene  Toxicity  557 

215.  A  Simple  and  Sensitive  Method  for  the  Measurement  of 
Endogenous  Cyclic  AMP  560 

216.  The  Use  of  the  Isolated  Lever  Cell  to  Study  Drug  Metabolism-   563 

217.  Mechanism  of  Indomethacin-induced  Ulcerations  of  the  Gastro- 
intestinal Tract 565 

218.  Bromobenzene-Induced  Hepatic  Necrosis:   Species  Differences 

and  Protection  by  SKF  525-A 567 

219.  Mechanism  of  Dimethylbenzanthracene-Induced  Bone  Marrow, 
Spleen,   Testis   and  Gut  Necrosis    569 

220.  Mechanisms    of  Drug- Induced  Bone  Marrow  Damage   571 

221.  Studies   on  the  Hepatotoxicity   of  Halogenated  Aryl  Hydro- 
carbons.     2.      Effect  of  Phenobarbital   and   3-Methylchol- 
anthrene  Induction  on   the  Hepatotoxicity  and   the  Pattern 

of  Metabolism  of  Bromobenzene   573 

222.  Studies   on  the  Hepatotoxicity   of  Halogenated  Aryl  Hydro- 
carbons.     1.      Studies   on   the  Relationship   of  Glutathione   to 

the  Hepatotoxicity   of  Bromobenzene  575 

Endocrinology   Branch 

Summary 577 

223.  Studies  on  the  Control  of  Calcium  Absorption:   Effect  of 

Sodium  Chloride  Loading  and  Depletion  585 

224.  Evaluation  of  Secondary  Hypothyroidism  with  Synthetic  Thyro- 
tropin Releasing  Factor  (TRF)  587 

225.  Pathophysiologic  Studies  in  Hypertension  589 

226.  The  Effect  of  Prostaglandins  upon  Cortisol  and  Aldosterone 
Secretion  of  Hypophysectomized-nephrectomized  Dogs  594 

227.  Studies  on  the  Etiology  of  the  Natriuresis  of  Fasting  596 

228.  The  use  of  17  a  Estradiol  in  the  Inhibition  of  the  Effect 

of  D-Penicillamine  on  Collagen  Solubility  in  Skin  598 

229.  The  Use  of  3-Methyl-2-(3-pyridyl) -indole  Methanesulfonate 

(GPA  2282)  in  the  Treatment  of  Hyperaldosteronism  600 


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230.  The  Circadian  Urinary  Excretion  of  Ketosteroids  in  Patients 

with  the  Adrenogenital  Syndrome  602 

231.  Calcium  Metabolism  in  Cystic  Fibrosis  603 

232.  The  Relationship  of  Central  Nervous  System  Degenerative  Dis- 
orders to  the  Adrenal  Hyper-  and  Hypoplasia.   Studies  in:  I. 
Subacute  Necrotizing  Encephalopathy  (SNE)  -  Bone  Marrow  and 
Electron  Microscope  Studies  on  Brain.   II.  Sudanophilic 
Leukodystrophy  and  Addison's  Disease  -  Brain  Electron 
Microscopy 605 

233.  Calcium  Metabolism  in  Idiopathic  Hypercalcemia  of  Infancy 
and  Childhood.   I.   The  Effect  of  Calcium  Infusion  on  Serum 
Calcium,  Thyrocalcitonin  (TCT)  and  Parathyroid  Hormone  (PTH) 
II.   24-Hour  Urinary  Cyclic  AMP  in  Two  Patients  with  the 
Disease 606 

234.  Effect  of  Prostaglandins  on  the  Dog  Kidney 608 

235.  Studies  on  the  Control  of  Sodium  Excretion.   I.  Mechanisms 

of  Sodium  Retention  610 

236.  Studies  on  the  Control  of  Renal  Sodium  Excretion.   II.   The 

Role  of  Adrenergic  Receptors  and  Cyclic  Nucleotides  612 

237.  Studies  on  Control  of  Jalcium  and  Magnesium  Excretion: 
Intestinal  Absorption  and  Renal  Excretion  of  Calcium  in 
Metabolic  Acidosis  and  Alkalosis  615 

238.  Studies  in  Calcium  and  Phosphorus  Metabolism.   I.  Ionic 
Interaction  with  Bone  Mineral  and  Biologic  Calcification  617 

239.  Studies  in  Calcium  and  Phosphorus  Metabolism.   II.  New  Con- 
cepts in  the  Pathogenesis  and  Treatment  of  Renal  Stones  of 
Calcium  Phosphate  Origin  619 

240.  Studies  in  Calcium  and  Phosphorus  Metabolism.  III.  Mode  of 
Action  of  Thyrocalcitonin  622 

241.  Studies  in  Calcium  and  Phosphorus  Metabolism.   IV.  Inter- 
action of  Hormones  at  the  Air-Water  Interface  624 

242.  Studies  in  Calcium  and  PhosphoriiS  Metabolism.  V.  Clinical 
Implications  of  25-Hydroxycholecalciferol  (25-HCC)  626 

243.  Studies  in  Calcium  and  Phosphorus  Metabolism.   VI.   Treat- 
ment of  Osteoporosis  with  Calcium  Infusion  and  Thyrocal- 
citonin     628 

244.  Studies  in  Calcium  and  Phosphorus  Metabolism.  VIII.  Clinical 
Applications  of  Thyrocalcitonin  (TCT)  630 

245.  Studies  in  Calcium  and  Phosphorus  Metabolism.  IX.  Effect  of 
Diuretics  632 

246.  Studies  in  Calcium  and  Phosphorus  Metabolism.   X.  Normo- 
calcemic  Primary  Hyperparathyroidism  634 

247.  Studies  in  Calcium  and  Phosphorus  Metabolism.  XI.  Calcium 
Infusion:   Diagnostic  Value,  Physiological  Consequences  and 
Effect  on  Calcium  Homeostasis  636 

Laboratory  of  Chemistry 

Summary 639 

248.  Mass   Spectrometry   on  Doubly-charged   Ions    643 

249.  Applications   of  Nuclear  Magnetic  Resonance   to   Biochemical 

or  Model   Bio-organic  Systems    644 


250.  Mass  Spectrometric  Methods  of  Analysis  646 

251.  X-ray  Crystallographic  Structural  Studies  of  Natural  and 
Synthetic  Compounds  648 

252.  The  Application  of  Mass  Spectrometry  to  Problems  in  Bio- 
chemistry    650 

253.  The  Chemical  Investigation  of  Biochemical  Reactions  651 

254.  The  Use  of  Digital  Computing  in  Problems  in  Biochemistry  652 

255.  Characterization  of  Natural  Products  654 

256.  The  Characterization  of  Natural  Materials  657 

257.  Structure  Elucidation,  Synthesis  and  Biosynthesis  of 

Natural  Products  659 

258.  Mass  Spectrometry  and  Structure  of  Natural  Products  664 

Laboratory  of  Technical  Development 

Summary 667 

259.  Indirect   Cardiac  Output   Computation  674 

260.  Microdialyzer   for  Continuous    Sampling  of   Small  Molecules 

from   Blood 675 

261.  Reflectance   Oximeter    for   Continuous  Measurement    of  Oxygen 
Saturation 6  77 

262.  Methodology   in  Fluorescence  Measurements   679 

263.  Fluorescent   Complexes  of  Proteins   681 

264.  Applications    of   Fluorescence   in  Biochemistry    684 

265.  Countercurrent   Chromatography:      Liquid-Liquid  Partition 
Chromatography  without    Solid   Support   688 

266.  Countercurrent    Chromatography:      Liquid-Liquid  Partition 
Chromatography  without   Solid   Support,    (Part   II)    690 

26  7.        Development    of   the   Spiral   Coil  Membrane   Blood  Oxygenator   and 
Systems    for  Long-term  Temporary   Support   of   Pulmonary   and 
Cardiovascular   Systems    — 693 

268.  Dual-Frequency   Ultrasonic   Flowmeter   698 

269.  Peltier-Seebeck  Equllibrator   700 

270.  An  Automated  Method   for  Rapid  Bacteriological  Assay    702 

271.  Blood  Flow  Measurement   Using  Nuclear  Magnetic  Resonance 
Techniques    706 

272.  An   Investigation  and  Development   of  Methods    for   the   Study 
of   the  Mechanism  of  Enzyme   Action  and   Function   in  Cellular 
Systems    and   in   Solution 709 

273.  Development   of   Stopped-Flow  Micro-Calorimetry   for   the   Study 

of   Biochemical   and  Cellular  Reactions   718 

Molecular  Disease   Branch 

Summary 721 

274.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins:   The 
Contribution  of  Carbohydrates  to  the  Structure  and  Function 

of  the  Plasma  Lipoproteins  731 

2  75.    The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins:   The 

Turnover  and  Function  of  Very  Low  Density  Lipoproteins  734 

276.    The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins:   The 

Functional  Roles  of  Plasma  High  Density  and  Low  Density 

Lipoproteins  738 


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277.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins: 
The  Metabolism  and  Structure  of  Plasma  Lipoproteins  in  the 

Rat 742 

278.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins: 
Studies  on  a  New  Lipoprotein  Complex  in  Plasma,  its 

Structure  Function  and  Genetic  Control  744 

279.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins: 
Post-heparin  Lipolytic  Enzymes  and  their  Role  in  Normal  and 
Abnormal  Lipid  Transport  and  Clearance  748 

280.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins: 

Studies  of  Familial  Hyperlipoproteinemia  752 

281.  The  Biochemistry^  and  Metabolism  of  Plasma  Lipoproteins:  The 
Structure  of  Very  Low  Density  Lipoproteins  760 

282.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins:  The 
Structure  and  Genetic  Control  of  Lipoproteins  762 

283.  The  Biochemistry  and  Metabolism  of  Plasma  Lipoproteins:  The 
Structure  and  Genetic  Control  of  Alpha  Lipoprotein  767 

284.  Glycolipids  and  Other  Lipid  Constituents  of  Normal  Human 

Liver 772 

285.  Tissue  Lipidoses:   Abnormal  Biochemistry  in  Tissue  Lipid 
Storage  Diseases  774 

286.  1.  Chemistry  and  Physical  Properties  of  Bovine  Parathyroid 

Hormone  (PTH) :  (a)  isolation  and  characterization  of 
bovine  PTH;  (b)  complete  amino  acid  sequence  of  bovine 
PTH;  (c)  physiochemical  properties  of  bovine  PTH 

2.  Chemistr>'  and  Immunological  Properties  of  Thyrocalcitonin 
(TC) :  (a)  comparison  of  the  physical  chemical  properties 
of  procine,  bovine,  human  and  salmon  TC;  (b)  characteri- 
zation of  the  amino  acid  residues  involved  in  the  immuno- 
logical reactivity  of  porcine  TC 

3.  Chemistry  and  Conformation  of  the  Carboxyl  Terminal 
Alanine  Peptide  (apoVLDL-ala)  Derived  from  the  Very  Low 
Density  Lipoprotein  Particle:  (a)  primary  structural 
studies  of  apoVLDL-ala;  (b)  physiochemical  properties 

of  apoVLDL-ala 779 

287.  Sterol  Synthesis  in  Mammalian  Arteries  787 

288.  Regulation  of  Cholesterol  Synthesis  in  Mammalian  Cells  in 

vitro 789 

289.  Mechanism  of  Release  of  Histamine  and  other  Vasoactive  Sub- 
stances from  Mast  Cells 791 

290.  Effects  of  Hormones  on  Metabolism  of  Adipose  Tissue 

Studied  in^  vitro 794 

291.  Formation,  Metabolism  and  Action  of  Cyclic  AMP  and  Cyclic 

GMP 796 

292.  Metabolism  of  Lung  Tissue  and  Phagocytic  Cells: 

1.  Synthesis  of  dipalmitoyl  lecithin  by  lung  and  alveolar 
macrophages , 

2.  Characterization  of  isolated  phagocytic  vesicles  from 
guinea  pig  polymorphonuclear  leukocytes  and  rabbit 
alveolar  macrophages, 

3.  Studies  of  alveolar  macrophage  functions  and  effects  of 
oxidant  gases  798 


293.  Metabolism  of  Phagocytic  Cells  

294.  Studies  on  the  Lipolytic  Enzymes  of  Adipose  Tissue  - 

295.  The  Mechanism  of  Hemoglobin  Biosynthesis  in  Rabbit 
Reticulocyte  Cell-Free  Systems  

296.  Evolutionary  Homology  of  Components  of  the  Protein- 
Synthesizing  Machinery  

297.  Regulation  of  Hemoglobin  Chain  Synthesis  in  Beta 
Thalas  semia 


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ANNUAL  REPORT  OF  THE 
CARDIOLOGY  BRANCH 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1970  through  June  30,  1971 


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In  the  Cardiology  Branch  two  new  major  areas  of  research  interests 
have  been  developed  over  the  past  three  years.   These  relate,  first,  to  the 
investigation  of  the  enzymatic  and  molecular  mechanisms  responsible  for 
modulating  the  contractile  state  of  the  myocardium,  and  second,  to  the  study 
of  myocardial  ischemia,  myocardial  hypoxia,  and  coronary  artery  disease. 
Our  investigative  efforts  in  these  two  areas  have  been  considerably  expanded 
this  past  year  with  emphasis  on  both  the  physiologic,  pharmacologic,  and  sub- 
cellular alterations  caused  by  myocardial  ischemia,  and  on  how  the  ischemia - 
induced  changes  can  be  modified  by  interventions  that  may  have  therapeutic 
potential. 

MOLECULAR  MECHANISMS  RESPONSIBLE  FOR  CARDIAC  CONTRACTION  AND 
FOR  THE  PHYSIOLOGICAL  EFFECTS  OF  INOTROPIC  AGENTS 

Normal  Mechanisms 


The  positive  inotropic  effects  of  catecholamines  have  been  postulated 
to  result  from  increases  in  intracellular  levels  of  cyclic  AMP  (C-AMP) „  Al- 
though considerable  evidence  exists  that  is  compatible  with  such  a  concept, 
it  is  still  uncertain  whether  stimulation  of  the  adenyl  cyclase-C-AMP  system 
and  enhancement  of  contractility  are  causally  related  or  merely  coincidental 
events.   For  example,  in  a  recent  study  we  have  shown  that  while  10"^  nor- 
epinephrine had  a  positive  inotropic  effect  within  10  seconds,  a  significant 
increase  in  C-AMP  concentration  could  not  be  demonstrated  until  20  seconds 
had  elapsed.  Moreover,  preliminary  data  appear  to  indicate  that  lower  con- 
centrations of  norepinephrine,  while  still  having  an  appreciably  positive 
inotropic  effect,  do  not  alter  myocardial  C-AMP  concentration.   Although 
there  are  several  explanations  for  this  apparent  dissociation  of  the  inotropic 
and  en23nnatic  effects  of  norepinephrine,  these  findings  do  raise  the  possi- 
bility that  the  norepinephrine -induced  stimulation  of  the  adenyl  cyclase-C- 
AMP  system  is  not  causally  related  to  the  coincidental  enhancement  of  myo- 
cardial contractility  that  occurs. 

Two  additional  studies  bearing  on  this  relationship  are  now  in  progress, 
If  there  is  a  causal  relationship  between  intracellular  C-AMP  concentration 
and  contractility,  inhibition  of  phosphodiesterase,  the  enzyme  responsible 
for  inactivating  C-AMP,  would  be  expected  to  enhance  the  cardiac  effects  of 
norepinephrine  and  glucagon.   This  was  ostensibly  verified  when  we  found  that 
while  theophylline  (a  phosphodiesterase  inhibitor)  did  not  alter  the  inotropic 
effects  of  calcium  (an  agent  not  believed  to  exert  its  inotropic  effects 
through  the  C-AMP  system),  it  caused  marked  potentiation  of  the  inotropic  ef- 
fects of  norepinephrine  and  glucagon.   However,  the  preliminary  results  of 
another  investigation  have  shown  that  at  a  concentration  of  theophylline  that 
increases  papillary  muscle  tension  by  approximately  507o,  no  increase  in  intra- 
cellular levels  of  cyclic  AMP  occurs.  Thus,  if  the  direct  inotropic  effects 


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of   theophylline   are  not  mediated   by   the   C-AMF   system,    it    is   possible    that 
its   potentiation   of   the    inotropic   effects   of  norepinephrine   and   glucagon   is 
also  caused  by  a   mechanism   independent    of  C-AMP„      To   further  elucidate    this 
problem,   we   are    in  the    process   of  determining  whether  an  increase    in   intra- 
cellular   levels   of  C-AMP   occurs  when  the    inotropic   effects   of  norepinephrine 
are  enhanced  by  theophylline. 

Current   theory  regarding  excitation-contraction  coupling   in  cardiac 
and    skeletal  muscle   centers   around    the    concept    that   calcium,    released   by   the 
sarcoplasmic   reticulum    (SR)    following  electrical  depolarization,    diffuses 
from   SR  to  a    receptor   located   on   the   actin   filament.      This   event    is   then  be- 
lieved  to  eventuate    in   the   interaction   of  actin  and  myosin    filaments,    thereby 
initiating  muscle    shortening.      Implicit   in   this   concept    is   the   assumption   that 
the    rate    of  diffusion   of  calcium   is    sufficient    to  account    for   the    rapid    se- 
quences  of  contraction  and   relaxation   characteristic    of  cardiac   and    skeletal 
muscle.      However,    the    only  data    relating   to  the   rate    of   calcium   ion  movement 
in  muscle    cytoplasm   indicate    that  mobility   is  at    least   50   to    100   times    slower 
than   in  water,   a    rate    probably   too   slow  to  be    compatible   with   the  above  hypo- 
thesis.    We   recently  constructed  an  analog  circuit    for   the    solution   of  diffu- 
sion problems  and   our   results  also   suggest   that   the    classical  hypothesis   for 
excitation-contraction   coupling  may  be    in  error.      Further   studies  are   being 
performed   in   skeletal  muscle    to  determine    the    feasibility  of   the    calcium   theory 
for  excitation-contraction   coupling. 

We  have    shown  previously   that   ouabain   causes  negative    inotropic   re- 
sponses  in   cat   papillary  muscles  when  external   calcium   concentration   is    low. 
Recent    studies  have   demonstrated   that    this  negative    inotropic   effect    is   con- 
centration-dependent and   is    specific    for   the   digitalis   glycoside    insofar  as 
norepinephrine,    isoproterenol,   and  angiotensin   II  produce    their  usual    inotropic 
responses   independent   of  external  calcium  concentration.      These   data    could   be 
explained  as    follows:      ouabain   results   in   greater  availability   of  Ca    to   the 
contractile   apparatus,    either  by  mobilizing  a    particular   intracellular   compart- 
ment  of   calcium  or   by  enhancing  calcium  entry   into   the    cell;    this   results    in 
enhanced   contractility.      However,    in  an  external  environment    of   low  calcium, 
either  part   of   the  mobilized   intracellular  calcium  moves   out   of   the   cell   or 
the   net    flux   into   the    cell  becomes  negative,    thereby  causing  a   decrease    in 
contractility.      These    results  would    seem   to  be    further  evidence    that    the   posi- 
tive   inotropic  effect   of   ouabain   is   related   to  alteration   of   intracellular 
calcium   stores. 

It  has   been   suggested   that  one    of   the   actions   of  digitalis  essential 
for   its   positive    inotropic   properties   is    inhibition   of  Na"*",    K   -dependent 
ATPase.      It  also  has   been    suggested   that  diphenylhydantoin    (DPH)    exerts  a 
protective   effect    on  digitalis-induced   arrhythmias  by   interfering  with   the 
digitalis-Na+,    K+-ATPase    interaction.      If   this    latter  hypothesis  were    correct, 
it  would   imply   that   the   mechanism   for   the    inotropic   activity  of  digitalis   is 
independent   of   inhibition  of  Na"*",    K"*'-ATPase,    since   DPH  does  not   interfere  with 
the    inotropic   effects   of  digitalis.     We    found,   however,    that    the   antiarrhythmic 
effects   of  DPH  cannot   be   attributed   to  prevention  of  digitalis-induced    inhibi- 
tion  of  Na+,   K+-ATPase.     Thus,    there   is   still  no  convincing  evidence   that 
seriously   questions   the    concept   that   the    interaction  between  digitalis  and 
Na+,   K^-ATPase    is   basic    to   the    inotropic  effects  exerted  by   this  agent. 


Myocardial  Failure 

Glucagon  exerts   a    positive    inotropic   effect   in  normal  hearts,    an 
effect  believed   to  be  mediated   by  C-AMP.     However,   we    previously  have    shown 
that   its   capacity   to   increase  myocardial   contractility  and    to   stimulate   adenyl 
cyclase   activity  are  markedly   impaired   in  animals  with  chronic   cardiac    failure, 
To  assess  directly   the    influeiice    of   chronic   heart    failure    on   the   effectiveness 
of  glucagon  as  an   inotropic   agent    in  human  myocardium,   we  measured   its   effect 
on   contractility  and  adenyl   cyclase   activity   in   left  ventricular  papillary 
muscles    obtained    from  patients  at  mitral  valve    replacement.      These    studies 
demonstrated   that   chronic   cardiac    failure    is   uniformly  associated  with  a    com- 
plete   loss   of   the    capacity   of   glucagon   to  enhance    contractility  and    stimulate 
adenyl   cyclase,    findings    that   probably  explain   the   disappointing  clinical 
results   of   this   drug   in   the    treatment   of  patients  with   prolonged   cardiac 
decompensation. 


Myocardial   Ischemia   and  Hypoxia 

To  determine    the  mechanisms  whereby   ischemia    results   in 
cardial   function,    studies   have   been   initiated   to  determine    the 
sequence   of  irreversible    subcellular  changes   occurring  during 
liminary  results   have   demonstrated   that    subcellular   systems   di 
in   their   sensitivity   to   ischemia.      For  example,   Na'^,   id'-ATPase 
stable,   whereas  mitochondrial  preparations   show  rapid   loss   of 
control  and   rates   of   oxygen  consumption;   adenyl  cyclase   respon 
monal   stimulation  appears   to  be    indetermediate „      Parallel   stud 
progress   in  which   ischemic -induced   reversible   and   irreversible 
cardial   contractility  will  be    correlated  with   subcellular   func 
After  defining   some    of  the    subcellular  mechanisms   responsible 
cardial   function  during   ischemia,   we   hope    to  develop   technique 
ia -induced   subcellular   injury,   and   therefore   physiologic    funct 
minimized. 


impaired  rayo- 

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Little    information   is  available   concerning   the   effects    of  hypoxia    or 
ischemia    on   the   capacity  of   the   heart   to  respond   to  various    inotropic   agents. 
During   the   course    of   studies   designed   to  investigate    this   problem,   we    found 
that   in  a    papillary  muscle   preparation   the    positive    inotropic   actions   of   two 
analogues   of  angiotensin  II  had  unique   characteristics,    i.e.,    inotropic 
potency  was  enhanced  by  hypoxia.      In  contrast,    the    concentration  response   curves 
cf  both  norepinephrine   and   ouabain  were   depressed  when   the   papillary  muscle 
was  exposed   to   low  oxygen   tensions.      In   order   to  evaluate    the   effects   of  hypoxia 
on  the    responsiveness   of  the    intact  heart   to  various   inotropic   agents,   a   dog 
heart-lung  preparation  was   developed   in  which  afterload,    preload,   and  heart 
rate   can  be   held   constant.      In   this  preparation,    in  contrast   to  cat   papillary 
muscle,    the    contractile    response    to  norepinephrine   was  augmented  during  hypoxia, 
the   dose-response    curve    shifting   to   the    left.      Furthermore,   no  deterioration   of 
the   preparation   occurred   after   the   highest   doses   of  norepinephrine   employed. 
Studies   are    in   progress   to  evaluate    the   possible   biochemical   or  physiologic 
basis   of   this   phenomenon.      The   effects   of  hypoxia,   as  well  as   ischemia,    on   the 
response   to  other   inotropic  agents  also  will  be   evaluated. 


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CORONARY  ARTERY  DISEASE 
Treatment   of  Coronary  Artery  Disease 

a)      Acute  Myocardial   Ischemia »     Approximately  40%  of   patients  who 
suffer  an  acute  myocardial   infarction  die,   and   two-thirds   of    these   deaths 
occur  within   the    first    few  hours,    before    the   patient    is   admitted   to  a   hospi- 
tal.     Death  under   such  circumstances    is   probably  due    to  cardiac  arrhythmias. 
To  evaluate    1)    the   type   of  arrhythmias   occurring  during  the   early  phases   of 
myocardial   infarction  and   2)    the   relative   efficacy  of  various  antiarrhythmic 
agents    in   this    setting,   we    studied   40  closed-chest   conscious   dogs    in  which 
acute  myocardial   ischemia   was   produced   by   inflating  a    balloon   cuff   previously 
implanted  around   the    left  anterior  descending   coronary  artery   just   distal   to 
its   first   diagonal  branch.      About   one -ha If   of  the   dogs   did  not   develop  arrhyth- 
mias  during   the    first   hour   of   occlusion „      In   60  per  cent    of   these,   however, 
sudden   release    of   occlusion  was    followed    15    seconds   to  3  minutes    later  by  ven- 
tricular  arrhythmias   which  progressed   rapidly   to  ventricular   tachycardia    (VT) . 
Reocclusion   of   the    coronary  artery  abolished    the  VT  with  a    return   to  a    sinus 
mechanism   in  all  dogs  with   release   arrhthmias.      Increasing   the   heart   rate   by 
either   pacing   or   the   administration   of  atropine    prevented   the    reappearance    of 
VT  when   the   occluding  balloon  was  again  released.     These   results   indicate   that 
1)    sudden   reperfusion   of  a   previously    ischemic   region   of  myocardium,    as  may 
occur   in  man   by   lysis   or  dislodgment    of  a    clot,   may  be    responsible    for   some 
of   the    serious  arrhythmias    seen   in  acute   myocardial   infarction  which   result 
in    sudden  death,    and   2)    increasing   the   heart   rate   by   the  administration   of 
atropine   may  be    successful   in   the   prevention   of  many,   but   not   all,    of   these 
arrhythmias.      In   contrast    to   this   group,    the   remainder   of   the   dogs   developed 
frequent   and   persistent  ventricular  ectopic   beats   or  episodes   of  VT  within 
the    first   one    to   two  hours   of   occlusion.      In  most   of   these   dogs   increasing 
the   heart   rate   with  atropine    or  administering    lidocaine  markedly  decreased   or 
completely  abolished   the   arrhythmias.      In   several   dogs,   however,    the   adminis- 
tration  of  atropine    or   lidocaine   alone   was   only  partially  effective,   and 
ectopic  activity  was  eliminated   only  after  administration   of   the    combination 
of   these    two  drugs. 

Although,    as    indicated   above,   atropine    is  effective    in   treating 
arrhythmias   occurring  during   the   onset   of  acute   myocardial   ischemia,    it    is  not 
known  whether   the   atropine-induced    increase    in  heart   rate   has   any   deleterious 
effect   on   the   degree   of  myocardial   ischemia.      We    therefore    studied    the    S-T 
segment   response    (obtained    from   10   to   12    implanted    intramyocardial  electrodes) 
to   repeated    five-minute    occlusions   of   the    left  anterior  descending  coronary 
artery   in   closed-chest   conscious   dogs   in  which  we   previously  had    implanted  an 
inflatable   balloon  around    the    left  anterior  descending   coronary  artery.      We 
found    there   was  a   direct    linear  relationship  between  heart   rate    (range    35-150 
beats/min)    and   the   degree    of    S-T   segment  elevation.     We    conclude    that    if   the 
degree    of   S-T  elevation   reflects    severity   or  extent   of   ischemia,    increases    in 
rate    in  experimentally  produced  acute   myocardial   ischemia   are   associated  with 
increases   in  the   degree   of  myocardial   ischemia.      Thus,   when  atropine    is 
administered   to  control  bradycardia -induced   arrhythmias   during  acute  myocardial 
ischemia,    the    lowest   effective   dose    should   be   used  and  excessive    increases    in 
rate   avoided.     There   also   seems   to  be   no   justification   for   the    routine   adminis- 
tration  of  atropine,    regardless   of   rate,    to  a    patient   already  admitted   to  a 


coronary  care  unit  and  under  constant  observation  since  arrhythmias  can  be 
treated  as  they  occur,  thereby  avoiding  the  deleterious  effect  of  unneces- 
sary  increases    in  heart   rate    on   the   degree    of  myocardial   injury. 

The  Montgomery  County  Heartmobile    is   a   mobile   coronary   care   unit   capa- 
ble   of  delivering  emergency  resuscitative    treatment   to  any  patient    in  a    defined 
geographic  area   within   5  minutes   of  alert.      We   are    currently  engaged   in  a 
collaborative   effort   to  determine   the   type   of  arrhythmias   that   occur  during 
the   prehospital  phase    of  acute  myocardial   infarction   in  man  and   to  determine 
the    relative   efficacy  of   atropine   and    lidocaine    in   the    treatment   of   such 
arrhythmias.      The   results    of   this   collaborative   effort  will  hopefully  yield 
additional   information   regarding   the    question  as   to  whether   the    self -adminis- 
tration  of  either   of   these    two  agents    is   desirable  when   sjmiptoms    of  acute 
myocardial   infarction  are   evident   but  when   some    time   will   be   required  before 
the    patient   can  be    seen  by  a    physician, 

b)      Chronic  Angina    Pectoris „     We   have    studied   the    symptomatic  and 
circulatory  effects   of  a    six -week  program  of  intense   physical  training   in 
patients  with  angina   pectoris  due   to  coronary  artery  disease.     After   training 
the    intensity   of  exercise   attained   before   angina    increased   57%.      The    triple 
product    (TP)    of  aortic    systolic   pressure,   heart    rate,   and  ejection   time   was 
calculated  and   used  as   an   index   of  myocardial   oxygen  consumption    (MVO2) o      TP 
(and  presumably  MVO2)   at  any   level   of  exercise  was   less  after   training,   thus 
accounting   for  part   of   the    improved  exercise    capacity.     However,    after   train- 
ing a   higher  TP  could   be   achieved  before    the   onset    of   ischemic   pain   in  over 
half   of   the   patients.      If   changes   in  TP  accurately  reflect   changes   in  MVO2, 
then  the    finding  that   ischemic   pain  occurred  at  a  higher  TP  suggests   that 
training,    in  addition  to  improving   the   efficiency   of   the    circulatory  response 
to  exercise,   might  also   improve  myocardial   oxygen  delivery. 

It  has  been   suggested   that  a  major  portion  of  the   beneficial  effects 
of  the  beta-blockers   in  treating  patients  with  angina   pectoris   could  be 
achieved  by  merely  depressing  the  heart   rate   response   to   sympathetic   stimu- 
lation.    Although  reports  have  appeared   suggesting  that   the  beta-blocking 
agent   practolol  has    such  effects,   we    found   in  dogs   that  a    given   reduction 
in  heart   rate   produced   by  propranolol  and   practolol  was  accompanied  by  the 
same   negative    inotropic   response.     We   also   found   that  while   practolol  may 
exert  a  modest  positive    inotropic  effect,   this  action   is  detectable   only  after 
full  beta-blockade   or  when  beta -receptor   stimulation  is  minimal;    such  an 
effect   is   inapparent   if  beta-receptor   stimulation   is   increased,   which  is   often 
the   case   when  beta-blockade    is  employed   clinically.      Thus,    practolol  as   used 
therapeutically  does  not  appear  to  act   on  heart   rate  more    selectively  than 
propranolol. 

Although  many   operative   attempts  have   been  employed   to  treat   patients 
with  coronary  artery  disease,   no  procedure   appears   to  be  more   promising  than 
the   saphenous  vein  bypass   operation.     We  are   currently  involved   in  a    collabora- 
tive effort  with   the   Surgical  Branch   to  assess   the   results   of  bypass  as  well 
as   to  determine   the   type   of  anatomic   disease   that  would  be  most  amenable   to 
such  a   procedure . 


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Characterization   of  Fibrinolytic  Mechanisms 

Fibrinolysis  may  play  a   role   in  retarding   the   development   of  coronary 
artery  disease   by  dissolving   fibrinous  deposits  at    the    site   of  endothelial  I 

injury.      Factors   that  alter   fibrinolysis    in  man  are    largely  unknown.     We 
have    shown  previously  that   the   magnitude    of   the    fibrinolytic   response    to  ] 

exercise   was   related   to  the   duration  and   intensity   of  exercise,   as  well  as   to 
the    time   of  day  it  was  performed.     We  also  observed   that    fibrinolytic  activity 
failed   to  increase   normally  during   intense   exercise   performed  by  a    group   of 
patients  with   type    IV  hyperlipoproteinemia.     More   recently  we   have   demonstrated 
that   the   diurnal  augmentation   of    fibrinolytic  activity  normally   occurring   in 
young   subjects    is   encountered    less    frequently   in   older   subjects.      Patients  with 
type   IV  hyperlipoproteinemia,   a    condition  associated  with   the   early  develop- 
ment  of  atherosclerosis,    show  an   impairment   that   is   greater   than   that   which 
can  be   attributed   to  age   alone.     Moreover,   patients  with  coronary  artery 
disease   but  with  normal   lipid   studies   demonstrate    smaller  diurnal   increases 
than  young  normal   subjects.     Although   the    latter  differences   can  be   attributed 
partly  to  age-related   factors,    there    is  a    tendency   for   further   impairment   that 
appears   to  be    independent    of  age.     Whether   these   defects   play  a    role    in  the 
early  development   of  coronary  artery  disease    remains   to  be   determined,   as   does 
the   cause    of   the   defective    response. 

Evaluation   of  Myocardial  Function 

Several   important   indices   of  myocardial   performance   depend  upon  accurate 
and   frequent  measurement   of  ventricular  volume.      To  make    such  measurements 
feasible,   we   have   developed  a    semi-automated  method    for   the    continuous   deter- 
mination  of  ventricular  volume    in  man.     When  volumes  determined   by   the   automated 
method  are   compared  with   those    obtained  by  manual  planimetry,    the    correlation 
coefficient    is   0.96;   volumes    of   test   objects  are   accurate   to  within   67o.     This 
automated   technique    thus   permits   rapid  and  accurate  measurement   of  ventricular 
volume    in  all   patients  having  diagnostic    left  ventriculograms. 

CIRCULATORY   PHYSIOLOGY  AND   CLINICAL 
CARDIOLOGY 

We   have    shown   that   the   heart   rate    response    to  baroreceptor  mediated 
enhancement   of   syropathetic   activity   is   impaired   in  patients  with  depressed 
cardiac   function  as   is   the  maximum  heart   rate   response   to  exhausting  exercise. 
Although   the   etiology   of  this   impairment   has  not  been  defined,    the   defect   does 
not  appear   to  be   due   either   to  decreased    sensitivity  of   the   adrenergic   receptor 
site   or  differences   in  background  vagal   tone.      Its   potential   for  reversibility 
also   is   unknown.      To  more    fully   characterize    this  defect,    the  heart   rate   re- 
sponses  to   1)    baroreceptor  enhancement   of   S3rmpathetic  activity  and   2)    exhaust- 
ing exercise   have   been  determined   in   functional  class   II  and   III  patients  about 
to  undergo  cardiac   surgery.      Samples   of  atrial  and   left  ventricular  papillary 
muscles  have    been  obtained  at   the   time    of   operation   for  analysis   of  norepine- 
phrine   content   in  an  attempt   to  correlate   depletion   of   these    stores  with   the 
impaired  heart   rate   responses.     The  preoperative   studies  will  be   repeated  post- 
operatively  to  determine    if   the   anticipated   improvement    in   cardiac    function   is 
associated  with  an   improved  capacity  of  the  heart   to  respond   to   sympathetic 
stimulation. 


-6- 


Individuals  with  severe  obstruction  to  outflow  of  either  the  right  or 
left  ventricles  may  experience  syncope  during  or  immediately  after  strenuous 
exercise.   In  order  to  clarify  the  hemodynamic  basis  for  this  phenomenon, 
several  circulatory  indices  were  measured  while  dogs  with  pulmonary  artery 
constriction  exercised  to  the  point  of  syncope „   Pulmonary  constriction  was 
achieved  by  the  inflation  of  a  balloon  cuff  previously  implanted  around  the 
pulmonary  artery.   Preliminary  results  show  that  syncope  is  almost  always 
caused  by  rapidly  progressive  arterial  hypotension  rather  than  by  arrhythmia. 
Decline  in  cardiac  output  generally  accompanies  the  fall  in  blood  pressure, 
suggesting  that  inadequacy  of  cardiac  output  rather  than  sudden  vasodilatation 
is  responsible  for  the  decline  in  blood  pressure  which  eventuates  in  circula- 
tory collapse. 


The  heart  rate  of  anesthetized  or  alert  hyperthyroid  animals  is  in- 
creased, as  is  the  intrinsic  frequency  of  contraction  of  hearts  isolated  from 
such  animals.   However,  we  have  found  that  the  basal  heart  rates  of  intact 
unanesthetized  cats  are  not  different  from  those  of  normal  cats.  To  explain 
these  differences,  we  have  examined  the  possibility  that  the  negative  chrono- 
tropic effect  of  the  vagus  is  enhanced  in  hyperthyroid  cats.   Preliminary  re- 
sults have  shown  that  several  cardiac  actions  of  acetylcholine  are  enhanced  in 
hearts  of  hyperthyroid  cats,  findings  compatible  with  the  hypothesis  that  the 
normal  basal  heart  rate  of  hyperthyroid  animals  is  due  to  increased  sensitivity 
of  the  sinus  node  to  acetylcholine. 

The  maximal  functional  capacity  of  the  hearts  of  patients  who  have 
undergone  "total  correction"  of  congenital  heart  defects  has  been  evaluated 
by  catheterization  at  rest  and  during  intense  treadmill  exercise.   Patients 
whose  atrial  septal  defects  have  been  completely  closed  and  whose  routine 
catheterization  studies  are  normal  may  have  residual  impairment  of  the 
cardiac  output  response  to  intense  upright  exercise  in  the  absence  of  pulmonary 
arterial  hypertension.  Patients  with  corrected  tetralogy  of  Fallot  have 
minimal  impairment  of  their  cardiac  output  response,  but  may  markedly  increase 
their  right  ventricular  outflow  gradient  and  right  ventricular  pressure 
during  exercise , 


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Annual  Report  of  the 

Section  on  Clinical  Biophysics 

Cardiology  Branch 

National  Heart  and  Lung  Institute 

July  1,  1970  through  June  30,  1971 

The  activities  of  this  Section  have  continued  along  the  lines  outlined  in 
last  year's  report.   Our  principle  efforts  have  centered  around  two  related 
areas  of  research:   Experimental  atherosclerosis  and  vascular  physiology.   In 
experimental  atherosclerosis  we  have  completed  the  first  major  phase  of  our 
program  which  was  designed  to  examine  the  detailed  topography  and  histology  of 
spontaneously-occurring  and  induced  atherosclerosis  in  swine  and  dogs.   We 
have  confirmed  the  findings  of  others  that  atherosclerosis  occurs  spontaneous- 
ly in  swine,  is  accelerated  by  feeding  a  high-fat,  high-cholesterol  diet,  and 
is  further  augmented  by  inducing  the  hypothyroid  state.   We  have  also  con- 
firmed the  fact  that  dogs  do  not  normally  develop  atherosclerosis  unless  the 
hypothyroid  state  is  induced.   Moreover,  we  have  found  that  the  porcine  ath- 
erosclerosis has  a  histologic  picture  which  closely  resembles  the  disease  in 
human  subjects,  i.e.,  it  is  usually  associated  with  a  marked  intimal  fibro- 
muscular  hyperplasia,  whereas  in  dogs  the  lesions  appear  more  xanthomatous. 
The  cytologic  characteristics  of  the  disease  process  in  both  species  using 
both  a  light  microscopy  and  electron  microscopy  was  also  found  to  be  consis- 
tent with  that  reported  in  the  literature. 

In  contrast  to  the  aho^re,    certain  new  observations  emerged  from  our 
experiments:   First  it  was  found  that  the  gross  topography  of  the  lesions  in 
early  disease  was  strikingly  similar  among  all  groups  of  animals  of  both 
species.   The  sites  of  greatest  occurrence  of  lesions  were  in  areas  of  vessel 
branch  points  and  their  associated  entrance  regions.   The  sites  of  highest 
lesion  incidence  were:   the  lower  aortic  trifurcations,  the  sinuses  of  Valsal- 
va, the  aortic  arch  orifices,  the  major  branch  orifices  in  the  upper  abdominal 
aorta,  the  entrance  regions  of  both  the  coronary  arteries,  as  well  as  the 
main  bifurcation  and  other  branch  points  of  the  extramural  coronary  arteries. 
The  presence  of  grossly  apparent  fibromuscular  intimal  thickening  in  the  older 
animals  did  not  significantly  alter  the  topography  of  lipid  deposition. 

Although  the  over-all  topographical  distribution  of  lesions  in  the  dif- 
ferent animal  preparations  and  different  species  were  strikingly  similar, 
careful  study  of  the  detailed  topography  at  each  anatomic  site  showed  interest- 
ing, consistent,  and  different  patterns  of  sudanophilia.   A  classification 
scheme  was  developed  so  that  these  patterns  could  be  quantified.   Analysis  of 
the  topography  using  this  scheme  showed  that  sudanophilia  patterns  appear  to 
be  unique  not  only  with  respect  to  anatomical  site  but  also  to  the  type  of 
animal  preparation.   Although  the  underlying  causes  for  these  intriguing 
differences  presently  remain  obscure,  they  are  of  obvious  etiologic  and  patho- 
genic significance,  pointing  to  the  presence  of  strong  localizing  factors  both 
of  a  hemodynamic,  histologic,  and  chemical  nature.   These  observations  have 
been  incorporated  into  the  experimental  design  of  the  second  phase  of  this 
program  and  will  be  pursued  as  appropriate  techniques  are  developed. 

We  have  also  found  that  lesions  which  have  the  histologic  appearance  of 


fatty  streaks  in  many  cases  go  on  to  develop  into  classical  raised  atheroma- 
tous lesions  indicating  that  the  fatty  streak  can  be  an  early  form  of  true 
atherosclerosis.  We  also  observed  an  inverse  relationship  between  the  pre- 
sence of  dense,  oriented  collagen  and  lipid  deposition.   This  was  seen  in  two 
circumstances:   at  branch  points  and  in  pre-existing  areas  of  f ibromuscular 
hyperplasia. 

At  branch  points  areas  of  collagen  are  laid  down  in  a  pattern  suggesting 
that  it  is  in  response  to  chronic  stress  exposure  related  to  the  hydro- 
mechanical  forces  in  these  regions.   For  example  a  dense  sheet  of  collagen  is 
developed  over  the  flow  divider  of  most  junctions ■ forming  a  tough  protective 
sheath  buttressing  the  endothelial  cells  against  the  increased  forces  required 
to  deflect  the  blood  flow  at  these  points.   Lipid  deposition  in  these  regions 
of  dense  collagen  is  seen  only  rarely  and  then  only  when  the  serum  lipid 
levels  have  been  elevated  to  very  high  values.   This  may  explain  the  unique 
detailed  topographic  distribution  of  lipid  that  was  described  above,  i.e., 
the  detailed  topographic  patterns  of  sudanophilia  may  be  related  to  the  dis- 
tribution of  pre-existing  less  permeable  fibrous  structures. 

While  fibromuscular  proliferation  and  collagen  deposition  could  also  be 
interpreted  as  a  result  of  lipid  infiltration  (i.e.,  in  response  to  the 
"toxic"  effects  of  lipid),  we  have  observed  a  number  of  instances  where  lipid 
deposition  and  fibromuscular  proliferation  were  observed  to  occur  separately. 
This  suggests,  at  least  under  certain  circumstances,  these  processes  are  in- 
dependent of  one  another.   For  example,  lipid  infiltration  commonly  was  seen 
in  the  absence  of  fibromuscular  changes  in  dogs  with  elevated  serum  lipids, 
and,  conversely,  fibromuscular  proliferation  was  frequently  observed  in  the 
absence  of  stainable  lipid  in  both  euthyroid  swine  and  dogs. 

Further  evidence  for  the  independence  of  these  processes  was  found  in  a 
study  designed  to  show  the  potentiating  effect  on  atherogenesis  of  increased 
blood  flow  (shear  stress) .   In  these  studies  arteriovenous  shunts  were  con- 
structed both  for  the  carotid  artery  and  for  the  iliofemoral  artery  such  that 
the  shunted  artery  in  each  case  carried  about  five  times  as  much  flow  as  the 
contralateral  control  artery.   The  shunted  iliofemoral  artery  responded  to 
this  increased  stress  with  a  marked  increase  in  atherogenesis,  whereas  the 
shunted  carotid  artery  responded  with  marked  fibromuscular  hyperplasia  but  no 
significant  increase  in  lipid  deposition.   These  studies  point  to  the  fact  the 
arterial  interface  does  respond' to  increased  hydromechanical  forces  and  de- 
pending on  local  tissue  factors  can  respond  with  increased  fibromuscular 
hyperplasia,  or  with  increased  lipid  deposition. 

Atheromatous  involvement  of  the  media  which  has  been  described  in  man 
also  was  observed  in  both  species  of  animals  studied  here.   Three  forms  of 
involvement  were  observed.   The  first  type  of  involvement,  which  is  similar  to 
that  reported  in  the  literature,  occurred  in  areas  of  matured  atheromatous 
lesions  and  appeared  to  represent  a  simple  spillage  of  the  atheromatous 
material  from  the  plaque  through  a  broken  internal  elastic  lamella  into  the 
medial  region.   In  this  situation  the  lipid  had  a  coarse,  granular  appearance 
and  appeared  to  evoke  strong  tissue  reaction  in  the  form  of  phagocytosis  and 
smooth  muscle  infiltration.   This  second  type  of  medial  involvement  occurred 
most  frequently  in  elastic  arteries  and  consisted  of  a  homogeneous  deposition 
of  oil  red  0  positive  material  which  was  diffuse  and  tended  to  fill  all  of 

9 


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the  interlamellar  spaces  of  the  wall.  This  type  of  involvement  appeared  to 
be  more  physiological  in  that  it  did  not  evoke  any  apparent  tissue  response. 
It  was  seen  most  commonly  in  the  ascending  aorta  and  frequently  would  extend 
from  media  to  the  adventitial  capillary  bed,  suggesting  that  it  might  repre- 
sent an  augmented  but  normal  flux  of  lipoproteins  across  the  arterial  wall  as 
a  result  of  hyperlipidemia.   The  third  type  of  involvement  appeared  to  be 
similar  to  the  second;  however,  it  usually  occurred  in  areas  with  the  pre- 
existing intimal  involvement  at  the  periphery  of  f ibromuscular  thickenings 
and  was  occasionally  associated  with  some  cellular  response. 

The  physiological  studies  which  bear  on  the  foregoing  observations  have 
been  carried  out  both  ijn  vivo  and  in  vitro.   In  an  effort  to  demonstrate 
further  that  the  vascular  interface  responds  to  the  adjacent  hydromechanical 
forces  another  study  was  done  in  addition  to  the  aforementioned  shunt  studies. 
In  this  study  it  was  observed  that  the  orientation  of  endothelial  nuclear 
patterns  appears  to  correspond  to  the  adjacent  shear  stress  distribution 
pointing  in  the  direction  of  the  streamlines  of  flow.   For  example,  endo- 
thelial cells  upstream  from  regions  of  orifices  all  tend  to  point  toward  the 
orifice  whereas  in  relatively  uniform  straight  sections  of  vessel  the  nuclei 
tend  to  have  a  longitudinally-oriented  pattern.   One  such  straight  segment  is 
the  descending  thoracic  aorta  which  invariably  has  elongated  nuclear  endo- 
thelial nuclei  all  pointing  parallel  to  the  axis  of  the  blood  vessel.   A  seg- 
ment of  the  descending  thoracic  aorta  was  removed,  opened  longitudinally,  and 
then  the  two  cut  circumferential  edges  were  sewed  together  to  form  a  tube 
again,  the  axis  of  which  now,  however,  was  90°  removed  from  its  original  axis, 
such  that  the  endothelial  cells  now  all  point  circumferentially.   This  tubular 
segment  was  then  resewn  into  its  original  bed  and  the  subsequent  changes  in 
endothelial  cell  pattern  observed  sequentially  over  the  ensuing  three  weeks. 
It  was  found  that  within  three  days  the  endothelial  cells  begin  to  turn  from 
their  circumferential  orientation  toward  a  longitudinal  orientation  and  by 
ten  days  all  cells  are  now  oriented  again  in  longitudinal  direction.   This, 
with  the  af oremantioned  A-V  shunt  data,  again  shows  that  the  vascular  inter- 
face is  responsive  to  the  adjacent  hydrodynamic  forces. 

A  series  of  in  vivo  studies  have  shown  that  the  topography  of  the  flux 
of  Evans  blue-tagged  albumin  appears  to  be  greatest  in  the  same  areas  that 
the  aforementioned  homogeneous  oil  red-0  staining  material  was  greatest. 
These  data  taken  together  with  the  studies  of  Duncan  who  showed  the  flux  of 
cholesterol  also  was  greatest  in  the  ascending  aorta,  lend  further  support  to 
the  aforementioned  suggestion  that  this  particular  type  of  medial  lipid  depo- 
sition may  be  related  to  a  normal  transmural  flux  of  lipoproteins. 

A  number  of  in  vitro  experiments  have  been  carried  out  to  study  the  fac- 
tors influencing  protein  transport  across  the  vascular  interface.  A  specially 
designed  device,  described  in  detail  in  previous  communications,  was  used  in 
which  the  thermal,  mechanical,  and  chemical  milieu  of  the  blood  vessel  could 
be  controlled  or  measured.   In  this  system  it  was  possible  to  estimate  the 
mass  transport  of  a  given  protein  species  (albumin  in  this  case)  as  a  function 
of  both  time  and  these  other  variables.   It  was  shown  that  the  excised  blood 
vessel  remains  "physiologically"  normal  for  periods  up  to  four  hours  provided 
it  is  kept  under  autologous  serum.  The  salient  aew  findings  were:   first,  a 
potent  barrier  to  albumin  flux  esists  in  a  local  region  associated  with  the 


endothelial  cells.   Removal  or  damage  of  the  endothelial  cells  causes  a  100- 
to  1000-fold  increase  in  the  flux  of  albumin  across  the  vascular  interface. 
Second,  transmural  pressure  differences  of  up  to  280  cm  of  water  pressure  do 
not  influence  the  flux  of  albumin,  either  in  the  presence  or  absence  of  endo- 
thelial cells.   This  suggests  that  protein  flux  across  the  arterial  wall  in 
the  presence  of  a  normal  media   is  not  influenced  by  the  pressure  drop.   Third, 
the  flux  of  albumin  across  the  interface,  however,  is  greatly  increased  when 
the  surface  is  stretched.   Finally,  a  wide  variety  of  solvents,  including 
saline,  and  particular  solutions  containing  certain  polar  solvents  such  as 
ethyl  alcohol  and  acetone  greatly  increase  the  permeability  of  the  interface 
to  protein. 

In  summary,  these  studies  indicate  that  the  major  barrier  to  albumin 
(probably  also  lipoprotein)  transport  exists  locally  in  the  region  of  the 
endothelial  surface;  it  depends  on  the  presence  of  normal  endothelial  cells; 
it  is  weakened  either  by  the  elution  of  some  material  or  by  decreasing  the 
oncotic  properties  of  the  adjacent  fluid;  it  is  decreased  by  stretching  and 
exposure  to  increased  shear  stress;  it  is  not  influenced  by  the  transmural 
pressure-  itself.   These  studies  are  being  continued  to  define  the  associated 
transport  mechanisms  in  greater  detail. 

A  number  of  theories  have  been  proposed  to  explain  the  pathogenesis  of 
atheromata.   The  essence  of  these  can  be  considered  under  three  major  head- 
ings; the  filtration,  the  intrinsic  and  the  thrombogenic  theories.  The  fil- 
tration theories  consider  the  increased  deposition  of  lipid  to  be  the  result 
of  an  imbalance  between  the  lipoprotein  flux  into  and  out  of  the  intimal 
tissues.   The  intrinsic  theories  consider  the  increased  lipid  deposition  to  be 
secondary  to  local  alterations  in  tissue  chemistries,  such  that  increased 
amounts  of  lipid  are  being  synthesized  in  situ  or  that  insoluble  lipid  accumu- 
lates from  accelerated  breakdown  of  lipoprotein  molecules.   The  thrombogenic 
theories  suggest  that  the  initiating  process  is  the  formation  of  microthrombi 
o.T  the  endothelial  surface  with  secondary  changes  in  local  lipid  transport  and 
metabolism. 

The  data  presented  in  this  report  are  not  at  odds  with  any  of  these  ideas 
and  in  fact  tend  to  reconcile  some  of  the  apparent  differences  in  viewpoint. 
All  of  these  ideas  can  be  related  at  least  in  theory  to  predisposing  hydro- 
m.echanical  events.  For  example,  the  flux  of  proteins,  including  lipoproteins 
into  or  out  of  the  animal  tissues,  can  be  altered  significantly  by  the  state 
of  stress  and  the  associated  chemical  milieu  as  was  shown  in  our  physiological 
studies.   Moreover,  protein  synthesis,  including  the  laying  down  of  oriented- 
structured  proteins,  such  as  collagen,  has  been  shown  to  be  related  to  chronic 
exposure  to  stress  in  studies  of  bone  and  tendon  by  other  workers.   Further- 
more the  rate  of  synthesis  of  cholesterol  by  fibroblasts  has  been  shown  by 
Avigan  to  be  increased  by  prior  mechanical  agitation.   Finally,  endothelial 
injury  caused  by  increased  exposure  to  shearing  stress  results  in  increased 
permeability  and  influx  of  serum  proteins,  as  well  as  an  increased  affinity 
for  platelet  adhesion  and  fibrin  deposition,  as  shown  by  previous  studies  from 
this  laboratory.   Thus,  when  the  foregoing  theories  of  atherogenesis  are 
viewed  together  with  the  present  data  they  appear  entirely  compatible.  •  It 
would  be  unwise  to  consider  any  one  of  them  the  sole  process  leading  to  the 
formation  of  atheroma.  The  present  groups  of  studies  would  suggest  that  each 


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of  these  theories  probably  represents  only  one  component  of  a  complex  system 
of  independent  and  often  interacting  atherogenic  processes,  many  of  which  are 
coupled  to  associated  mechanical  events. 

The  characteristic  topographic  patterns  of  lipid  deposition  and  con- 
sistent location  of  f ibromuscular  intimal  thickenings  described  in  these 
studies  point  to  local  factors  that  act  at  a  level  of  resolution  not  pre- 
viously appreciated.   If  we  are  to  try  to  establish  correlations  between 
mechanical  stresses,  local  rheologic  properties,  structural  protein  patterns, 
histochemical  properties  and  local  tissue  biochemistry,  we  cannot  rely  upon 
the  relatively  gross  techniques  currently  available  but  instead  must  evolve 
a  new  battery  of  more  sophisticated  microtechniques  which  implies  the  need 
for  acquiring  new  talent,  special  tools,  expanded  collaborative  efforts, 
contractural  help,  and  an  increased  budget  to  accommodate  these  needs. 


/X 


Annual  Report  of  the 

Section  of  Pathology 

National  Heart    and  Limg  Institute 

July   1,    19  70   to  June   30,    1971 

This  section  is    concerned  with   structural   changes  produced   in  heart, 
blood  vessels,    and   lung  by   cardiovascular  and  pulmonary   diseases.      This 
section  works  primarily  with  human    "material,"  much   of  which   is   submitted   from 
hospitals    and  institutions    outside    the  Clinical   Center   of   the  National 
Institutes   of  Health.      The  number   of  hearts   submitted   to    this   section  have 
progressively   increased:      in   1967,    the  number  was   159;    in  1970,    the  number  was 
298,      The  submitted  specimens    are   studied   routinely  by   gross   and  histological 
means    and   occasionally  by   electronmicroscopic   means.      The  number  of  paraffin 
blocks  prepared   in   the  section's   histology   unit   in   1970  were    3,789.      The 
number  of  hematoxylin  and   eosin  stains  performed  was   5,246;    the  number  of 
special  stains,    3,425;    and   the  number   of   unstained  slides,    10,764.      Thus, 
19,435   slides  were   cut   and   8,671   slides    stained   during   this    12-month   period. 
Approximately    4,000   of   the   19,000   slides    cut  were   large    (3x1.5   inches).      All 
gross   photography  performed    for   this   section  has  been   done   in   the  Laboratory 
of   Pathology,    National   Cancer   Institute. 

Coronary  Heart   Disease:      A   continuing  project   of   this   section  has  been 
the  study   of  major  extra-   and   intramural   coronary   arteries   of  patients    dying 
of  acute  myocardial  infarction.      During   the  past   2   years    92     patients  have 
been  studied  at  necropsy.      The   3  major  extramural   coronary   arteries  were 
excised  intact,    decalcified,    sectioned,    processed,    and    3  histologic  sections 
from  each   0.5    cm.    segment  were   examined.      Among   the      92  patients,    66  had 
transmural  myocardial   infarcts,    7  has   subendocardial   infarcts,    and   19    died 
suddenly    (<6  hours)   without  myocardial  necrosis.      Thrombi  were    found   in  extra- 
mural  coronary   arteries    in   33    (50%)    of   66   patients  with   transmural    infarcts, 
in  0   of    7  with   endocardial    infarcts,    in   2   of   19   patients  who   died  suddenly. 
The  number  of  major  extramural   coronary    arteries    (3  per  patient)    narrowed 
greater   than    75%  by  old   atherosclerotic  plaques  was    2.4   in   the   transmural 
cases,    2.0   in   the  subendocardial   cases,    and   2.3  in   the   sudden   death  patients. 
Of   the  many    thousands    of  slides   of   coronary    arteries    examined   in   the  92 
patients,    only    3  sections   showed  normal    coronary   arteries.      Although    the   degree 
of   luminal  narrowing   varies   considerably,    coronary    arterial   atherosclerosis   is 
a  diffuse   disease.      The   lumens   of   the    coronary   arteries   distal   to   sites   of 
thrombosis  were   >75%   narrowed  by   old  plaques   in    33   of   the    35  patients  with 
thrombi.      The    coronary   artery    distal   to   a  thrombus   has   never  been  studied 
previously.      Since   thrombi  were   rare   in  sudden   death    cases,    and   since    they 
occurred   only   in   the   coronary   arteries   of  patients  who   already  had  severely 
narrowed  vessels,    it   is  believed   that    thrombi   are   consequences    rather   than 
causes    of  myocardial    infarcts.      Thrombi  were    observed   almost   exclusively  in 
patients    dying  with    features    of   the  pump    failure   syndrome    (shock   or   severe 
congestive  heart    failure). 

Prosthetic  Cardiac  Valves:  A  continuing  effort  of  this  section  has  been 
the  morphologic  evaluation  of  hearts  and  other  organs  of  patients  dying  after 
replacement  of  one  or  more  cardiac  valves  with  prosthesis.  From  1963  through 
1970,    181  patients    died   after  valve    replacement:      107  within   2  months   of 


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operation,  and  74  at  later  periods  up  to  80  months.   Among  the  causes  of  death 
early,  prosthetic  dysfunction  was  responsible  in  30%,  no  anatomic  cause  was 
found  in  28%,  bleeding  and  technical  mishaps  19%,  infection  4%,  and  mis- 
cellaneous causes  5%.   Among  the  74  patients  who  died  late,  prosthetic  dys- 
function accounted  for  death  in  37%  and  factors  secondarily  related  to 
prostheses  in  24%.   All  patients  who  died  late  following  aortic  valve  replace- 
ment had  intimal  fibrous  proliferation  in  the  aortic  roots.   Whether  or  not 
the  coronary  arterial  ostia  will  become  narrowed  with  time  following  aortic 
valve  replacement  remains  to  be  seen.   The  degree  of  intimal  proliferation  in 
the  ascending  aorta  in  patients  who  died  late  was  proportional  to  the  degree 
of  renal  hemosiderosis,  indicating  that  the  aortic  lesion  is  due  to  turbulence 
of  blood  traversing  the  aortic  valve  and  that  this  turbulence  causes  intra- 
vascular hemolysis  which  is  apparent  in  the  kidney  by  deposition  of  iron  in 
renal  tubules,   l^ether  or  not  the  metallic  ball  now  used  in  Starr-Edwards 
prostheses  will  hold  up  over  many  years  remains  to  be  seen.   There  is  sug- 
gested evidence  now  that  the  cloth-covered  struts  will  show  evidence  of  wear 
after  about  3  years.   Although  additional  years  of  life  have  been  provided  to 
many  critically  ill  patients  froFi  severe  valvular  heart  disease  by  cardiac 
prostheses,  it  is  apparent  10  years  after  valve  replacement  began  that  the 
ideal  cardiac  valve  is  not  presently  available.   It  would  appear  from  this 
study  that  the  caged-ball  rigid  frame  prostheses  are  simply  not  capable  of 
functioning  properly  in  a  few  selected  hearts  with  either  small  left  ven- 
tricular cavities  or  small  aortas. 

Aortic  Valve  Disease:   Among  the  congenital  malformations  of  the  aortic 
valve,  the  least  understood  and  the  least  well  recognized  is  the  unicuspid 
unicommissural  valve.   This  valve  is  the  most  common  cause  of  aortic  stenosis 
during  the  first  year  of  life,  but  its  recognition  in  adults  has  been  in- 
frequent.  Twenty-one  adult  patients  found  at  necropsy  or  surgery  to  have 
unicommissural  valves  were  studied.   Although  this  basic  valve  structure  may 
render  it  inherently  stenotic,  the  long  duration  of  the  murmur  and  the  late 
age  of  onset  of  symptoms  of  left  ventricular  outflow  obstruction  in  these 
patients  strongly  suggest  that  stenosis,  at  least  in  part,  is  acquired. 
Since  these  valves  are  characterized  by  only  one  commissure,  valvotomy  is 
hazardous  and  valve  replacement  appears  indicated  if  operation  is  indicated. 

Endocarditis :   A  major  project  of  this  section  during  the  past  year  has 
been  the  study  of  necropsy  patients  with  infective  (I)  and  non-infective  endo- 
carditis (E) .   Of  55  necropsy  patients  with  active  valvular  IE,  58%  had 
vegetations  on  previously  anatomically  normal  valves.   Predisposing  factors 
allowing  entrance  of  virulent  or  unusual  organisms  or  alteration  of  host  de- 
fense mechanisms  occurred  in  70%  of  patients  with  IE  on  previously  normal 
valves.   Valvular  dysfunction  occurred  in  70%  of  patients  with  IE  on  pre- 
viously normal  valves.   Valvular  dysfunction  occurred  in  70%  of  the  55 
patients,  causing  congestive  heart  failure  in  all.   Myocardial  lesions  were 
present  in  92%  of  the  39  patients  in  whom  multiple  histologic  sections  of 
myocardium  were  examined.   A  new  observation  was  the  finding  of  papillary 
muscle  necrosis  in  75%  of  patients;  mitral  regurgitation  occurred  as  a  result 
of  this  necrosis  in  only  one.   Since  myocardial  inflammation  was  focal  in  all 
but  2  patients  myocardial  lesions  are  not  believed  to  be  a  primary  cause  of 
congestive  heart  failure  in  patients  with  IE. 


^^ 


Twenty-nine  necropsy  patients  with  healed  valvular  IE  were  reviewed. 
Active  IE  had  occurred  on  anatomically  abnormal  valves  in  24  patients,  9  of 
whom  had  congenitally  bicuspid  aortic  valves.   All  29  patients  had  evidence 
of  valvular  dysfunction  during  and  after  the  active  IE  and  2  7  had  overt  con- 
gestive heart  failure.   Active  IE  was  the  sole  or  contributory  cause  of 
valvular  dysfunction  in  23  of  the  29  patients.   The  presence  of  fibrosis  of 
the  papillary  muscles  in  83%  of  the  29  patients  supports  the  view  that  this 
lesion  is  the  result  of  healed  papillary  muscle  necrosis,  observed  frequently 
in  the  patients  with  active  IE. 


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3- 


Five  patients  with  active  IE  confined  to  mural  endocardium  were  studied. 
This  type  of  endocarditis  is  unique.  Each  patient  had  an  underlying  malignant 
disease  and  the  causative  organism  in  each  was  a  fungus.  The  vegetations  were 
located  on  right  ventricular  endocardium  in  3  patients  and  on  left  atrial 
endocardium  in  2 .  In  each,  the  mural  IE  was  not  primary  but  rather  the  result 
of  extension  of  an  infective  process  originating  at  another  site.  No  evidence 
of  cardiac  dysfunction  occurred  in  any  of  the  5  patients. 


The  clinical  and  necropsy  features  of  45  patients  with  active  non- 
infective  ("marantic")  endocarditis  also  were  studied.   This  type  of  endo- 
carditis is  characterized  by  the  lack  of  organisms  demonstrable  on  histologic 
section  of  the  vegetation.   Vegetations  occurred  on  anatomically  normal  valves 
in  40  patients  and  in  functionally  normal  valves  in  44  patients.   Malignancy 
was  present  in  39  patients .   A  precordial  murmur  occurred  in  55%  of  the 
patients.   Systemic  emboli  to  the  brain  caused  death  in  6  patients. 

Congenital  Aneurysm  of  the  Ductus  Arteriosus;   Although  patent  ductus 
arteriosus  is  a  well-recognized  congenital  malformation,  aneurysm  of  this 
structure  rarely  has  been  appreciated.   An  aneurysm  at  this  site,  however, 
was  observed  in  a  2-month-old  child.   The  literature  in  60  previously 
described  patients  with  ductal  aneurysm  was  reviewed.   It  is  apparent  that 
the  aortic  end  of  the  ductal  aneurysm  is  always  patent  and  that  the  pulmonary 
arterial  end  may  or  may  not  be  patent.   Nearly  half  of  the  ductal  aneurysms 
develop  complications  (rupture,  embolism  or  infection)  and  therefore  operative 
resection  appears  warranted  when  this  diagnosis  is  established. 

Papillary  Muscle  Dysfunction:   A  unique  opportunity  presented  itself  when 
a  71-year-old  man  was  admitted  to  a  local  hospital  with  what  turned  out  to  be 
a  silent  acute  myocardial  infarction.   This  patient  presented  evidence  of 
.severe  congestive  heart  failure,  but  since  the  cause  of  the  heart  failure  was 
unknown  cardiac  catheterization  was  undertaken.   At  the  first  study,  when  no 
precordial  murmur  was  apparent,  hemodynamics  indicated  that  the  mitral  valve 
functioned  abnormally.   Two  weeks  later,  when  a  loud  murmur  of  mitral  re- 
gurgitation was  present,  hemodynamics  reconfirmed  severe  mitral  regurgitation. 
The  uniqueness  of  this  case  lies  in  the  fact  that,  for  the  first  time,  serial 
cardiac  catheterization  was  performed  in  a  patient  during  the  period  when 
papillairy  muscle  necrosis  was  taking  place.   Also,  hemodynamic  mitral  re- 
gurgitation was  demonstrated  before  auscultatory  mitral  regurgitation  was 
apparent. 


b  -s 


Electron  Microscopy 

The  electron  microscopic  unit  of  this  section,  which  became  fully 


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operational  in  April  19  70,  yielded  several  studies.   The  spleen  in  type  I 
hyperlipoproteinemia  was  studied  ultrastructurally .   Foam  cells  were  observed 
that  contained  a  material  identified  as  ceroid.   The  ceroid  was  organized  in 
the  form  of  granules  and  the  process  of  formation  of  these  granules  was 
described.   The  ceroid  was  considered  to  represent  non-digestible  end  products 
of  the  metabolism  of  chylomicrons  taken  up  by  macrophages  in  splenic  sinusoids. 
Several  cardiac  myxomas  were  studied  ultrastructurally.   The  electron  micro- 
scopic observations  support  the  concept  that  the  tumor  cells  are  derived  from 
endocardial  endothelial  cells.   Ultras tructural  observations  were  made  in  12 
patients  who  had  left  ventricular  outflow  myocardium  excised  at  time  of 
operation.   Each  of  these  patients  had  idiopathic  hypertrophic  subaortic 
stenosis  and  electron  microscopy  disclosed  that  the  ultras tructural  features 
of  this  entity  are  distinctive  and  are  characterized  by  severe  disorientation 
of  myocardial  fibers.   The  patterns  observed  in  the  muscle  excised  from  the 
left  ventricular  outflow  tract  were  compared  to  muscle  observed  at  left 
ventricular  apex  and  the  severe  disorientation  pattern  was  not  observed  in 
the  apical  biopsies.   The  features  of  the  muscle  in  the  left  ventricular  out- 
flow tract  are  similar  to  muscle  observed  in  newborns  and  suggest  that  this 
disorientation,  and  thus  the  basic  disease,  is  present  from  the  time  of  birth. 

Ultras tructural  studies  were  made  on  the  acute  and  chronic  effects  of 
normothermic  anoxia  on  canine  hearts.   All  16  dogs  subjected  to  45  minutes 
of  cardiac  anoxia  showed  extensive  myocardial  damage  ultrastructurally  where- 
as all  7  dogs  subjected  to  3  minutes  of  cardiac  anoxia  showed  only  minimal 
myocardial  damage.   These  observations  would  not  have  been  apparent  on  light 
microscopy  whereas  they  were  clearly  apparent  by  electron  microscopy.   The 
effects  of  hyperosmotic  perfusate  on  ultras tructure  and  function  of  the  iso- 
lated canine  heart  were  studied.   Eleven  dogs  were  studied:   5  were  perfused 
with  filtered  plasma,  and  6  with  filtered  p las ma -de xt ran.   Interstitial  edema, 
swelling  of  sarcoplasmic  reticulum,  and  mitochondrial  damage  were  observed  in 
each  of  the  5  hearts  perfused  by  filtered  plasma.   In  contrast,  interstitial 
edema  was  absent  in  each  of  the  6  hearts  perfused  by  filtered  plasma-dextran 
and  swelling  of  sarcoplasmic  reticulum  and  mitochondrial  damage  occurred  in 
only  2.   Thus,  the  osmolarity  of  the  perfusate  is  important  in  preventing 
edema  in  perfused  hearts  . 

A  continuing  study  of  this  section  has  been  the  ultrastructure  of  myo- 
cardium removed  at  biopsy  in  patients  with  primary  myocardial  disease.   The 
cardiac  biopsies  are  being  done  at  either  D.C.  General  Hospital  or  at  the 
Veterans  Administration  Hospital  in  Washington,  D.C.   Observations  at  this 
point  indicate  that  mitochondrial  alterations,  swelling  of  sarcoplasmic 
reticulum  and  dilatation  of  the  transverse  tubular  system  are  the  main 
alterations  present.   These  alterations,  however,  are  not  specific  for  primary 
myocardial  disease.   None  of  the  observations  has  proved  useful  from  a 
diagnostic  standpoint. 


4S 


Project  Title; 


Serial   No.     NHLI-1 

1,  Cardiology 

2,  Clinical  Physiology 

3,  Bethesda,  Md. 

PHS-NIH 
Indivudual  Project  Report 
July  1,  1970  through  June  30,  1971 

Potentiation  of  the  Inotropic  Effects  of  Norepinephrine, 
Glucagon,  and  Dibutyryl  Cyclic  AMP  by  Theophylline 


Previous  Serial  Number:   None 

Principal  Investigator:   C.  Lynn  Skelton,  M,  D. 

Other  Investigators:     Melvin  L.  Marcus,  Mo  D, 

Fred  E.  Karch 
Thomas  J.  Hougen 
Stephen  E.  Epstein,  M.  D. 


Cooperating  Units; 


None 


Project  Description:   The  positive  inotropic  response  to  catecholamines  and 
glucagon  has  been  postulated  to  result  from  an  increase  in  the  intracellular 
level  of  cyclic  AMP,  produced  by  activation  of  adenyl  cyclase.   If  this  hy- 
pothesis is  valid,  inhibition  of  phosphodiesterase,  the  enzyme  responsible 
for  inactivating  cyclic  AMP,  would  be  expected  to  enhance  the  cardiac  effects 
of  these  agents.   We  therefore  examined  the  effects  of  theophylline,  an  ef- 
fective inhibitor  of  phosphodiesterase,  on  the  isometric  response  of  cat 
papillary  muscles  to  varying  concentrations  of  norepinephrine  (10"11  to  10"" 
M),  glucagon  (10"^  to  10-^),  and  of  dibutyryl  cyclic  AMP  (DBC)  (lO'S  to  lO'^ 
M),  a  more  lipid  soluble  derivative  of  cyclic  AMP  which  is  thought  to  mimic 
the  intracellular  effects  of  cyclic  AMP.  At  a  theophylline  concentration 
(2.5  X  10"^  M)  which  caused  little  increase  in  baseline  contractile  function 
(avg,  less  than  107o) ,  a  marked  potentiation  of  the  inotropic  effects  of  nor- 
epinephrine, glucagon  and  DBC  was  observed.   Thus,  the  concentration  of  nor- 
epinephrine producing  half  maximal  activity  decreased  from  1  x  10"'  M  to  1  x 
10"%:  that  of  DBC  decreased  from  8  x  10""^  to  4  x  10"'^  M.   In  addition,  the 
peak  tension  development  produced  by  glucagon  increased  from  0.7  +  .25  g/mm^ 
to  3.0  +  .72  g/mm'^  (p<.01).   In  contrast,  theophylline  did  not  alter  the 
inotropic  effects  of  calcium  (2.5  x  10"^  -  1.0  x  10"^),  an  agent  not  believed 
to  exert  its  inotropic  effects  through  the  cyclic  AMP  system.   These  findings 
provide  further  evidence  that  the  inotropic  effects  of  norepinephrine,  gluca- 
gon, and  DBC  are  mediated  via  increases  in  the  intracellular  level  of  cyclic 
AMP. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publication:   Submitted  for  publication 


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Serial  No.     NHLI"2(c) 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Md, 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Comparison  of  Left  Ventricular  and  Pulmonary  Arterial 

Injection  Sites  in  the  Determination  of  Cardiac  Output  by 
the  Indicator  Dilution  Technique 

Previous  Serial  No»:   None 

Principal  Investigator:   Richard  L.  Shepherd,  M.D. 

Other  Investigators:     D.  Luke  Clancy,  M.D„ 

Lawrence  M.  Higgs,  M.D, 


Stephen  E,  Epstein,  M.D. 


Cooperating  Units:  None 


Project  Description:   The  validity  of  the  indicator  dilution  technique  for 
measuring  cardiac  output  has  been  established  by  comparing  the  values 
obtained  with  those  determined  simultaneously  by  the  Pick  method.   In  such 
studies  indicator  was  injected  in  the  lesser  circulation  and  sampled  in  a 
systemic  artery.   Because  the  adequacy  of  the  left  ventricle  as  a  mixing 
chamber  has  been  questioned,  left  ventricular  injections  seldom  have  been 
used.  This  study  was  undertaken  to  assess  the  accuracy  and  reproducibility 
of  cardiac  output  measurements  made  from  left  ventricular  indicator  dilution 
curves. 

In  58  patients  with  a  variety  of  hemodjmamic  abnormalities,  four  indi- 
cator dilution  curves  were  inscribed  in  rapid  succession  alternating  the 
site  of  injection  between  pulmonary  artery  and  left  ventricle  and  using  a 
single  systemic  arterial  sampling  site.   Indocyanine  green  was  the  indicator. 

The  average  cardiac  output  calculated  using  the  left  ventricular  curves 
exceeded  that  using  the  pulmonary  arterial  curves  by  0„037  L/min  (standard 
error  +  0.052  L/min).   The  estimated  standard  error  of  a  left  ventricular 
measurement  of  cardiac  output  was  0.41  L/min  and  that  of  a  pulmonary  arterial 
measurement  was  0.35  L/min.   Thus,  cardiac  output  determined  from  left 
ventricular  curves  did  not  differ  significantly  from  determinations  made  from 
pulmonary  arterial  curves,  and  the  reproducibility  of  the  two  methods  was 
essentially  the  same. 

Data  from  individual  patients  with  the  largest  differences  between 
cardiac  output  determinations  by  the  two  methods  suggest  that  in  the  presence 
of  severe  mitral  regurgitation  left  ventricular  curves  tend  to  overestimate 
output  while  pulmonary  arterial  curves  tend  to  underestimate  output.  We 

-1-  ft 


Serial  No.   NHLI-2(c) 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

conclude  that  left  ventricular  indicator  dilution  curves  give  accurate  and 
reproducible  measurements  of  cardiac  output  except  in  occasional  patients 
with  severe  mitral  regurgitation. 

Proposed  Course:   Completed 

Honors  and  Awards:  None 

Publications:   In  preparation. 


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Serial  No.   NHLI-3(c) 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Acquired  Right  Ventricular  Infundibular  Stenosis  in  Patients 
with  Ventricular  Septal  Defect 

Previous  Serial  Number:   None 

Principal  Investigator:    Richard  L.  Shepherd,  M.D. 

Other  Investigators:      D.  Luke  Clancy,  M.D. 

Richard  B,  Jaffe,  M.D. 
Joseph  Perloff,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Radiology  Dept.  ,  Clinical  Center;  Division  of  Cardiology, 
Dept.  of  Medicine,  Georgetown  Univ.  School  of  Medicine 

Project  Description:   With  the  possible  exception  of  bicuspid  aortic  valve, 
ventricular  septal  defect  (VSD)  is  the  commonest  congenital  cardiovascular 
malformation  seen  in  children.   Although  many  patients  with  this  defect  die 
in  infancy  of  left  ventricular  failure  or  develop  severe  pulmonary  vascular 
disease  and  die  as  young  adults,  the  drop-out  rate  due  to  these  complications 
is  not  sufficient  to  explain  the  observation  that  it  is  relatively  uncommon 
to  see  an  adult  with  uncomplicated  VSD.   The  observation  that  many  patients 
with  a  VSD  experience  spontaneous  closure  of  the  defect  explained  the  dis- 
crepancy in  prevalence  of  this  entity  in  children  and  adults.   However, 
clinical  studies  have  suggested  that  development  of  right  ventricular  ob- 
struction is  another  possible  cause  for  the  infrequency  with  which  isolated 
VSD  is  seen  in  adults.   By  serial  hemodynamic  studies  we  have  recently  docu- 
mented the  development  of  acquired  right  ventricular  infundibular  stenosis  in 
five  children  with  VSD. 

When  first  studied  at  ages  six  months  to  four  years,  each  child  had 
pulmonary  arterial  hypertension,  a  trivial  or  absent  pressure  gradient  across 
the  right  ventricular  outflow  tract,  and  a  large  left- to-right  shunt  at 
ventricular  level.   When  restudied  1  1/2  to  five  years  later,  each  was  found 
to  have  a  normal  pulmonary  arterial  pressure,  a  large  pressure  gradient  across 
the  right  ventricular  inf undibulum,  and  a  reduced  left-to-right  shunt.   Two 
of  the  patients  had  large  right-to- left  shunts  at  ventricular  level,  and  like 
patients  with  tetralogy  of  Fallot  presented  on  their  second  admission  with 
cyanosis  and  squatting.   Both  have  undergone  corrective  operations. 

This  study  thus  demonstrates  that  severe  right  ventricular  infundibular 
stenosis  can  develop  in  children  with  uncomplicated  VSD.   It  also  indicates 
that  while  the  acquisition  of  right  ventricular  infundibular  stenosis  is 
beneficial  in  some  patients  with  VSD  (since  pulmonary  arterial  pressure  and 

-1-  SLO 


J 


Serial  No.   NHLI-3(c) 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

flow  are  thereby  reduced),  the  infundibular  narrowing  can  become  excessive 
and  result  in  a  physiologic  situation  identical  to  that  seen  in  Fallot  s 
tetralogy. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   In  preparation 


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Serial  No.   NHLT-4(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


1 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Hemodynamic  Assessment  of  Kay  Shiley  Prosthetic  Valves 

Previous  Serial  Number:   None 

Principal  Investigator:   Richard  L.  Shepherd,  M.  D. 

Other  Investigators:     D.  Luke  Clancy,  M.  D. 

R.  Reis,  M.  D. 
Stephen  E.  Epstein,  M.  D. 
Andrew  G.  Morrow,  M.  D. 


Cooperating  Units: 


Clinic  of  Surgery 


Project  Description:   Previous  studies  have  assessed  the  in  vivo  hemodynamic 
function  of  Starr  Edwards  valve  prostheses  but  there  is  no  information  avail- 
able relating  to  the  performance  of  the  low  profile  disc  type  (Kay  Shiley) 
valve  prosthesis. 

Since  January  1966,  fifty-one  Kay  Shiley  valves  have  been  used  to  re- 
place diseased  mitral  and/or  tricuspid  valves  in  48  patients.   Twenty-three 
of  the  48  patients  had  mitral  valve  replacement;  three  of  the  23  patients 
also  had  tricuspid  annuloplasties  and  two  others  had  myotomy  for  IHSS.   In 
18  of  the  48  patients  both  mitral  and  tricuspid  valves  were  replaced;  4  of 
the  18  patients  had  Kay  Shiley  protheses  in  both  mitral  and  tricuspid  areas 
and  the  others  had  a  Starr  Edwards  mitral  prothesis  and  a  Kay  Shiley  tri- 
cuspid prosthesis.   Three  patients  had  Starr  Edwards  aortic  protheses  and 
Kay  Shiley  mitral  prosthesis  and  4  patients  had  Starr  Edwards  aortic  and  mitral 
prostheses  and  Kay  Shiley  triscupid  prostheses. 

There  have  been  22  postoperative  deaths  and  8  patients  who  have  been 
lost  to  follow  up.   There  are  18  survivors  who  have  had  postoperative  cathe- 
terizations.  In  patients  with  tricuspid  valve  replacement,  the  right  atrial 
mean  pressure  was  8  mm  Hg  or  more  in  every  case.   In  patients  with  mitral 
valve  replacement,  the  left  atrial  mean  pressure  was  18  mm  Hg  (average). 
Calculated  valve  areas  in  vivo  were  66  -  757o  of  the  in  vitro  valve  areas 
provided  by  the  manufacturer. 

The  operative  mortality  in  this  group  of  patients  is  considerably  higher 
than  the  mortality  in  this  institution  using  the  caged  ball  prosthesis.  The 
reasons  for  the  higher  mortality  are  not  clear.   Many  of  these  patients  had 
more  than  one  valve  replaced  and  the  low  profile  valve  was  usually  selected 
when  the  ventricular  cavity  would  not  accept  a  caged  ball  prosthesis.   As  has 
been  found  in  the  caged  ball  prosthesis,  the  Kay-Shiley  prosthesis  causes  mild 
to  moderate  obstruction  to  atrial  emptying. 


S4 


Serial  No.   NHLI-4(c) 


PHS-NIH 
lndi\iidual  Project  Report 
July  1,  1970  through  June  30,  1971 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   Manuscript  in  preparation. 


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Serial  No.  NHLI-5(c) 


1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Reversibility  of  Impaired  Heart  Rate  Response  to  Sympathetic 
Stimulation  in  Patients  with  Cardiac  Decompensation 

Previous  Serial  Number:    None 

Principal  Investigator:   Douglas  R.  Rosing,  M.  D. 

Other  Investigators:      G.  David  Beiser,  M.  D. 

David  R.  Redwood,  M.  D. 
Eldon  R.  Smith,  M.  D. 
Stephen  E.  Epstein,  M.  D. 

Cooperating  Unit:         None 

Project  Description:   We  have  shown  that  the  heart  rate  response  to  barore- 
ceptor  mediated  enhancement  of  sympathetic  activity  is  impaired  in  patients 
with  depressed  cardiac  function,  as  is  the  maximal  heart  rate  response  to  ex- 
hausting exercise.   Although  the  etiology  of  this  impairment  has  not  been  de- 
fined, the  defect  does  not  appear  to  be  due  to  either  decreased  sensitivity 
of  the  adrenergic  receptor  site  or  differences  in  background  vagal  tone.  Its 
potential  for  reversibility  also  is  unknown.   To  more  fully  characterize  this 
defect,  the  heart  rate  responses  to  l)  baroreceptor  enhancement  of  sympathetic 
activity  produced  by  lowering  mean  arterial  pressure  with  nitroglycerin  and 
2)  exhausting  exercise  have  been  determined  in  functional  class  II  and  III 
patients  about  to  undergo  cardiac  surgery.   Samples  of  atrial  and  left  ven- 
tricular papillary  muscles  have  been  obtained  at  the  time  of  operation.  These 
samples  will  be  analyzed  for  norepinephrine  content  in  an  attempt  to  correlate 
depletion  of  these  stores  with  the  impaired  heart  rate  response.   In  addition, 
the  preoperative  studies  will  be  repeated  after  operation  to  determine  if  the 
anticipated  improvement  in  cardiac  function  results  in  an  improved  heart  rate 
response  to  sympathetic  stimulation. 

Proposed  Course  of  Project:   As  outlined  above 

Honors  and  Awards:   None 

Publications:   None 


^X^ 


Serial  No.   NHLI-6(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:  Effects  of  Aging,  Type  IV  Hyperlipoproteinemia,  and 
Coronary  Artery  Disease  on  the  Diurnal  Pattern  of 
Fibrinolytic  Activity 

Previous  Serial  Number:  NHLI  -  29(c) 

Principal  Investigator:  Douglas  R,  Rosing,  M.D, 


Other  Investigators; 


Cooperating  Units: 


Pieter  Brakman,  M„D. 
David  R.  Redwood,  M.D. 
Stephen  E,  Epstein,  M.D, 

The  James  F„  Mitchell  Foundation,  Washington,  D.C, 


Project  Description:  The  fibrinolytic  system  is  important  in  maintaining  an 
equilibrium  between  fibrin  deposition  and  dissolution,  and  it  has  been 
postulated  that  a  defect  in  this  system  may  play  a  role  in  the  pathogenesis 
of  arteriosclerosis.  Therefore  diurnal  patterns  of  plasma  euglobulin 
fibrinolytic  activity,  estimated  by  the  Astrup  fibrin  plate  method  and 
expressed  as  mm^  lysis,  were  determined  in  1)  young  normal  subjects  -  mean 
age  20;  2)  older  normals  -  mean  age  44;  3)  patients  with  type  IV  hyperlipo- 
proteinemia -  mean  age  44;  and  4)  patients  with  coronary  artery  disease  and 
normal  lipid  studies  -  mean  age  52.  Because  fibrinolytic  activity  gradually 
increases  during  the  day  in  young  normal  subjects  at  bed  rest  and  peaks 
between  5  and  8  p.m.,  samples  collected  at  8  a.m.,  5  p.m.,  and  8  p.m. 
were  selected  to  charactierize  and  compare  the  four  groups.  Mean  peak  fibrin- 
olytic activity  was  also  compared  among  the  groups.  At  5  p.m.  fibrinolytic 
activity  in  18/19  young  normals  increased  at  least  75  mm^ ;  however,  only 
13/24  older  normals  (P  <.05) ,  5/20  patients  with  endogenous  hypertrigly- 
ceridemia, and  3/16  patients  with  coronary  artery  disease  demonstrated  similar 
increase.  At  8  p.m.  fibrinolytic  activity  in  19/19  young  normals  increased 
at  least  100  mm^ ;  only  14/24  older  normals  (P  <.005),  3/20  patients  with 
triglyceride  abnormalities,  and  6/16  patients  with  coronary  artery  disease 
exhibited  this  increase.   The  difference  between  the  increases  in  fibrinolytic 
activity  in  the  age -matched  older  normals  and  type  IV  patients  were 
statistically  significant  at  8  p.m.  but  not  at  5  p.m.   The  differences  between 
the  older  normals  and  coronary  artery  disease  patients  were  not  statistically 
significant.  Absolute  values  of  mean  peak  fibrinolytic  activity  were: 
young  normals  =  254  +  14  mm'^,  older  normals  =  202  +  22  mm^,  type  IV  patients 
137  +  14  mm2,  and  patients  with  coronary  artery  disease  164  +  17  mm  .  The 
differences  in  absolute  values  between  the  young  and  older  normals  (P  <.05), 
and  the  older  normals  and  type  IV  patients  (P  <  .01)  were  statistically 
significant,  but  the  differences  between  the  older  normals  and  patients  with 


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Serial  No.   NHT.T-6(c) 

PHS-NIH 
Indivudual  Project  Report 
July  1,  1970  through  June  30,  1971 

coronary  artery  disease  did  not  achieve  statistical  significance.  Thus,  the 
data  demonstrate  that  the  diurnal  augmentation  of  fibrinolytic  activity  norm- 
ally occurring  in  young  normal  subjects  is  encountered  less  frequently  in 
older  subjects.   Patients  with  type  IV  hyperlipoproteinemia,  a  condition 
which  has  been  associated  with  the  early  development  of  atherosclerosis,  show 
an  impairment  that  is  greater  than  that  which  can  be  attributed  to  age  alone. 
Patients  with  coronary  artery  disease  but  with  normal  lipid  studies  demonstrate 
smaller  diurnal  increases  than  older  normal  subjects,  although  the  differences 
are  not  statistically  significant. 

Honors  and  Awards :   None 

Publications:  Manuscript  in  preparation. 


31^ 


J 


Serial   No.  NHLI-7 


1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 

Indivudual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Propranolol  in  the  Treatment  of  Arrhythmias  Occurring  During 
Acute  Myocardial  Ischemia  in  Conscious  Dog 

Previous  Serial  Number :   None 

Principal  Investigator:   Douglas  R.  Rosing,  M.  D. 

Other  Investigators:     James  Talano,  M.  D. 

Richard  Karsh,  M.  D. 
G.  David  Beiser,  M.  D. 
Stephen  E.  Epstein,  M.  D. 


Cooperating  Units: 


None 


Project  Description:   Approximately  407o  of  patients  who  suffer  an  acute  myo- 
cardial infarction  die,  and  2/3  of  these  deaths  occur  within  the  first  few 
hours,  before  the  patient  is  admitted  to  a  hospital.   Since  the  mechanism  of 
death  under  such  circumstances  is  probably  due  to  cardiac  arrhythmias,  survival 
following  acute  myocardial  infraction  might  be  increased  by  the  initiation  of 
a  program  in  which  high  risk  individuals  receive  an  effective  anti- arrhythmic 
agent  on  a  chronic,  prophylactic  basis  or  in  certain  acute  situations  where 
there  is  a  strong  suspicion  that  an  acute  myocardial  infarction  has  occurred. 
Propranolol,  administered  chronically  to  patients  with  angina  pectoris,  has 
antiarrhythmic  effects  and  has  been  shown  to  decrease  the  size  of  ischemic 
areas  after  acute  coronary  occlusion  in  anesthetized  dogs.  This  drug  might 
therefore  prove  of  benefit  to  the  patient  suffering  an  acute  myocardial  in- 
farction. On  the  other  hand,  since  bradycardia  decreases  the  fibrillation 
threshold  of  the  myocardium  and  favors  the  development  of  re-entry  type  arrhyth- 
mias, the  decrease  in  heart  rate  that  occurs  with  propranolol  administration 
may  predispose  to  the  development  of  malignant  arrhythmias. 

To  determine  whether  propranolol  has  a  beneficial  or  deleterious  effect 
during  acute  myocardial  infarction  on  arrhythmias,  we  are  studying  its  effects 
during  acute  ischemia  produced  in  closed  chest  conscious  dogs.  This  is  accomp- 
lished by  emplacing  inflatable  balloons  around  the  left  anterior  descending 
coronary  arteries  in  dogs  as  a  first  stage  procedure.  At  least  one  week  after 
recovery  from  operation,  acute  myocardial  ischemia  is  produced  in  the  conscious 
dog  by  inflating  the  balloon  and  thereby  occluding  the  coronary  artery.  Stable 
arrhythmias  that  develop  are  treated  with  .05  mg/kg  doses  of  intravenous  pro- 
pranolol administered  every  five  to  ten  minutes. 

In  six  dogs  that  developed  ventricular  arrhythmias  after  coronary  occlusion, 
propranolol  had  no  consistent  beneficial  or  detrimental  effects. 


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Serial  No.      NHLI-7 

PHS-NIH 
Individual   Project   Report 
July   1,    1970   through  June   30,    1971 

In   three   dogs    that  had  marked   ST   segment   changes   but   no   consistent 
arrhythmia   after   occlusion,   administration   of  propranolol  did  not  produce 
arrhythmias.      These   preliminary  results    suggest    that  while   propranolol 
administered   chronically   to  patients  with  angina   pectoris  may  not   have   any 
detrimental  effects    if  acute    ischemia    occurs,    there    is  also    little    likeli- 
hood   that    the   drug  will  prevent   arrhythmias   occurring  during    such  episodes. 

Proposed   Course    of   Project:      1)      The   number   of  dogs    studied   during   occlusion 
of   the    coronary  artery  will  be    increased;    2)    the   effect   of   propranolol 
on  arrhythmias  which   develop  after   release    of  coronary  artery  occlusion 
will   be    studied    (these  arrhythmias  are   often  more  malignant   than   occlusion 
arrhythmias);    3)    the   effect   of   practolol,   a    "cardio-specif ic"  beta    receptor 
blocking  agent  with   little   tendency  to   lower  resting  heart   rate,   will  be 
evaluated . 

Honors  and  Awards:      None  ^ 

Publications:      None 


^S 


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Serial  No.     NHLI-8(c) 

1.  Cardiology 

2.  Clinical   Physiology 
3„      Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  The  Effects  of  Training  in  Patients  with  Coronary  Artery 
Disease  and  Angina  Pectoris 

Previous  Serial  Number:   NHLI-25(c) 

Principal  Investigator:   David  R,  Redwood,  MoD„ 


Other  Investigators; 


Cooperating  Units; 


Douglas  R„  Rosing,  M.D, 
Stephen  E„  Epstein,  M„D. 

None 


Project  Description:   In  the  last  few  years  there  has  been  increasing  aware- 
ness of  the  beneficial  effects  of  exercise  in  patients  with  coronary  artery 
disease.  Thus,  several  recent  studies  have  shown  that  physical  training 
increases  exercise  performance  in  patients  with  angina  and  that  at  least 
part  of  this  improvement  is  due  to  a  more  efficient  circulatory  response  to 
exercise  such  that  myocardial  oxygen  demands  (MVO2)  for  any  given  intensity 
of  exercise  presumably  are  reduced.   Nevertheless,  it  is  not  known  whether 
training  also  leads  to  an  enhanced  capacity  of  the  coronary  vessels  to 
deliver  oxygen. 

We  therefore  studied  the  effects  of  a  six -week  program  of  intense 
training  in  seven  patients  with  angina  pectoris  due  to  coronary  artery 
disease.  Aortic  systolic  pressure,  heart  rate,  and  ejection  time  were 
recorded  continuously  during  upright  bicycle  exercise  before,  at  3  weeks, 
and  at  the  conclusion  of  training.  After  six  weeks  exercise  capacity  marked- 
ly increased:   time  to  onset  of  angina  rose  an  average  of  6.8  +  1.5  minutes 
(P  <  .01)  and  the  intensity  of  exercise  (measured  by  total  body  O2  consump- 
tion) attained  before  angina  increased  57  +  19%  (P  <  .005).  The  greatest 
change  in  exercise  capacity  occurred  during  the  first  3  weeks  of  training. 
The  triple  product  (TP)  of  aortic  systolic  pressure,  heart  rate,  and  ejection 
time  was  calculated  and  used  as  an  index  of  MVO2 ,  TP  (and  presumably 
MVO2)  at  any  level  of  exercise  was  less  after  training,  thus  accounting  for 
part  of  the  improved  exercise  capacity.  However,  after  training  a  higher  TP 
could  be  achieved  before  the  onset  of  ischemic  pqin  (4885  vs.  4290,  P  <  .05). 
If  changes  in  TP  accurately  reflect  changes  in  MVO2,  then  the  finding  that 
ischemic  pain  occurred  at  a  higher  TP  suggests  that  training,  in  addition  to 
improving  the  efficiency  of  the  circulatory  response  to  exercise,  might  also 
improve  myocardial  oxygen  delivery. 

Proposed  Course  of  Project:   Completed. 

Honors  and  Awards:   None 

Publications:  Manuscript  in  preparation, 

-1-  ^ 


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Serial  No.       NHLI-9 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,   Md. 

PHS-NIH 

Individual  Project   Report 

July   1,    1970   through  June   30,    1971 

Project  Title:      The  Effects   of  Atropine    on   the   Degree    of  Myocardial   Ischemia 

During  Coronary  Occlusion   in   the   Conscious   Dog 
Previous   Serial  Number:      None 
Principal   Investigator:      David  R.   Redwood,  M„D„ 

Other   Investigators:  Eldon  R.    Smith,   M.D, 

Stephen  E.  Epstein,   M.Do 

Cooperating  Units:      None 

Project  Description:      Atropine    is    frequently  employed   in  patients  with  acute 
myocardial   ischemia    to  treat  bradycardia -induced  arrhythmias,   and  we   have 
shown  experimentally   that  atropine    is  effective    in   treating  arrhythmias   oc- 
curring during   the    onset   of  myocardial   infarction.      It   is  not   known,   however, 
whether   the   atropine -induced   increase    in  heart   rate   has  any  deleterious 
effect   on   the   degree    of  myocardial   ischemia.      To  avoid   the    tachycardia   present 
in   open-chest   anesthetized   dogs,   we    studied   the    S-T    segment   response    (obtained 
from   10    -    12    implanted   intramyocardial  electrodes)    to   repeated   five -minute 
occlusions   of   the    left  anterior  descending  coronary  artery  at  hourly   intervals 
in   closed-chest   conscious   dogs   in  which  we   previously  had   implanted  an   in- 
flatable  balloon  around   the    left  anterior  descending  artery.     Occlusions  were 
conducted    in  random  order    1)    at   control  heart   rate    (avg.    95),    2)   with  atropine 
pretreatment    (,005    -    ,05  mg/kg/iv) ,   and   3)    during  atrial  pacing.      Repeated 
control   occlusions   caused  no  change    in  degree    of   S-T   response.     When   compared 
to   control   occlusion   there   was  a    significant   correlation  between   the    per 
cent    increase    in  heart   rate   produced  by  atropine   and   per  cent   increase    in 
total   S-T  elevation    (y  =  0.89  x  +  5.00,    r  =    .95,    P  <   .001).      There   was  no 
significant   difference   between   the    increase    in   S-T  elevation  with   occlusion 
following  atropine  and    that    following  occlusion  during   rate-matched  atrial 
pacing. 

To  determine   if  an  increase   in  heart   rate    from  even   slower  control 
rates    (as  might   occur   in   some   patients  with  acute  myocardial   infarction) 
also  was   deleterious,   various   degrees   of  bradycardia  were    induced  by  vagal 
stimulation  during  episodes   of  myocardial   ischemia.      Similar   results  were 
obtained:      i.e.,    there  was  a   direct  relationship  between  increases   in  heart 
rate    from  40  to   95  beats/min  and   increases   in   S-T   segment  elevation. 

We   conclude    that    if   the   degree    of   S-T  elevation   reflects    severity  or 
extent   of   ischemia,    increases   in  rate    in  experimentally  produced  acute   myo- 
cardial  ischemia   are   associated  with  proportional   increases   in   the   degree    of 
myocardial   ischemia.      Thus,    when  atropine    is  administered   to  control  brady- 
cardiac-induced  arrhythmias  during  acute  myocardial   ischemia,   the   lowest 
effective   dose    should  be   used  and  excessive   increases   in  rate  avoided, 

-1-  3e> 


Serial  No.  NHLI-9 


PHS-NIH 
Individual  Project   Report 
July   1,    1970  through  June   30,    1971 

Proposed  Course   of  Project:      Continuing 

Honors  and  Awards:     None 

Publications:     None 


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Serial   No.      NHLI-10 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970   through  June  30,  1971 

Project  Title:   The  Role  of  Cyclic  AMP  in  Myocardial  Contractility 

Previous  Serial  Number:  None 

Principal  Investigators:  Melvin  L.  Marcus,  M.  D. 

Leonard  E.  Grauer,  M.  D. 

Other  Investigators:     Copal  Krishna,  Ph.  D. 

Stephen  E.  Epstein,  M.  D. 

Cooperating  Units:       Laboratory  of  Clinical  Pharmacology 

Project  Description:   The  effects  of  catecholamines  on  myocardial  contract- 
ility are  thought  to  be  mediated  via  increases  in  the  intracellular  level  of 
cyclic  AMP  which  result  from  stimulation  of  adenyl  cyclase.   In  an  attempt 
to  examine  this  hypothesis  more  thoroughly,  the  effects  of  norepinephrine  on 
myocardial  contractility  and  on  tissue  levels  of  cyclic  AMP  were  studied 
employing  the  isometr ically  contracting  cat  papillary  muscle.   All  experiments 
were  performed  at  30°C  with  the  muscles  at  the  peak  of  their  length-tension 
curve  (Lmax)-   The  effects  of  two  concentrations  of  norepinephrine  (1  x  10"^ 
and  1  X  10"")  were  studied.   At  each  concentration,  the  effects  of  norepineph- 
rine.  The  papillary  muscles  were  then  quickly  frozen  with  cold  isopentane, 
measured,  weighed,  and  assayed  for  cyclic  AMP  by  the  Gilmore  method. 

Control  levels  of  cyclic  AMP  in  the  cat  papillary  muscle  were  found  to 
be  1.7  +  0.1  pcM/mg  tissue.   A  concentration  of  1  x  10"*^  norepinephrine  pro- 
duced an  increase  in  both  myocardial  contractility  and  cyclic  AMP  within  5 
seconds.   A  concentration  of  1  x  10""  norepinephrine  had  a  positive  inotropic 
effect  within  10  seconds,  but  a  significant  increase  in  cyclic  AMP  concentra- 
tion could  not  be  demonstrated  until  20  seconds  had  elapsed. 

There  are  several  explanations  for  the  dissociation  of  the  inotropic  and 
enzymatic  effects  of  norepinephrine  found  in  these  preliminary  studies.   It 
is  possible  that  activation  of  the  adenyl  cyclase-cyclic  AMP  system  and  en- 
hancement of  contractility  are  coincidental  and  causally  unrelated  events.  On 
the  other  hand,  it  is  possible  that  our  technique  is  not  sensitive  enough  to 
detect  small  changes  in  tissue  levels  of  cyclic  AMP  which  nevertheless  may  be 
physiologically  significant.  It  is  also  possible  that  the  adenyl  cyclase- 
cyclic  AMP  system  is  responsible  for  mediating  several  intracellular  functions, 
each  function  being  compartmentalized  into  a  separate  pool.   Thus,  each  pool 
may  be  sufficiently  small  so  that  assaying  tissues  for  total  cyclic  levels 
becomes  too  insensitive  a  technique  to  discern,  for  example,  changes  in  the 
pool  specifically  responsible  for  altering  myocardial  contractility. 


3x 


I 


Serial   No.      NHLI-10 


PHS -NIH 
Individual    Project   Report 
July   1,    1970     through   June   30,    1971 


Proposed  Course   of    Project:    Continuing 
Honors    and  Awards:      None 
Publications:      None 


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33 


Serial  No.   NHLI-ll(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project   Report 
July   1,    1970  through  June   30,    1971 

Project  Title:      A  Completely  Automated  Video-Tracking  Technique    for   the 
Determination   of  Dynamic   Changes   in  Ventricular  Volume 

Previous   Serial  Number:     NHLI-22(c) 

Principal  Investigator:     Melvin  L.  Marcus,   M.D. 

Other   Investigators:      William  H.    Schuette 
Willard  Whitehouse 
James   Bailey,   M„D„ 
D.   Luke   Clancy,  M„D. 
Stephen  E.   Epstein,   M„D. 

Cooperating  Units:      Biomedical  Engineering  and   Instrumentation  Branch, 
Division   of  Research   Services; 

laboratory   of  Applied   Studies,   Division   of  Computer 
Research  and  Technology; 
Administrative   Branch,   Clinical  Center 

Project  Description;      Several   important   indices    of  myocardial  performance 
depend   upon  accurate   and   frequent  measurement   of  ventricular  volume.      Studies 
employing   such  measurements  have   been    limited   because    of   the   difficulty   of 
manually  measuring  and    calculating  volumes    frequently  enough   to   obtain 
meaningful  data.     We    therefore   have   developed  a    completely  automated  method 
for  determination   of  ventricular  volume    in  man.      Left  ventricular  cineangio- 
grams    (16  mm)    taken   in   the   RAO  position  at   60   frames/sec  are   projected  with 
a    flickerless   projector   onto  a   Plumbicon   television   camera.     A    second   tele- 
vision  camera    is   used   for  masking  out  noncontributory  portions    of   the    film 
and    for   shading   selected  areas   to   facilitate   accurate    recognition   of   the 
opacified   chamber.     An  electronic  video-tracking  device    then   simultaneously 
determines   the   area   and   the  maximum   length  of   the    opacified   chamber   in  each 
cine    frame;    these   data   are   recorded  as  analog   signals   on  magnetic   tape. 
Volumes  are    calculated   by   computer  and  plotted  against    time.     When  volumes 
determined  by   this  automated  method   are   compared  with   those    obtained  by 
manual  planimetry   the   correlation   coefficient   is   r  =    .96;   volumes   of   test 
objects    (20-360  cc)   are  accurate   to  within   6.0  +  3.6%,     This  automated 
technique    thus  permits   rapid  and   accurate  measurement   of  ventricular  volume 
in  all  patients  having  diagnostic    left  ventriculograms. 

Proposed  Course   of  Project:      Completed 

Honors  and  Awards:     None 

Publications:     Manuscript   submitted   for  publication. 


3^" 


I 

J 


Serial  No.        NHLI-12 


1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Mechanism  of  Action  of  the  Inotropic  Effects  of  Theophylline 

Previous  Serial  Number:   None 

Principal  Investigator:   Melvin  L.  Marcus.  M.  Do 


Other  Investigators: 


C.  Lynn  Skelton,  M.  D. 
Leonard  E.  Grauer,  M.  D. 
Gopal  Krishna,  Ph.  D. 
Stephen  E.  Epstein,  M.  D. 


Cooperating  Units:   Laboratory  of  Chemical  Pharmacology 

Project  Description:   Theophylline  is  thought  to  exert  its  positive  inotropic 
effect  on  the  heart  by  increasing  the  intracellular  levels  of  cyclic  3',5'- 
AMP  through  inhibition  of  phosphodiesterase.   To  investigate  this  hypothesis, 
the  effects  of  theophylline  on  the  contractile  function  of  isolated  cat 
papillary  muscles  were  studied.   In  isotonic  experiments,  theophylline  (2.5  x 
10"3m)  caused  a  marked  shift  in  the  force-velocity  curve  upward  and  to  the 
right  from  control  levels.   In  quick  release  experiments,  the  modulus  of 
series  elasticity  was  unchanged  by  2.4  x  10"-^M  theophylline.   In  isometric 
experiments,  theophylline  (2.5  x  10"°  -  2.5  x  10"%)  caused  concentration 
related  increases  in  tension  and  rate  of  tension  development  that  averaged 
2.8  ±  0.3  gm/mm2  (P  <  0.001)  and  9.7  ±  1.4  gm/mm2/sec  (P  <  0.001)  at  the 
peak  concentration.   Unlike  norepinephrine  and  dibutyryl  cyclic  AMP  (agents 
believed  to  work  through  the  cyclic  AMP  system),  time  to  peak  tension  devel- 
opment (TTP)  was  increased.   Propranolol  (1  x  lO'^M)  caused  a  shift  in  the 
concentration-response  curve  to  the  right,  and  prior  reserpinization  decreased 
the  peak  inotropic  effect  of  theophylline  by  more  than  407o.   Therefore,  the 
inotropic  effects  of  theophylline  are  partly  due  to  a  direct  effect  and 
partly  to  norepinephrine  release.   Since  theophylline  lengthens  TTP,  its 
direct  inotropic  effects  either  must  not  be  mediated  by  cyclic  AMP,  or  if 
they  are,  then  the  drug  must  have  additional  effects  on  the  contractile 
mechanisms. 

Further  investigations  have  involved  the  effects  of  theophylline  on  the 
intracellular  levels  of  cyclic  AMP  in  the  papillary  muscle.   The  basal  level 
of  cyclic  AMP  in  the  papillary  muscle  is  1.7  ±  0.1  pcM/mgm.   Theophylline 
1  x  10"%  increased  tension  in  the  papillary  muscle  by  approximately  50%  but 
had  no  significant  effect  on  the  intracellular  level  of  cyclic  AMP.   These 
data  suggest  that  the  inotropic  effects  of  theophylline  are  probably  not  medi- 
ated by  changes  in  the  intracellular  level  of  cyclic  AMP. 


Proposed  Course  of  Project: 


Continuing  investigation  of  the  effects  of  the- 
ophylline on  cyclic  AMP  levels. 

1  3S- 


iLii 


D  -s 


Serial   No.      NHLI-12 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


Honors  and  Awards:   None 
Publications:   None 


1 


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Project  Title: 


Serial  No.    NHT.I-13 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Enzyme  and  Receptor  Activities  of  Subcellular  Systems  Isolated 
from  Ischemic  Rabbit  Hearts 


Previous  Serial  Number:   None 

Principal  Investigator:   George  E.  Lindenmayer,  M.  D, ,  Ph.  D. 

Other  Investigator:      Stephen  E.  Epstein,  M.  D, 


Cooperating  Units 


None 


Project  Description:   The  present  investigation  was  designed  to  determine  the 
time-dependent  sequence  of  irreversible  subcellular  changes  in  the  ischemic 
myocardium.   Male  rabbits  were  sacrificed  by  cervical  dislocation  (CD)  and 
the  hearts  were  left  in  situ  for  varying  times.   After  CD,  no  respirations 
were  observed,  cardiac  contractions  ceased  within  5  minutes,  and  body  temper- 
ature dropped  about  5°C  in  the  150  minutes  after  CD.   Although  this  model 
does  not  represent  pure  ischemia  (since  hypoxia  probably  predominates  until 
cardiac  arrest),  it  is  a  simple,  reproducible  one  which  will  permit  us  to 
select  subcellular  systems  £^r  study  in  more  physiologic  models  of  pure  ische- 
mia.  Preliminary  results  already  have  demonstrated  that  subcellular  systems 
differ  in  their  sensitivity  to  ischemia.   For  example,  Na"*",  K+-ATPase  is  re- 
markably stable.   Activities  150  minutes  after  CD  were  90%  of  values  obtained 
1  minute  after  CD.   Conversely,  mitochondrial  preparations  (isolated  in  KCl- 
EDTA  medium)  showed  rapid  loss  of  respiratory  control  (i.e.  507o  decrease  with- 
in 15  minutes  after  CD)  and  rates  of  oxygen  consumption  (State  3) .   The  sen- 
sitivities of  these  systems  to  appropriate  ligands  will  be  determined  (e.g. 
Km's  for  Mg'ATP,  sodium  and  potassium,  and  Ki  for  ouabain  of  Na  ,  BC^-ATPase 
preparations) .   Other  subcellular  systems  that  will  be  examined  are  adenyl 
cyclase,  myofibrils, calcium  binding  by  membranes  and  lysosomes.   Eventually, 
components  of  ischemia  (e.g.  hypoxia,  substrate  lack,  etc.)  will  be  analyzed 
with  respect  to  subcellular  changes.   An  attempt  also  will  be  made  to  correlate 
ischemic- induced  reversible  and  irreversible  changes  in  myocardial  contractility 
with  subcellular  functional  aberrations. 

The  eventual  goals  we  hope  to  achieve  with  this  approach  to  the  study  of 
myocardial  ischemia  are  to  determine  the  subcellular  mechanisms  responsible 
for  iiiipaired  myocardial  function  during  ischemia,  and  to  develop  techniques 
whereby  ischemia- induced  subcellular  injury,  and  therefore  physiologic  func- 
tion, can  be  rainized. 

Proposed  Course  of  Project:   Outlined  in  abstract;  now  in  progress. 

Honors  and  Awards :   None 

Publications:   None  1  ^^ 


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Serial  No.   nhT.T-14 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS -NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Titel:   Physiological  and  Biochemical  Characteristics  of  a  New  Group 
of  Inotropic  Agents:   Analogues  of  Angiotensin  II. 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.  D.,  Ph.  D. 

Other  Investigators:     Theodore  Goodfriend,  H.  D. 

Theodore  Cooper,  M.  D. ,  Ph.  D. 

Cooperating  Units:       Department  of  Pharmacology,  University  of  Wisconsin, 

Madison,  Wisconsin 

Project  Description:   Angiotensin  II  and  several  of  its  analogues  have  direct 
positive  inotropic  effects.   In  the  course  of  investigating  the  effects  of 
hypoxia  on  the  activity  of  these  agents  we  found  that  the  inotropism  of  two 
of  the  angiotensin  II  analogues  has  a  unique  characteristic,  i.e.,  potenti- 
ation by  hypoxia.   Thus,  angiotensin  II,  des-1,  val-5  produced  a  507o  increase 
in  isometric  contractile  force  in  isolated  cat  papillary  muscles  at  2  x  10"'M 
and  a  maximum  increase  in  contractile  force  of  80%  at  10~"M;  when  the  muscles 
were  made  hypoxic  by  equilibrating  the  Kreb's  solution  with  an  oxygen  concen- 
tration of  57o  instead  of  95%,  control  tension  decreased  32  +  YL   but  the  con- 
centration-response curve  for  this  compound  shifted  to  the  left  and  maximum 
response  increased.   The  507o  increase  in  tension  occurred  at  5  x  10"9m  and 
the  maximum  increase  in  contractile  force  (at  10""M)  was  1907o.   The  responses 
to  angiotensin,  des-1,  ile-5  were  also  greater  in  hypoxic  papillary  muscles. 
However,  this  compound  was  found  to  be  less  potent  than  the  former. 

The  inotropic  responses  are  independent  of  catecholamine  stores  since 
identical  responses  were  obtained  in  papillary  muscles  from  catecholamine- 
depleted  muscles  of  chronically  denervated  cats.   The  potentiation  produced 
by  hypoxia  is  also  independent  of  extracellular  (Ca-H-).   Present  studies  are 
being  performed  to  determine  the  inotropic  properties  of  these  compounds  when 
administered  to  animals  with  an  intact  coronary  circulation. 

In  assessing  the  biochemical  properties  of  these  polypeptides,  we  have 
found  that  they  bind  specifically  to  submitochondrial  phosphorylating  parti- 
cles (ETPjj)  from  beef  heart.   They  increase  the  rate  of  phosphorylation,  the 
tightness  of  coupling  (respiratory  control),  and  resistance  to  "aging"  con- 
ditions of  intact  heart  mitochondria.   Phosphorylation  rate  is  increased  407, 
by  5  X  10~°g  polypeptide/mg  mitochondrial  protein.   Although  the  metabolic 
responses  to  these  polypeptides  improve  oxidative  metabolism,  it  is  unclear 
whether  this  is  the  mechanism  for  their  unique  inotropic  return  in  hypoxia. 


1^ 


Serial  No.   NHLI-14 


PHS -NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  19  71 


Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   Submitted  for  publication. 


I 


if 


Serial   No.       NHLI-15 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Supersensitivity  of  Hyperthyroid  Myocardium  to  Decreased 
Extracellular  Calcium 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.  D.,  Ph.  D. 

Other  Investigators:     Peter  J.  Dempsey,  M.  D. 

Theodore  Cooper,  M.  D. ,  Ph.  D. 

Cooperating  Units:       None 

Project  Description:   Previous  workers  have  demonstrated  that  the  contractile 
state  of  both  the  intact  heart  and  isolated  myocardial  preparations  obtained 
from  hyperthyroid  animals  is  increased.   However,  the  cellular  mechanisms 
responsible  for  the  enhanced  contractile  state  produced  by  thyroid  hormone 
remains  unknown.   The  purpose  of  this  study  was  to  determine  the  sensitivity 
of  hyperthyroid  myocardium  to  extracellular  calcium  concentration  LCa''\J  . 
Two  different  processes  presumed  in  part  to  reflect  calcium  transport  in 
myocardial  cells  was  measured:  first,  developed  isometric  tension  (T)  and 
rate  of  tension  development  (dT/dt);  and  second,  the  transmembrane  action 
potential  (TAP).   At  0.45  mM  [ca++]e  T  decreased  32  +  3%  in  control  and 
51  +  57o  in  hyperthyroid  myocardium.   At  0.15  mM  Lca++Jg  the  decrease  in  T 
was  54  +  47o  in  control  and  76  +  37o  in  hyperthyroid  muscles.   Although  the 
sensitivity  of  the  contractile  apparatus  to  [Ca++J  of  hyperthyroid  myocar- 
dium was  thus  marked,  the  effect  of  Ca"*"*"  on  TAP  in  hyperthyroid  myocardium 
was  similar  to  that  in  normal  myocardium.   These  findings  may  provide  some 
clue  as  to  the  mechanism  of  increased  contractility  of  hyperthyroid  myocar- 
dium. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   None 


4c 


Serial  No.    NHLI-16 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Control  of  Heart  Rate  in  ?Iyperthyroid  Cats 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.  D.,  Ph.  D. 

Other  Investigator:      Theodore  Cooper,  M.  D. ,  Ph.  D. 


Cooperating  Units; 


None 


Project  Description:   The  heart  rates  of  anesthetized  or  alert  hyperthyroid 
arJ.mals  are  increased,  as  are  the  intrinsic  frequencies  of  contraction  of 
hearts  isolated  from  such  animals.   However,  we  have  found  that  the  basal 
heart  rates  of  intact  unanesthetized  cats,  in  which  the  electrocardiogram  was 
monitored  by  telemetry,  were  not  different  from  those  of  normal  cats.   Basal 
heart  rates  were  obtained  by  telemetry  from  the  cats  in  the  early  morning 
hours  while  the  cats  remained  in  their  cages.  Any  disturbance  of  the  cats 
caused  exaggerated  increases  in  heart  rates  as  compared  to  control  animals. 
However,  when  the  hearts  were  removed  from  these  hyperthyroid  cats  and  studied 
on  a  Langendorff  apparatus  isolated  from  neural  or  hormonal  influences,  the 
inherent  sinus  node  rates  were  40-50^  higher  than  the  rates  of  normal  cats. 
These  observations  can  be  explained  by  the  hypothesis  that  the  negative  chrono- 
tropic effect  of  the  vagus  is  enhanced  in  hyperthyroid  cats.  This  enhancement 
could  have  two  mechanisms.   First,  the  frequency  of  vagal  nerve  discharge  could 
be  increased,  or  second,  the  hyperthyroid  hearts  could  be  more  sensitive  to 
acetylcholine  (Ach),  the  neurotransmitter.  The  latter  hypothesis  is  being 
tested  in  isolated  perfused  hearts  from  hyperthyroid  cats.   Preliminary  results 
have  shown  that  several  cardiac  actions  of  Ach  are  enhanced  in  hearts  from 
hyperthyroid  cats.   Thus,  we  have  found  that  the  direct  negative  inotropic  ef- 
fect of  Ach,  a  muscarinic  action,  is  enhanced  in  hyperthyroid  myocardium,  as 
is  the  release  of  norepinephrine  from  the  ventricular  myocardium,  a  nicotinic 
response  to  Ach.   In  addition,  decreased  conduction  through  the  A-V  node 
caused  by  Ach  occurs  at  lower  doses  of  Ach  in  hearts  from  hyperthyroid  cats. 
Studies  are  in  progress  to  determine  the  sensitivity  of  the  sinus  node  to  Ach 
in  hearts  from  hyperthyroid  cats. 

Proposed  Course  of  Project:   Project  continuing. 

Honors  aind  Awards  :   None 

Publications:   None 


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Serial   No.      NHTJ-17 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Reports 
July  1,  1970  through  June  30,  1971 

Project  Title:   Effects  of  Hypoxia  on  the  Cardiac  Response  to  Inotropic 
Interventions 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.  D.,  Ph.  D. 

Other  Investigators:     Stephen  E.  Epstein,  M.  D. 

Theodore  Cooper,  M.  D. ,  Ph.  D. 

Cooperating  Units:       None 

Project  Description:   To  evaluate  the  effects  of  hypoxia  on  the  capacity  of 
the  heart  to  respond  to  various  inotropic  agents,  cat  papillary  muscles  were 
made  hypoxic  by  changing  the  equilibrating  gas  mixture  from  95%  to  5%   oxygen; 
p02  of  the  Krebs'  solution  decreased  from  440  +  8  to  122  +  6  mm  Hg.   Baseline 
contractile  force  decreased  32  +  37o,  and  the  concentration-response  curve  of 
norepinephrine  (NE)  was  depressed.   Following  the  maximum  response  to  NE,  de- 
terioration of  developed  tension  occurred  within  10  minutes.   Maximum  response 
to  ouabain  was  less  in  hypoxia  than  under  control  conditions  but  no  deteriora- 
tion occurred  after  maximum  response  was  achieved.   The  concentration-response 
curve  of  angiotensin  II  was  unchanged  under  hypoxic  conditions. 

In  order  to  determine  if  a  similar  hypoxia  induced  depression  in  ino- 
tropic activity  occurred  in  intact  hearts  perfused  with  blood  through  their 
coronary  arteries,  a  dog  heart -lung  preparation  has  been  developed  in  which 
afterload  and  heart  rate  are  held  constant  and  preload  is  controlled  by  a 
resistance  clamp.   Left  ventricular  pressure,  dp/dt,  left  and  right  coronary 
arterial  flow  and  circuit  flow  are  measured.   Induction  of  hypoxia  is  accom- 
plished by  decreasing  the  inspired  percentage  of  oxygen. 

When  arterial  p02  was  decreased  from  100  to  20-25  mmHg  (Hb  saturation 
407o)  at  a  constant  pH  and  pC02 ,  there  was  an  initial  transient  increase  in 
maximum  dp/dt.   After  return  to  control  value,  dp/dt  remained  stable  for  up 
to  two  hours.   This  stable  hypoxic  state  was  associated  with  an  approximately 
four-fold  increase  in  coronary  flow.   In  this  preparation,  in  contrast  to  cat 
papillary  muscles,  the  contractile  response  to  NE  was  augmented  during  hypoxia, 
the  dose  response  curve  shifted  to  the  left.   Furthermore,  no  deterioration 
of  the  preparation  occurred  after  the  highest  doses  of  NE  employed. 

Studies  are  in  the  process  to  evaluate  both  the  biochemical  basis  of 
this  phenomenon  and  the  effects  of  hypoxia  on  the  response  of  this  preparation 
to  other  inotropic  agents. 

1  vi 


J 


Serial   No. 


NHLI-17 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 


Proposed  Course  of  Project:   Continuing 
Honors  and  Awards :   None 
Publications:   None 


IV 


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.   —  - 

Serial  No.  NHLI- 18(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1960  through  June  30,  1971 

Project  Title:   The  Natural  History  of  the  Floppy  Mitral  Valve  Syndrome 

Previous  Serial  Number:   None 

Principal  Investigator:   Samuel  B.  Itscoitz,  M.  D, 

Other  Investigators;     Stephen  E.  Epstein,  M.  D. 

D.  Luke  Clancy,  M.  D. 

Cooperating  Units:       None 

Project  Description:   Until  1963,  midsystolic  clicks  and  late  systolic  murmurs 
were  believed  to  be  extracardiac  in  origin  and  of  no  clinical  importance.   In 
1963,  however.  Barlow  and  co-workers  first  introduced  evidence  that  these  sounds 
were  due  to  mitral  valve  disease  with  accompanying  mitral  regurgitation.  This 
has  been  documented  repeatedly  by  other  investigators,  and  during  the  past 
eight  years  additional  observations  have  been  made.   The  disorder  clusters  in 
families  and  is  probably  congenital,  although  the  murmur  is  not  present  at 
birth.   It  is  related,  in  unclear  fashion,  to  both  Marfan' s  and  Turner's  syn- 
dromes.  Anatomic  material,  when  available,  usually  reveals  a  large  redundant, 
slightly  thickened  posterior  mitral  valve  leaflet  with  long  thin  chordae  ten- 
dineae.  Endocarditis  has  been  reported  and  may  result  in  chordae  rupture  in 
severe  mitral  regurgitation.   ECC  abnormalities  are  common  and  suggest  inferior 
wall  ischemia.  Arrhythmias  occur  frequently,  and  sudden  death  has  been  reported. 

Because  of  its  rather  recent  recognition,  however,  the  natural  history  of 
this  disorder  is  not  well  known.  Since  the  tension  exerted  on  chordae  tendineae 
is  directly  related  not  only  to  the  intraventricular  pressure  but  also  to  the 
area  of  the  mitral  valve  leaflets  to  which  it  is  attached,  (i.e.  the  value  leaf- 
lets act  as  an  amplifier)  a  large  redundant  leaflet  would  cause  greater  than 
normal  tension  to  be  exerted  on  the  chordae.  Although  one  might  thus  anticipate 
that  spontaneous  chordal  rupture  would  commonly  complicate  this  abnormality, 
there  is  only  one  such  case  report  in  the  literature  at  the  present  time. 

In  the  past  two  years,  two  patients  have  been  seen  on  the  Cardiology  ward 
who  required  mitral  valve  replacement  for  severe  mitral  regurgitation  secondary 
to  ruptured  chordae  tendineae.   The  ruptured  chordae  had  attached  to  a  billow- 
ing, redundant,  floppy  posterior  mitral  valve  leaflet.   Of  interest,  the  typi- 
cal auscultatory  features  of  the  floppy  valve  were  not  present  in  either  of 
these  patients;  presumably  the  mechanism  of  regurgitation  was  no  longer  re- 
lated to  the  floppy  valve  per  se,  but  rather  to  the  ruptured  chordae.  These 
additional  cases  provide  further  evidence  that  this  complication  may  constitute 


«^^ 


Serial  No.  NHLI-18(c) 


PHS-NIH 
Individual  Project  Report 
July  1,  1970   through  June  30,  1971 


a  part  of  the  natural  history  of  this  entity. 
Proposed  Course  of  Project:   Completed 
Honors  and  Awards :   None 
Publications:   In  preparation 


a  ' 


ti 


I 


^ 


•KMtwMUHMHfSH 


Serial  No.  NHLI-19(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


\ 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Disseminated  Intravascular  Coagulation  (DIVC)  Complicating 
Severe  Congestive  Heart  Failure  (CHF) 

Previous  Serial  Number:  None 

Principal  Investigator:  Samuel  B.  Itscoitz,  M.  D. 

Other  Investigators:  Stephen  E.  Epstein,  M.  D. 

Cooperating  Units:  None 

Project  Description:   The  syndrome  of  DIVC,  an  uncommon  complication  of  a 
wide  variety  of  disease  states,  has  received  increasing  attention  in  the 
past  5-10  years.   It  is  now  commonly  accepted  that  conditions  in  which 
thromboplastic  substances  gain  access  to  the  circulation  (i.e.,  amniotic 
fluid  embolism)  or  conditions  in  which  regional  blood  flow  is  stagnant  (i.e., 
shock  of  any  origin)  provide  the  milieu  in  which  DIVC  may  occur.   Nonetheless, 
reports  of  DIVC  complicating  severe  CHF  are  very  rare. 

Despite  its  supposed  rarity,  in  the  past  three  years  ten  patients  on  the 
Cardiology  ward  have  had  relatively  well  documented  episodes  of  DIVC.   All 
patients  were  in  severe  congestive  heart  failure,  usually  biventricular  in 
origin.   The  etiology  of  their  heart  disease  was  variable,  including  rheumat- 
ic heart  disease,  myocardiopathy,  healed  endocarditis,  and  idiopathic  chordae 
tendineae  rupture.   Diagnosis  of  DIVC  syndrome  was  based  on  the  following 
laboratory  studies:  platelet  count,  presence  of  circulating  fibrinogen  degra- 
dation products,  prothrombin  time,  thrombin  time,  partial  thromboplastin  time, 
factor  V  level,  factor  VIII  level,  and  bleeding  time. 

Severity  of  the  DIVC  was  quite  variable,  ranging  from  severe  thrombocyto- 
penia in  one  patient  (who  was  found  to  have  70  gm.  of  fresh  thrombus  in  the 
left  atrium  at  operation)  to  mild  transient  thrombocytopenia  in  another. 
Clinical  deterioration  accompanied  the  DIVC  episode  in  some,  but  not  all, 
patients.   Response  to  heparin,  when  administered,  was  variable.   Evidence 
strongly  suggestive  of  pulmonary  embolic  disease  was  commonly  noted.   One 
patient  has  been  lost  to  followup.   Of  the  seven  known  survivors,  variable 
degrees  of  cardiac  disability  persist  ranging  from  functional  Class  II  to 
functional  Class  IV. 

Proposed  Course  of  Project:   Completed 
Honors  and  Awards :   None 

Publications:   In  preparation. 

1  <<^ 


I 


Serial  No.  NHTJ-20(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   A  Radioisotope  Imaging  Technique  for  the  Identification  of 
Myocardial  Scar 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  E.  Goldstein,  M„  D. 

Other  Investigators:     Douglas  R.  Rosing,  M.  D. 

Gerald  Schall,  M.  D. 
Steven  Larson,  M.  D. 
Stephen  E.  Epstein,  M.  D. 

Cooperating  Units:       Department  of  Nuclear  Medicine 

Clinical  Center,  N.I.H. 

Project  Description:   The  recent  advent  of  direct  surgical  approaches  to 
ischemic  heart  disease,  utilizing  either  myocardial  revascularization  or 
resection  of  noncontracting  portions  of  the  ventricular  wall,  has  made  the 
distinction  of  viable  contractile  tissue  from  scar  of  crucial  importance. 
At  present,  scarred  regions  of  ventricular  myocardium  are  identified  as 
areas  of  abnormal  wall  motion  on  ventriculography.   This  technique,  however, 
is  indirect  and  insensitive.   At  the  time  of  surgery  the  heart  may  be 
examined  visually  and  surface  electrograms  obtained.   But,  aside  from  the 
necessity  of  thoracotomy,  these  methods  also  require  that  the  epicardial 
layers  are  representative  of  the  entire  ventricular  wall.   Thus,  a  safe, 
sensitive  radioisotope  technique  which  would  allow  detailed  mapping  of  living 
myocardial  tissue  in  man  without  necessitating  thoracotomy  would  be  an  impor- 
tant contribution  to  preoperative  evaluation.  The  use  of  rubidium-84  in  com- 
bination with  the  Auger  positron  camera  configuration  may  represent  such  a 
technique. 

Rubidium  (Rb)  is  an  alkali  metal  whose  chemical  and  biological  behavior 
closely  mimics  that  of  potassium.   When  administered  intravenously  Rb  accumu- 
lates rapidly  in  heart  and  skeletal  muscle  (and  other  organs)  so  that  it  is 
almost  entirely  removed  from  the  blood  stream  within  two  minutes.   This  prop- 
erty has  been  utilized  to  measure  coronary  flow  in  man.   After  intravenous  or 
intracoronary  administration  of  the  positron-emitting  isotope  Rb-84,  a  coin- 
cidence counting  scintillation  camera  is  used  to  identify  those  areas  of  the 
myocardium  which  fail  to  take  up  Rb-84  due  to  replacement  of  muscle  tissue  by 
scar.   This  technique  has  reportedly  been  successful  in  localizing  areas  of 
infarction  in  dogs.   Coincidence  counting  should  provide  a  high  resolution 
image,  theoretically  resolving  0.5  cm  points  apart.   Positron  emission  also 
allows  "focusing"  of  the  camera  so  that  its  tomographic  "cuts"  of  the  chest 
can  be  obtained.   Thus,  a  three-dimensional  array  of  radioactivity  can  be 

1  <^7 


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A- 


Serial  No.  NHLI-20(c) 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


mapped  and  "focused"  further,  using  a  digital  computer.   Preliminary  studies 
with  open-chest  dog  preparations  have  demonstrated  the  ability  of  Rb-84  to 
delineate  the  myocardium  even  on  intravenous  injection.   Intravenous  adminis- 
tration of  Rb-84  in  two  patients,  however,  failed  to  permit  adequate  imaging 
of  the  myocardium.   We  currently  plan  to  determine  whether  intracoronary  ad- 
ministration will  result  in  sufficient  myocardial  concentration  to  result  in 
a  satisfactory  scan.   We  also  plan  to  evaluate  the  imaging  properties  of 
potassium-A3,  an  isotope  reported  to  be  useful  as  a  myocardial  scanning  agent. 

Proposed  Course  of  Project:   Project  continuing. 

Honors  and  Awards :   None 

Publications:   None 


1 


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Serial  No.      NHLI-21 

1,  Cardiology 

2,  Clinical  Physiology 
3o  Bethesda,  Md„ 


Project  Title; 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Correlation  of  Antiarrhythmic  Effects  of  Diphenylhydantoin 
with  Digoxin-induced  Changes  in  Contractility,  Na-K  ATPase 
and  K+  Efflux 


Previous  Serial  Number:  None 

Principal  Investigator:   Robert  E„  Goldstein,  M.D, 

Other  Investigators:     S,  C„  Penzotti,  M.D, 

Karen  S„  Kuehl,  M.D. 
Kirk  Ho  Prindle,  M.D, 
Clifford  A,  Hall,  M,D. 
Elwood  0„  Titus,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Laboratory  of  Chemical  Pharmacology,  NHLI 

Project  Description:  To  clarify  the  suppressant  action  of  diphenylhydantoin 
on  digitalis -induced  arrhythmias  we  studied  the  effects  of  diphenylhydantoin 
and  digoxin,  alone  and  in  combination,  on  contractile  force,  Na-K  ATPase 
activity  and  K^  efflux  in  Krebs-Ringer  perfused  dog  hearts.  Neither  control 
perfusion  nor  diphenylhydantoin  alone  (3  x  10~%)  altered  Na-K  ATPase  or 
produced  K^  efflux,  Diphenylhydantoin  alone  depressed  contractile  force  an 
average  of  29%  at  15  min.  Digoxin  alone  (10"%)  in  7  dogs  caused  a  59% 
rise  in  contractile  force  at  onset  of  arrhythmia  (toxicity)  (avg.  15.9  min  of 
perfusion  +  0,7  SE)  along  with  net  ¥^   efflux  of  50  +  12  pM/min  and  decrease 
in  Na-K  ATPase  from  13.8  to  5.2  |jM  Phos/mg  protein/hr.  (P  <  0.001).  Per- 
fusion with  diphenylhydantoin  and  digoxin  combined  (5  dogs)  delayed  toxicity 
to  28,9  +2,8  min,  at  which  time  contractile  force  was  higher,  917,  above 
control  (P  <  0.05),  K*"  efflux  tended  to  be  greater  (87  +  20  uM/min)  and  Na-K 
ATPase  was  lower  (2,5,  P  <  0,05)  than  with  digoxin  alone.  Combined  diphenyl- 
hydantoin-digoxin  perfusion  lasting  only  until  the  time  toxicity  appeared 
with  digoxin  alone  (7  dogs),  however,  yielded  higher  Na-K  ATPase  (7.7,  P  "^ 
0.02)  compared  with  digoxin  alone;  K*"  efflux  was  unchanged  and  contractile 
force  decreased  (26%  above  control,  P  <  0.05).  Thus  diphenylhydantoin 
appears  to  diminish  the  rate  at  which  digoxin  inhibits  Na-K  ATPase.   Neverthe- 
less, diphenylhydantoin  ultimately  permits  digoxin  to  produce  greater  inhi- 
bition of  Na-K  ATPase,  greater  increase  in  contractile  force,  and  a  tendency 
toward  greater  K*  efflux  than  possible  without  diphenylhydantoin.  This  sug- 
gests that  the  antiarrhythmic  effects  of  diphenylhydantoin  cannot  be  attri- 
buted to  prevention  of  inhibition  of  Na-K  ATPase  or  to  diminution  of  K*" 
efflux,  two  changes  characteristically  accompanying  digoxin  administration. 
Proposed  Course  of  Project:   Completed 
Honors  and  Awards:   None 
Publications:  Manuscript  in  preparation  ^^ 


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Serial  No.     NHLI-22 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


Project  Title: 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Hemodynamic  Changes  During  Exertional  Syncope  in 
Dogs  with  Pulmonary  Artery  Constriction 


Previous  Serial  Number:   None 

Principal  Investigator:   Robert  E.  Goldstein,  M„  D. 

Other  Investigators:     Al«n  Nimetz,  M.  D. 

Joseph  Pierce,  D.  V.  M. 
Stephen  E.  Epstein,  M.  D. 


Cooperating  Units; 


None 


Project  Description:   Individuals  with  severe  obstruction  to  outflow  of 
either  the  right  or  left  ventricles  may  experience  syncope  during  or  immedi- 
ately after  strenuous  exercise.   In  order  to  clarify  the  hemodynamic  basis 
for  this  phenomenon,  several  circulatory  indices  were  measured  while  dogs 
with  pulmonary  artery  constriction  exercised  to  the  point  of  syncope.   Pul- 
monary constriction  was  achieved  by  the  inflation  of  a  Jacobsen  balloon  cuff 
previously  sewn  around  the  pulmonary  artery.   During  the  course  of  treadmill 
exercise,  continuous  recordings  were  made  of  systemic  arterial,  right  ventric- 
ular, and  distal  pulmonary  artery  pressures  and  the  electrocardiogram.   In 
several  animals  aortic  blood  flow  was  measured  by  a  circumferential  electro- 
magnetic flowmeter  cuff.   Preliminary  results  showed  that  syncope  was  almost 
always  accompanied  by  rapidly  progressive  arterial  hypotension  rather  than  by 
arrhythmia.   Decline  in  cardiac  output  generally  accompanied  the  fall  in 
blood  pressure,  suggesting  that  inadequacy  of  cardiac  output  rather  than  sud- 
den vasodilatation  was  responsible  for  the  decline  in  blood  pressure  which 
led  to  circulatory  collapse.   Further  experimental  studies  will  be  performed 
in  animals  with  implanted  flowmeters  in  order  to  substantiate  these  prelimi- 
nary findings. 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 


Publications : 


None 


S» 


Project  Title; 


Serial   No.    NHLI-23 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Comparison  of  Simultaneously  Determined  Inotropic  and  Chrono- 
tropic Effects  of  Practolol  and  Propranolol 


Previous  Serial  Number:  None 

Principal  Investigator:  Robert  E.  Goldstein,  M.D, 

Other  Investigators: 


Cooperating  Units; 


Clifford  A.  Hall,  M,D, 
Stephen  E,  Epstein,  M.D. 

None 


Project  Description:      Recent     reports  have   differed   concerning   the   relative 
negative   inotropic  effects   of  the   P-blockers  practolol  and  propranolol.     To 
resolve   these   conflicting   findings,    simultaneous  changes   in   contractile    force 
and  heart   rate  were  measured  when  increasing  doses   of  either  propranolol   or 
practolol  were   given  to  dogs   receiving  infusions   of   isoproterenol.      Infusion 
was  adjusted   initially   to  produce   a  mean  heart   rate   of   155;   blood  pressure  was 
held  constant.     Contractile   force  was  measured  by  a   right  ventricular   strain 
gauge  arch.     When  drug  dosages  producing  equal  decreases   in  heart   rate  were 
compared,   the  associated   decrement   in  contractile   force  was  not   significantly 
different;    for  example,   when  heart   rate   decreased   15%,   contractile   force  was 
lowered   37%  with  practolol  and  41%  with  propranolol    (NS) .     Thus,   a   given  re- 
duction in  heart  rate  was  accompanied  by   the   same   negative   inotropic   response 
with  both  drugs.      Effective   P-blocking  doses   of  propranolol    (up   to   1  mg/Kg) 
given  after  3-blockade   had  been  produced  by   large   doses   of  practolol   caused 
no   further  depression   in  contractile   force.     These   findings   suggest   that 
depression  by  propranolol   in  doses   less   than   1  mg/Kg  is   largely  related   to 
P-blockade;    primary  depressant  effects  were    seen  only  with  doses  above    1  mg/ 
Kg,      Practolol  after   large   doses   of  propranolol,   however,    increased   contrac- 
tile  force   and  heart  rate:      at   2-5  mg/Kg  contractile    force   increased   33%,  and 
hefirt   rate   6%.     Thus  practolol  may  exert  a  modest  positive   inotropic  effect 
detectable   after   full   P-blockade    or  possibly  when   p-receptor   stimulation   is 
minimal.     Nevertheless,    such  an  effect   is   inapparent   if  P-receptor   stimula- 
tion  is    substantial,   which   is   often  the   case  when  P-blockade    is  employed 
clinically.     Thus,   practolol  as  used  therapeutically,   does  not  appear  to  act 
on  heart   rate  more   selectively  than  propranolol. 

Proposed  Course    of   Project:      Continuing 

Honors  and  Awardsj.None 

Publications:     None 


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Serial  No,  NHLI-24(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,   Md. 


1 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Effects  of  Chronic  Heart  Failure  on  the  Capacity  of  Glucagon 
to  Enhance  Contractility  and  Adenyl  Cyclase  Activity  of 
Human  Papillary  Muscles 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  E,  Goldstein,  M.D, 

Other  Investigators:     C„  Lynn  Skelton,  M.D, 

Gerald  S„  Levey,  M.D. 
D„  Luke  Clancy,  M.D. 
Go  David  Beiser,  M.D, 
Stephen  E.  Epstein,  M.D. 


Cooperating  Units: 


None 


Project   Description:      Glucagon  exerts   a    positive    inotropic   effect    in  normal 
hearts   but   is   ineffective    in  animals  with   chronic   cardiac    failure.      To 
assess   directly   the    influence    of   glucagon   on  human  myocardium,    we   measured 
contractility  and  activation   of  adenyl   cyclase,    the   enzyme    thought   to 
mediate   the   inotropic  action  ot   glucagon,    in   left  ventricular  papillary 
muscles   obtained    from   12   patients  at  mitral  valve    replacement.      On   the   basis 
of  preoperative  ventricular  end-diastolic   pressures  and   cardiac   output 
(independent   of  papillary  muscle   data)    patients  were    classified   in   3   groups: 
normal,   with   cardiac    failure,    or   indeterminate.      Concentration-response 
curves    showed   that   glucagon   caused   a  mean   11%  rise    in  peak  papillary  muscle 
tension  and    127,  rise    in   peak   rate    of   tension  development    in   the   normal 
patients;   myocardial  adenyl   cyclase   activity   from  each  patient   rose   after 
glucagon    (average   =   87,).      In   the   papillary  muscles   of   the   patients  with 
cardiac    failure,    glucagon  produced  no  augmentation    in  either   tension   or 
adenyl   cyclase   activity.      In   contrast,    contractility  and  adenyl   cyclase 
activity   increased  after  norepinephrine    in  both  normal   patients  and   those 
with   cardiac    failure.      The    indeterminate    group  had    two  patients  whose 
papillary  muscles   responded   to  glucagon   and   two  whose    papillary  muscles  did 
not   respond.     Thus,    direct    study   of  human  papillary  muscles    shows    that 
chronic    cardiac    failure    is   uniformly  associated  with  a    complete    loss    of   the 
normal  enhancement   of  contractility  and  associated  activation   of  adenyl 
cyclase   after   glucagon,    perhaps  explaining   the    inefficacy   of   this   drug   in 
treating  patients  with  chronic   cardiac    failure. 

Proposed  Course:      Completed 


Honors  and  Awards:      None 

Publications:     Manuscript    submitted    for  publication. 

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Serial  No„     NHLI-25 

1,  Cardiology 

2.  Clinical  Physiology 
3o      Bethesda,   Md, 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Comparison  of  the  Inotropic  and  Chronotropic  Activity  of 
Glucagon  and  Isoproterenol  in  the  Intact  Canine  Heart 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  E.  Goldstein,  M„D„ 


Other  Investigators; 


Cooperating  Units: 


David  R„  Redwood,  M,D. 
G,  David  Beiser,  M„D„ 
Alan  Nimetz,  M.D, 
Joseph  Pierce,  D.V„M. 
Stephen  E.  Epstein,  M,D„ 

Division  of  Research  Resources,  Bureau  of  Health 
Professions  Education  &  Manpower  Training 
Laboratory  of  Kidney  and  Electrolyte  Metabolism, 
National  Heart  and  Lung  Institute 

Project  Description:   Although  glucagon  and  isoproterenol  are  both  known  to 
be  powerful  inotropic  agents,  the  relative  potency  of  these  two  drugs  has 
not  been  systematically  assessed.  We  therefore  compared  the  cardiac  effects 
of  glucagon  and  isoproterenol  in  each  of  10  dogs.  Using  a  strain  gauge  sewn 
to  the  right  ventricle,  dose-response  curves  were  obtained  for  each  drug 
while  arterial  pressure  was  held  constant.  Cardiac  output  was  measured  at 
the  peak  dose  (maximal  strain  gauge  deflection)  of  each  drug.  Results  were 
similar  whether  glucagon  was  infused  continuously  or  given  as  a  bolus.  Peak 
doses  of  both  glucagon  and  isoproterenol  increased  strain  gauge  deflection 
from  a  mean  of  15  to  36  mm  (paired  comparison  of  strain  gauge  deflections  at 
peak  glucagon  and  isoproterenol  showed  a  mean  difference  of  0.24_'f2,0  mm  SE)  , 
Heart  rate  at  peak  dose  averaged  217  beats/min  for  glucagon  and  195  beats/min 
for  isoproterenol  (P  <  .005).  Cardiac  output  averaged  4.9  L/min  during  peak 
glucagon  and  6.6  during  peak  isoproterenol  (P  <  ,05).   In  conclusion,  direct 
comparison  of  glucagon  and  isoproterenol  in  the  normal  canine  heart  showed 
the  maximal  inotropic  effect  of  glucagon  to  be  indistinguishable  from  that  of 
isoproterenol.   In  contrast  to  previous  impressions,  peak  doses  of  glucagon 
produced  marked  tachycardia,  exceeding  that  of  peak  isoproterenol  when  pres- 
sure was  held  constant.  Under  these  same  circumstances  peak  glucagon  also 
increased  cardiac  output  considerably,  although  less  than  peak  isoproterenol. 
Studies  in  three  dogs  with  chronic  congestive  heart  failure  due  to  pulmonary 
artery  constriction  showed  a  marked  reduction  in  glucagon  responsiveness  con- 
sistent with  diminished  glucagon  responsiveness  observed  in  chronically  fail- 
ing myocardium  of  cat  and  man. 
Proposed  Course  of  Project:   Completed 
Honors  and  Awards:   None 
Publications:  None 

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Serial  No.   NHTJ-26(c) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   The  Dj-^namic  Nature  of  Left  Ventricular  Outflow  Obstruction 
in  Idiopathic  Hypertrophic  Subaortic  Stenosis 

Previous  Serial  Number:   None 

Principal  Investigator:   D.  Luke  Clancy,  M.  D. 

Other  Investigators:     Richard  L.  Shepherd,  M.  D. 

G.  David  Beiser,  M.  D. 
Stephen  E.  Epstein,  M.  D. 

Cooperating  Units:   None 

Project  Description:   Left  ventricular  outflow  obstruction  in  idiopathic  hy- 
pertrophic subaortic  stenosis  (IHSS)  is  caused  by  midsystolic  apposition  of 
the  disproportionately  hypertrophied  cephalad  portion  of  the  ventricular  sep- 
tum with  the  free  margin  of  the  anterior  mitral  valvular  leaflet,  and  the 
degree  of  obstruction  is  variable.   Unusual  findings  during  hemodynamic 
studies  in  three  patients  support  the  hypothesis  that  the  degree  of  obstruc- 
tion in  this  condition  is  determined  by  three  factors:   the  force  of  ventric- 
ular contraction,  ventricular  volume,  and  ventricular  afterload.   In  one 
patient  spontaneous  atrioventricular  dissociation  resulted  in  spontaneous 
changes  in  left  ventricular  end-diastolic  pressure,  and  presumably  in  end- 
diastolic  volume.   With  increases  in  end-diastolic  pressure  and  volume  the 
degree  of  left  ventricular  outflow  obstruction  decreased,  whereas  with  de- 
creases in  end-diastolic  pressure  and  volume  the  degree  of  obstruction  in- 
creased.  During  left  ventricular  pulsus  alternans  in  this  same  patient,  the 
more  forceful  ventricular  contractions  resulted  in  an  increase  in  the  degree 
of  obstruction, and  peak  pressure  in  the  systemic  arteries  did  not  alternate. 

In  the  second  patient  PVC  responses,  which  initially  were  typical  of 
IHSS,  transiently  became  normal  following  the  injection  of  angiographic  con- 
trast material,  suggesting  that  an  increase  in  ventricular  volume  and  depres- 
sion of  myocardial  contractility  temporarily  relieved  the  left  ventricular 
outflow  obstruction.   In  the  third  patient  a  spontaneous  increase  in  systemic 
arterial  pressure,  and  thus  in  left  ventricular  afterload,  completely  abolished 
a  100  mm  Kg  subaortic  systolic  gradient. 

Proposed  Course  of  Project:   Completed. 

Honors  and  Awards:   None 

Publications:   Submitted  for  publication. 


Serial  No.    NHLI-27 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 


Project  Title: 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Dissociation  of  Cardiac  Inotropic  and  Membrane  Effects  of 
Ouabain 


Previous  Serial  Number:   NHLI  -  21 


Principal  Investigator:   Peter  J.  Dempsey,  M.  D, 


Other  Investigators: 


Cooperating  Units; 


Kenneth  M.  Kent,  M.  D. ,  Ph.  D. 
Theodore  Cooper,  M.  D. ,  Ph.  D. 

None 


Project  Description:   Ouabain  causes  negative  inotropic  responses  in  cat 
papillary  muscles  when  the  external  calcium  concentration,  [.'-'^''"^Je'  ^^  0.45 
mM  or  lower.   This  negative  inotropism  occurs  with  shortening  of  phase  2  of 
the  transmembrane  action  potential  (TAP),  the  latter  being  the  characteristic 
response  to  ouabain. 

Further  studies  have  shown  that  the  negative  inotropism  was  dose  depend- 
ent.  Ouabain  4  x  10"^M  at  [Ca++Jg  =  0.15  mM  produced  at  12  +  1%   decrease  in 
isometric  contractile  force  whereas  ouabain  3  x  lO'^M  caused  a  41  +  6%  de- 
crease.  Norepinephrine,  isoproterenol,  and  angiotensin  II  produced  their 
usual  inotropic  responses  independent  of  the  [^a++Jg. 

When  ouabain  was  added  to  cat  papillary  muscles  at  L^a  J  lower  than 
0.45  mM,  there  was  an  initial  lengthening  of  phase  2  of  the  TAP.   This  was  a 
transient  response  for  as  the  negative  inotropic  response  to  ouabain  became 
established,  phase  2  shortened  in  the  characteristic  manner  for  ouabain. 

A  possible  explanation  for  these  events  is  a  ouabain-induced  loss  of  Ca"*"^ 
"rrom  the  cell  into  the  perfusing  solution  which  has  a  low  [S'Si^^   .   This  loss 
of  Ga"""  would  explain  the  lengthening  of  the  TAP  and  the  negative  inotropic 
response. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 


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Publications ; 


ARTICLE  PUBLISHED  IN  PERIODICAL: 

Dempsey,  P.J. ,  McCallum,  Z.T. ,  Kent,  K.M. ,  and  Coopei;  T,:  Dissociation  of 
cardiac  inotropic  and  membrane  effects  of  ouabain.  J.  Pharm.  Exp.  Therap. 
176:  78  ,  1971. 

1  rir 


Serial   No.     NHLI-28 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Calcium  Ion  Movement  in  Skeletal  Muscle 

Previous  Serial  Number:   None 

Principal   Investigator:  Chester  E.  Clark,  M.  D. 

Other  Investigators:     None 

Cooperating  Units:       None 

Project  Description:  Current  theory  regarding  excitation-contraction  cou- 
pling in  cardiac  and  skeletal  muscle  centers  around  the  concept  that  calcium, 
released  by  the  sarcoplasmic  reticulum  (SR)  following  electrical  depolariza- 
tion, diffuses  from  SR  to  a  receptor  located  on  the  actin  filament.  Then, 
through  a  series  of  as  yet  unknown  transformations,  Ca  is  believed,  in  this 
location,  to  cause  the  interaction  of  actin  and  myosin  filaments,  thereby 
initiating  muscle  shortening.  Implicit  in  this  hypothesis  is  the  assumption 
that  the  rate  of  diffusion  of  Ca  is  sufficient  to  account  for  the  rapid  se- 
quences of  contraction  and  relaxation  characteristic  of  cardiac  and  skeletal 
muscle.  However,  the  only  data  relating  to  the  rate  of  calcium  ion  movement 
in  muscle  cytoplasm  indicate  that  mobility  is  at  least  50  to  100  times  slower 
than  in  water,  a  rate  probably  too  slow  to  be  compatible  with  the  above  hypoth- 
esis. If  the  slow  rate  measured  in  muscle  cytoplasm  can  be  attributed  to  up- 
take by  the  SR,  then  faster  rates  would  be  expected  in  muscle  subcompartments 
where  SR  envelops  the  contractile  apparatus  and  is  structured  to  produce  min- 
imal interference  with  calcium  mobility.  It  would  also  be  expected  that 
electrical  depolarization  would  minimize  calcium  uptake  by  SR. 

In  order  to  ascertain  if  the  intracellular  movement  of  calcium  between 
subcellular  compartments  is  rapid  enough  to  control  contraction  of  striated 
muscle,  an  analogue  circuit  for  the  solution  of  diffusion  problems  was  con- 
structed. Using  the  known  binding  constants  for  SR  it  was  found  that  uptake 
of  calcium  by  SR  could  not  account  for  the  slow  movements  of  calcium  intra- 
cellularly.  This  finding  thus  raises  the  possibility  that  the  classical  hy- 
pothesis for  excitation-contraction  coupling  may  be  in  error. 

To  confirm  this  finding  experimentally  in  living  tissue,  individual 
myofibrils  were  isolated  by  homogenation  and  centrifugation,  and  the  measure-  _ 
ments  of  the  delay  in  onset  of  contraction  after  exposure  to  calcium  was  at-   fl 
tempted  by  measuring  alterations  in  optical  density.   This  technique  was  em- 
ployed since  it  has  been  reported  that  optical  density  changes  in  parallel  to 
the  degree  of  myofibrillar  contraction.   However,  no  correlation  was  discovered 
between  myofibrillar  contraction  and  optical  density,  a  finding  calling  into 

1  ci 


Serial   No,     NHLI-28 


July 


PHS-NIH 
Individual  Project  Report 
1,  1970  through   June  30,  1971 


question  many  studies  published  in  the  literature  based  on  such  methodology. 

Two  other  approaches  of  measuring  Ca  diffusion  rates  in  vivo  are  being 
developed.   First,  we  are  in  the  process  of  developing  techniques  to  isolate 
single  muscle  fibers  from  the  frog  semitendinosus  muscle.   The  fiber  will  be 
cut  across  one  end  and  exposed  to  calcium^^.   After  varying  periods  of  time 
the  muscle  will  be  sliced  and  the  distribution  of  calcium^^  determined.  From 
this  data  a  diffusion  rate  can  be  calculated. 

Finally,  we  are  trying  to  utilize  high  speed  photomicrography  to  detect 
the  delay  of  myofibrillar  contraction  after  exposure  to  calcium. 

Proposed  Course  of  Project:   Project  continuing. 

Honors  and  Awards :   None 

Publications:   None 


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S-7 


Serial  No.     NHLI-29 


1„      Cardiology 

2,  Clinical   Physiology 

3.  Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   The  Treatment  of  Ventricular  Arrhythmias  Occurring  During 
Acute  Coronary  Artery  Occlusion  in  Conscious  Dogs: 
Efficacy  of  Atropine  and  Lidocaine 

Previous  Serial  Number:   None 

Principal  Investigator:   G.  David  Beiser,  MoD, 

Other  Investigators:     Douglas  R.  Rosing,  M„D„ 

Richard  B.  Karsh,  M„D. 
James  Talano,  M.D„ 
Martin  Miller 
James  Bailey,  M„Do 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Division  of  Computer  Research  and  Technology 

(Computer  Systems  Laboratory  and  Laboratory  of 
Applied  Studies) 

Project  Description:  Ventricular  arrhythmias  associated  with  acute  myo- 
cardial infarction  may  seriously  compromise  cardiac  output  or  result  in  a 
fatal  episode  of  ventricular  fibrillation  or  cardiac  standstill.  In  order 
to  evaluate  the  treatment  of  such  arrhythmias,  we  studied  40  closed-chest 
conscious  dogs  in  which  acute  myocardial  ischemia  was  produced  by  inflating 
a  balloon  cuff  previously  implanted  around  the  left  anterior  descending 
coronary  artery  just  distal  to  its  first  diagonal  branch.   Of  the  40  dogs, 
15  developed  frequent  and  persistent  ventricular  ectopic  beats  and/or 
episodes  of  ventricular  tachycardia  within  the  first  one  or  two  hours  of 
occlusion.   In  5  dogs  the  arrhythmia  progressed  rapidly  to  ventricular  fibril 
lation  and  death.   In  the  remaining  10  dogs  increasing  the  heart  rate  with 
the  intravenous  administration  of  atropine  markedly  decreased  or  completely 
abolished  the  arrhythmia.  However,  in  some  of  the  dogs  it  was  necessary  to 
increase  the  heart  rate  to  as  high  as  140  -  160/min  to  suppress  the 
arrhythmia.   The  intravenous  administration  of  lidocaine  in  a  dose  of 
2  mg/kg  either  decreased  the  number  of  premature  beats  or  abolished  the 
arrhythmia  completely.  In  three  dogs  the  administration  of  atropine  to  a 
heart  rate  of  120/min,  or  lidocaine  at  a  dose  of  2  mg/Kg,  were  only 
partially  effective  in  the  treatment  of  the  arrhythmia.  However,  the  combina- 
tion of  these  two  drugs  eliminated  all  ectopic  activity. 

Proposed  Course  of  Project:   Additional  dogs  will  be  studied  to  further  define 
the  efficacy  of  atropine  and  xylocaine,  alone  and  in  combination,  in  the 
treatment  of  arrhythmias  associated  with  acute  experimental  myocardial  in- 
f«rc;tion„ 

-1-  SS 


♦ 


I 


I 


Serial  No.     NHLI-29 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 


Honors  and  Awards:  None 
Publications:  None 


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Serial  No.  ^rHT  .T-.^n/r) 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   The  Effects  of  Operative  Correction  of  Congenital  Heart 
Disease  on  the  Functional  Capacity  of  the  Heart 

Previous  Serial  Number:   NHLI-34(c) 

Principal  Investigator:   G.  David  Beiser,  M.  D. 

Other  Investigators:     Stephen  E.  Epstein,  M.  D. 

Robert  E.  Goldstein,  M.  D. 
Douglas  R.  Rosing,  M.  D. 
David  R.  Redwood,  M.  D. 
Andrew  G.  Morrow,  M.  D. 

Cooperating  Units:       Clinic  of  Surgery,  NHLI 

Project  Description:   In  the  past  decade  great  strides  have  been  made  in  the 
operative  treatment  of  patients  with  congenital  heart  disease.   However, 
progress  in  the  methods  used  to  evaluate  the  functional  results  of  such  op- 
erations have  lagged  far  behind.   Thus,  while  we  know  that  many  patients  with 
severe  cyanotic  congenital  heart  disease  experience  marked  symptomatic  im- 
provement after  operative  correction,  we  have  little  information  as  to  whether 
the  functional  capacity  of  the  heart  is  completely  normal.   We  also  have  no 
information  relating  to  the  effects  on  cardiac  function  of  a  ventriculotomy  or 
of  the  insertion  of  a  patch  in  the  right  ventricular  outflow  tract.   With  the 
recent  development  in  this  laboratory  of  a  sensitive  technique  to  evaluate 
the  maximal  pumping  capacity  of  the  heart,  a  study  was  undertaken  with  the 
purpose  of  answering  these  questions.   In  brief,  the  cardiac  output,  oxygen 
uptake,  and  right  ventricular  and  pulmonary  arterial  pressures  were  determined 
during  maximal  treadmill  exercise  in  patients  in  whom  an  atrial  septal  defect 
or  tetralogy  of  Fallot  had  been  repaired  six  months  to  three  years  previously. 

Seven  of  eleven  asymptomatic  patients  with  closed  atrial  septal  defects   | 
had  a  significant  impairment  in  their  cardiac  output  response  to  exercise.  In 
addition,  one  of  three  patients  who  had  pulmonary  arterial  hypertension  pre- 
operatively  developed  abnormally  high  pulmonary  pressures  during  intense  exer- 
cise.  Of  the  ten  patients  with  correction  of  tetralogy  of  Fallot  who  have 
been  studied,  eight  showed  a  small  but  definite  impairment  in  the  pumping  ca- 
pacity of  the  heart;  two  had  a  normal  cardiac  output  at  intense  exercise.  Al- 
though it  might  have  been  expected  that  patients  with  tetralogy  of  Fallot 
would  have  a  reduced  capacity  of  the  pulmonary  vascular  bed  and  thus  develop 
pulmonary  hypertension  during  maximal  exercise,  none  of  the  ten  patients  devel- 
oped abnormal  pulmonary  arterial  pressures. 


4a 


I 


Serial  No.   NHLI-30(c) 


PHS-NIH 
Indivudual  Project  Report 
July  1,  1970  through  June  30,  1971 

However,  right  ventricular  systolic  pressure,  measured  in  six  patients, 
increased  in  four  from  normal  or  minimally-elevated  levels  at  rest  to  greater 
than  85  mmHg  during  intense  exercise.   This  elevation  was  due  to  increases  in 
right  ventricular  outflow  gradients  ranging  from  50  to  6  5  mmHg.   Finally,  the 
presence  of  significant  pulmonic  regurgitation  in  five  patients  with  corrected 
tetralogy  of  Fallot  did  not  appear  to  compromise  their  cardiac  performance 
relative  to  the  other  patients. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications :   Manuscript  in  preparation. 


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Serial  No,        NHLI-31 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Ventricular  Tachycardia  Following  Release  of  Coronary 

Artery  Occlusion  in  Conscious  Dogs  and  the  Antiarrhythmic 
Effects  of  Atropine 

Previous  Serial  Number:   None 

Principal  Investigator:   G.  David  Beiser,  M.D„ 

Other  Investigators:     Douglas  R.  Rosing,  M.D. 

Richard  B.  Karsh,  M.D. 
James  Talano,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:  None 

Project  Description:   Arrhythmias  occurring  in  the  acute  stages  of  myocardial 
infarction  generally  have  been  attributed  to  persistent  myocardial  ischemia 
secondary  to  coronary  artery  occlusion.   To  evaluate  the  mechanisms  and 
treatment  of  such  arrhythmias,  we  studied  40  closed-chest  conscious  dogs 
in  which  acute  myocardial  ischemia  was  produced  by  inflating  a  balloon  cuff 
previously  implanted  around  the  left  anterior  descending  coronary  artery 
just  distal  to  its  first  diagonal  branch.  Of  the  40  dogs,  18  did  not 
develop  arrhythmias  during  the  first  hour  of  occlusion.   In  11  of  these  18, 
however,  sudden  release  of  occlusion  was  followed  15  seconds  to  3  minutes 
later  by  ventricular  arrhythmias  which  progressed  rapidly  to  ventricular 
tachycardia.   In  all  11  dogs  reocclusion  of  the  coronary  artery  abolished 
the  ventricular  tachycardia  within  30  seconds  with  a  return  to  the  sinus 
mechanism.   Reocclusion  for  10  to  15  minutes  followed  by   successive  periods 
of  release  and  reocclusion  consistently  reproduced  this  phenomena.   In  5  of 
6  dogs  atrial  pacing  at  rates  of  105  to  130  beats  per  minute  was  successful 
in  over-riding  re  lease -induced  ventricular  tachycardia  or  preventing  its 
appearance.  Likewise,  the  administration  of  atropine  in  10  dogs  just  prior 
to  release  of  occlusion  in  doses  that  increased  heart  rate  from  an  average 
of  76  +  6  (standard  error  of  the  mean)  to  112  +  3  beats  per  minute  prevented 
ventricular  tachycardia  in  all  dogs. 

Release  arrhythmias  were  also  studied  in  an  additional  group  of  5  dogs 
in  which  transient  ventricular  arrhythmias  occurred  during  the  first  hour 
of  occlusion.   Release  of  occlusion  in  these  animals  resulted  in  very 
malignant  appearing  ventricular  tachycardias  which  on  the  first  or  subse- 
quent releases  progressed  rapidly  to  ventricular  fibrillation  in  3  dogs 
despite  therapeutic  attempts. 


(£2 


Serial  No.      NHLI-31 


l?"< 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 


These  results  indicate  that  1)  sudden  reperfusion  of  a  previously 
ischemic  region  of  myocardium,  as  may  occur  in  man  by  lysis  or  dislodgment 
of  a  clot,  may  be  responsible  for  some  of  the  serious  arrhythmias  seen  in 
acute  myocardial  infarction  which  result  in  sudden  death;  2)  increasing  the 
heart  rate  by  the  administration  of  atropine  may  be  successful  in  the  pre- 
vention of  many  but  not  all  of  these  arrhythmias. 


Proposed  Course  of  Project: 


To  further  define  the  character  of  these 
arrhythmias  in  relation  to  the  onset  of 
occlusion  and  to  study  the  effects  of  other 
therapeutic  agents  in  preventing  their 
appearance  with  release  of  occlusion. 


Honors  and  Awards:  None 


Publications:  None 


IW 


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Serial  No.         NHTJ-32 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


1 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Mechanical  behavior  of  large  coronary  arteries. 

Previous  Serial  NuTiber:   NHLI-47 

Principal  Investigator:   Dali  J.  Patel,  M.D.,  Ph.D. 

Other  Investigators:   Thomas  E.  Carew,  Ph.D. 

Project  Description: 

Objective:   To  study  1)  the  rheology  and  2)  the  effect  of  increased  stress 
on  the  protein  permeability  of  the  endothelial  surface  of  the  left  cir- 
cuTiflex  coronary  artery  (LCCA)  and  the  anterior  descending  coronary 
artery  (ADCA)  . 

Methods  Employed:   1)   The  study  evaluating  the  static  elastic  properties 
of  LCCA  has  been  completed  (see  publication) .   2)   Left  coronary  artery 
was  perfused  in  situ  at  various  perfusion  pressures  and  flows  with 
o.cygenated  blood  containing  known  amounts  of  Evans  blue  dye  for  approxi- 
mately 1  hour.   At  the  end  of  the  experiment  the  endothelial  surface  of 
both  LCCA  and  ADCA  was  exposed  and  examined  under  a  ref lectoraeter  for 
blue  staining.   The  permeability  of  the  endothelium  to  albumin  was 
evaluated  by  quantifying  the  blue  staining  of  the  endothelial  surface. 
The  amount  of  dye  bound  albumin  that  had  entered  the  surface  was  corre- 
lated with  the  shearing  stress  as  well  as  the  pressure  to  which  the 
artery  was  subjected  during  the  perfusion  experiment. 

Major  Findings:   The  data  was  grouped  into  4  groups:   1)  High  shearing 
stress  (average  value  30  dynes/cm  )  and  high  pressure  (average  value 
170  cm  H2O) ;  2)  high  shearing  stress  and  low  pressure;  3)  low  shearing 
stress  and  high  pressure  and  4)  low  shearing  stress  and  low  pressure. 
Preliminary  results  suggest  that  high  shearing  stress  led  to  a  greater 
dye  concentration  in  the  intima  than  did  high  pressure. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
Although  the  importance  of  hydrodynamic  and  rheologic  factors  in  early 
vessel  damage  has  been  recognized,  its  role  has  not  been  critically 
evaluated  in  the  coronary  vascular  bed  to  date.   The  present  studies 
are  designed  toward  better  understanding  of  the  role  of  mechanical 
stress  in  producing  vascular  diseases. 

Proposed  Course  of  the  Project:   1)   Studies  to  evaluate  the  dynamic  vis- 
co-elastic  properties  of  the  LCCA  are  in  progress.   2)   Studies  are  in 
progress  a)  to  delineate  the  role  of  shearing  stress  vs  normal  stress  in 
increasing  the  permeability  of  the  endothelial  surface,  and  b)  to 
define  the  critical  stress  at  which  the  endothelium  deteriorates. 

Honors  and  Awards:   None. 


^^ 


« 


Serial  No.       NHLI-32 


PHS-NIH 

Individual  Project  Report 

July  I,    1970  through  June  30,  1971 


Publications: 


Patel,  D.J.,  and  Janicki,  J.S.:   Static  elastic  properties  of  the  left 
coronary  circumflex  artery  and  the  common  carotid  artery  in  dogs. 
Circulation  Res.  2J_:    149-158,  1970. 


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Serial  No.   NHT.T-.'^.'^ 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Blood  velocity  profiles  and  wall  shear  in  the  aorta  and  its 
major  branches. 

Previous  Serial  Number:   NHLI-48 

Principal  Investigator:   Dali  J.  Patel,  M.D.,  Ph.D. 

Other  Investigators:   Donald  L.  Fry,  M.D. 

Joseph  S.  Janicki,  M.S. 

S.  C.  Ling,  Ph.D. 

H.  Bulent  Atabek,  Ph.D. 

Cooperating  Units:   The  Department  of  Space  Science  and  Applied  Physics, 
The  Catholic  University  of  America 

Project  Description: 

Objective:   To  measure  and  quantify  1)  the  blood  velocity  fields,  2) 
turbulence,  and  3)  the  interfacial  shearing  stress  on  the  endothelial 
surface  along  the  major  arteries  of  dogs  through  the  cardiac  cycle 
under  various  conditions. 

Methods  Employed:   A  constant-temperature  heated  film  anemometer  system 
has  been  adapted  for  direct  measurement  of  in  vivo  aortic  velocity 
fields.   Although  the  method  is  promising  all  technical  problems  are 
not  yet  resolved  and  effort  in  the  past  year  was  directed  towards  these. 
Also  progress  is  made  towards  automatic  data  processing  without  which 
considerable  time  would  be  spent  in  plotting  velocity  fields  from  se- 
quentially obtained  velocity  curves  within  the  cross-section  of  the 
aorta. 

Major  Findings:   Preliminary  results  indicate  that  velocity  profiles  along 
the  descending  thoracic  aorta  are  essentially  blunt;  slight  skewing 
is  observed  which  may  be  due  to  flow  being  diverted  to  intercostal 
arteries. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  method  provides  a  powerful  tool  in  quantitative  investigations  of 
the  vascular  system.   It  will  help  assess  the  role  of  hydrodynamic 
factors  in  potentiating  the  development  of  atheroma  in  the  major  arter- 
ies. 

Proposed  Course  of  the  Project:   A  numerical  technique  has  been  developed 
and  verified  in  models  at  Catholic  University  to  compute  velocity  pro- 
files and  wall  shearing  stress  from  measurement  of  pressure,  pressure 
gradient  and  radius  in  a  straight  segment  of  a  tube.   Attempts  will  be 
made  to  test  this  indirect  but  easier  method  vs  direct  measurements  of 
wall  stress  and  velocity  profiles  in  a  dog. 


64 


Serial  No.       NHLI-33 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


PKS-NIH 

Individual  Project   Report 

July   1,    1970   through  June   30,    1971 


Honors   and  Awards; 


None. 


Publicati£)ns; 


None. 


I  PH.t 


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67 


Serial  No.       NHLI-34 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30;,  1971 

Project  Title:   Vascular  mechanics:   arterial  wall  properties 

Previous  Serial  Number:   NHLI-49 

Principal  Investigator:   Dali  J.  Patel,  M.D.,  Ph.D. 

Other  Investigators:   Joseph  S.  Janicki,  M.S.,  R.  N.  Vaishnav,  Ph.D. 

Cooperating  Unit?:   Department  of  Civil  and  Mechanical  Engineering,  The 
Catholic  University  of  America,  Washington,  D.  C 

Project  Description: 

Objective:   To  study  the  nonlinear,  visco-elastic  properties  of  the  blood 
vessel  under  physiologic  conditions  with  special  emphasis  on  measurement 
of  "strain  energy  density." 

Methods  Employed:   A  segment  of  the  middle  descending  thoracic  aorta  was 
studied  in  dogs.   The  pressure-radius-longitudinal  force  relationship  was 
studied  (using  specially  designed  transducers)  by  connecting  the  segment 
to  a  reservoir  filled  with  oxygenated  blood  and  adjusting  the  height  of 
the  reservoir  to  cover  the  physiologic  range  of  pressure.   In  addition, 
a  flow  pump  was  connected  to  this  assembly  to  superimpose  sinusoidal 
pressures  at  various  frequencies  on  the  segment  to  study  its  dynamic 
properties.   From  these  data  it  was  possible  to  compute  1)  the  values  of 
elastic  constants  to  describe  the  non-linear  behavior  of  the  vessel  wall 
and  2)  the  incremental  visco-elastic  constants  in  the  three  principal 
directions  (radial,  circumferential  and  longitudinal) . 

Major  Findings:   1)   Previous  studies  in  the  laboratory  have  shown  that  the 
blood  vessel  wall  is  essentially  incompressible  and  demonstrates  aniso- 
tropic behavior  with  a  certain  elastic  symmetry.   2)   The  values  of  in- 
cremental visco-elastic  coefficients  in  the  three  principal  directions 
increased  with  frequency.   3)   The  values  of  the  elastic  part  of  these 
coefficients  (real  part  of  the  complex  coefficient)  were  much  greater 
than  their  viscous  counterpart.   4)   The  nonlinear  elastic  behavior  of 
the  vessel  wall  could  be  described  adequately  in  the  physiologic  range 
of  pressures  by  7  constants.  From  these  data  it  was  possible  to  predict 
the  values  of  incremental  elastic  constants  as  well  as  strain  energy 
density  at  any  desired  value  of  pressure.   This  latter  finding  was  ex- 
tremely useful  for  precise  comparison  of  data  among  different  animals. 

Significance  to  Bio-medical  research  and  the  Program  of  the  Institute: 
Detailed  experimental  studies  like  this  are  essential  for  building  a 
mathematical  theory  to  describe  the  vascular  system  which  in  turn  would 
complement  our  Section's  program  on  experimental  atherosclerosis.  The 
reasons  for  this  are  twofold:   1)  The  dynamic  behavior  of  the  vascular 

1  ^2 


I 


Serial  No.       NHLT-34 


PHS-NIH 

Individual  Project  Report 

July  I,    1970  through  June  30,  1971 

system  depends  critically  upon  the  physical  properties  of  the  vessel 
wall  and  its  tethering  mechanism.   These  properties  had  not  been  ade- 
quately quantified  prior  to  these  studies.   2)   Recent  studies  in  our 
laboratory  have  indicated  that  an  increased  strain  energy  density  in 
the  vessel  wall  may  act  as  a  driving  force  to  increase  the  flux  of  lipo- 
proteins across  the  vessel  wall.   Thus,  it  becomes  important  to  esta- 
blish the  nature  and  magnitudes  of  the  vessel  wall  elastic  moduli  so 
that  the  strain  energy  density  of  the  wall  can  be  calculated  and  com- 
pared to  the  distribution  of  early  experimental  atheroma- 
Proposed  Course  of  the  Project:   The  studies  will  be  continued  1)  to  in- 
clude j^  vivo  dynamic  anisotropic  properties  of  the  blood  vessel  wall; 
and  2)  to  provide  an  experimental  basis  for  a  nonlinear  visco-elastic 
theory  of  the  blood  vessel  wall. 

Honors  and  Awards :   None 

Publications: 

Patel,  D.J.,  and  Vaishnav,  R.N. :   Rheology  of  large  blood  vessels » 
Cardiovascular  Fluid  Dynamics.  Academic  Press,  Inco   Londouo   In  press. 


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Serial  No.      NHLI-35 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  I,    1970  through  June  30,  1971 

Project  Title:   In  vitro  studies  of  the  influence  of  mechanical  factors  on 
transvascular  albumin  flux. 

Previous  Serial  Numbers:   None 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objective:   To  study  the  effect  of  various  mechanical  factors  on  the  flux 
of  albumin  across  the  vascular  interface. 

Methods  Employed:   Freshly  removed  canine  aortic  tissue  was  placed  in  a 
specially  designed  device  (described  in  previous  reports)  such  that  the 
interface  could  be  exposed  to  varying  predetermined  degrees  of  stretch 
and  trnasmural  pressure,  while  being  covered  with  autologous  serum  con- 
taining Evans  blue  tagged  albumin.   Intact  endothelial  surfaces,  as  well 
as  surfaces  that  had  been  denuded  by  gentle  stroking  with  a  camel's  hair 
brush  were  studied. 

Major  Findings:  It  was  found  that  the  flux  of  albumin  across  the  vascular 
interface  was  increased  by  stretching  the  surface.  In  contrast  to  this 
transmural  pressure  drops  up  to  280  cm  of  water  pressure  caused  no 
change  in  the  flux  across  the  surface,  either  in  the  intact  preparation 
or  in  that  in  which  the  endothelial  cell  surface  had  been  removed.  The 
removal  of  endothelial  cells  increases  the  permeability  of  the  vascular 
interface  by  at  least  2  orders  of  magnitude. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
These  studies  shed  light  on  the  mechanical  factors  which  influence  the 
transport  of  albumin  across  the  vascular  interface.   They  indicate  that 
the  greatest  barrier  to  protein  flux  resides  in  a  small  region  associa- 
ted with  the  endothelial  cell  layer.   The  fact  that  pressure  does  not 
influence  the  flux  of  albumin  into  the  wall,  either  in  the  presence  of  a 
normal  barrier,  or  in  its  absence,  suggests  that  the  arterial  wall 
buttresses  the  endothelial  cells  and  their  associated  barrier  against 
the  forces  of  pressure  rendering  pressure  ineffective  as  a  driving  force 
for  protein  transport.   Mechanical  events  analogous  to  the  foregoing  may 
also  be  operative  in  the  vascular  system  and  of  significance  in  the 
transport  of  p-lipoprotein  in  the  pathogenesis  of  atherosclerosis. 

Proposed  Course  of  the  Project:   Studies,  such  as  the  above,  will  be  re- 
peated with  various  other  proteins  and,  in  particular,  with  p-lipopro- 
tein when  appropriate  tagging  techniques  become  available  for  their 
quantification. 

1  7tf 


Serial  No.     NHTJ-35 

1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda^  Maryland 


PHS-NIH 
Individual  Project  Report 
July  I,    1970  through  June  30,  1971 


Honors  and  Awards:   None 


Publications: 

Fry,  D.L.:   Localizing  factors  in  arteriosclerosis.   Ch.  II.  Athero- 
sclerosis and  Coronary  Heart  Disease.   Hahnemann  Symposium,  1971. 
In  press. 


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Serial  No.     NHLI-36 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   In  vitro  study  of  the  influence  of  temperature  on  trans- 
vascular  albumin  flux. 

Previous  Serial  Numbers:   None 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objective:  To  qviantify  the  effective  temperature  on  protein  flux  into  the 

vascular  wall. 
Methods  Employed:   Freshly  removed  canine  aortas  were  placed  in  a  spe- 
cially designed  device  such  that  various  portions  of  the  vascular  seg- 
ment could  be  held  at  different  prescribed  temperatures.   Each  of  these 
areas  was  then  exposed  to  autologous  serum  containing  Evans  blue  tagged- 
albumin  for  specified  periods  of  time  and  the  subsequent  flux  of  albumin 
into  the  wall  measured. 

Major  Findings:   Below  300°  K  the  flux  of  albumin  into  the  vascular  inter- 
face appears  to  follow  an  exponential  relationship  suggesting  that  it 
could  be  an  activated  diffusion  process.   Above  312°  K  the  resistance  of 
the  diffusion  barrier  virtually  disappears  resulting  in  massive  influx 
of  protein  into  the  wall.  Histologic  studies  suggest  that  the  endo- 
thelial cells  belcw  this  temperature  remain  normal  and  above  this  tem- 
perature suffer  changes  in  staining  properties  and  become  pyknotic. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
There  has  been  evidence  from  this  laboratory  suggesting  that  the  per- 
meability of  the  vascular  interface  may  be  related  to  its  internal 
energy  density.   If  so,  it  would  be  expected  also  to  show  the  character- 
istic temperature-flux  relationship  of  an  activated  diffusion  process. 
While  the  foregoing  data  are  of  a  preliminary  nature  and  as  yet  show 
too  much  scatter  to  prove  the  hypothesis,  they  are  nevertheless  sugges- 
tive. The  data  also  suggest  that  at  temperatures  in  excess  of  300  K 
the  vascular  interface  appears  suddenly  to  "melt,"  which  is  consistent 
with  the  idea  that  part  of  the  barrier  may  reside  in  the  cell  membranes 
which  are  known  to  have  a  transition  in  this  range. 

Proposed  Course  of  the  Project:   Further  studies  will  be  done  to  refine  the 
measuring  techniques  so  that  the  functional  relationships  between  tem- 
perature and  flux  can  be  quantified  more  accurately.   Electron  micro- 
scopic and  histochemical  studies  of  the  tissue  will  be  done  in  an 


1%. 


Serial   No.       NHTJ-36 

1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  I,    1970  through  June  30,  1971 

effort  to  identify  changes  in  composition  of  the  wall  with  temperature, 
particularly  those  associated  with  the  "melting"  process.   Finally, 
these  studies  will  be  repeated  with  various  lipoproteins  when  suitable 
tagging  techniques  for  their  quantification  become  available. 

Honors  and  Awards:   None 

Publications:   None 


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Serial  No.      NHTJ-37 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,    1970  through  June  30,  1971 

Project  Title:   In  vivo  studies  of  aortic  intimal  histologic  and  chemical 
response  to  acutely  induced  mechanical  stress. 

Previous  Serial  Numbers:   NHLI-41 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.D. 

Cooperating  Units:   Section  on  Pathology,  Cardiology  Branch 

Project  Description: 

Objective:   To  continue  in  vivo  studies  of  the  intimal  histologic  and 

chemical  response  to  acutely  induced  mechanical  stress  in  shear,  tension, 
and  compression. 

Methods  Employed:   A  variety  of  mechanical  devices  have  been  devised  which 
allow  controlled  mechanical  stress  to  be  applied  to  the  vascular  intima 
in  a  variety  of  configurations  in  the  acute  animal  preparation.   The  de- 
tails of  these  methods  have  been  described  in  previous  reports.   Addi- 
tional methodology  was  developed  to  quantify  the  tissue  response  to  the 
above  induced  stresses.   Two  new  techniques  were  developed  for  estimat- 
ing the  flux  of  Evans  blue  tagged  albumin  across  the  vascular  interface. 
The  first  was  a  direct  ref lectometric  approach  in  which  the  freshly 
opened  stretched  vascular  interface  is  mounted  in  an  evenly  illuminated 
thermally  and  chemically  controlled  bath.   The  image  of  the  submerged 
interface  is  projected  by  a  lens  system  to  a  large  ground  glass  plate, 
one  point  of  which  is  scanned  by  a  photocell.   With  an  appropriate  se- 
lection of  filters  it  is  possible  to  measure  the  amount  of  light  absorbed 
in  the  blue-stained  regions  as  a  function  of  the  Evans  blue  concentra- 
tion in  the  vessel  surface.   The  second  method  is  a  fluorescence  tech- 
nique based  on  an  observation  which  we  have  made  that  Evans  blue  bound 
albumin  fluoresces  at  about  630  nanometers  when  excited  by  the  green 
band  of  the  mercury  spectrum.   The  amount  of  red  light  emitted  can  then 
be  quantified  using  appropriate  filter  systems  such  that  the  concentra- 
tion of  Evans  blue  may  be  related  to  the  fluorescence  intensity. 

Major  Findings:   The  above  studies  have  corroborated  the  results  of  pre- 
vious studies  in  this  laboratory  in  that  the  vascular  interface  has 
been  found  to  be  responsive  to  applied  mechanical  stress:   endothelial 
cells  yield  at  about  400  dynes/cm^  and  the  permeability  of  the  vascular 
interface  to  protein  increases  at  stresses  below  this  valueo   The  re- 
flectometric  measurement  of  staining  density  distribution  of  Evans  blue 
dye  on  the  opened  vascular  interface  was  found  to  correlate  well  with 
the  intensity  of  Evans  blue  albumin  fluorescence  elicited  from  the 
corresponding  histologic  section. 

1  7^ 


* 


Serial  No. 


NHLI-37 


PHS-NIII 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
Both  experimental  as  well  as  naturally-occurring  atherosclerosis  is  a 
discrete  process  occurring  in  the  vascular  intima,  the  topography  of 
which  suggests  that  the  development  of  atherosclerotic  lesions  tends 
to  be  accelerated  in  regions  exposed  to  increased  mechanical  stress. 
If  the  mechanism  of  this  process  is  to  be  studied,  then  it  is  essential 
to  develop  techniques  for  studying  discretely  the  transport  mechanics 
of  the  vascular  interface. 

Proposed  Course  of  the  Project:  This  project  has  been  completed  and  data 
is  being  analyzed  and  prepared  for  publication. 

Honors  and  Awards :   None 

Pub lications:   None 


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Serial  No.   NHLI-38 


1.  Cardiology  Branch 

2o  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


1 


PHS-NIH 
Individual  Project  Report 
July  I,    1970  through  June  30,  1971  £ 

Project  Title:   The  develop.Tient  of  methods  for  the  in  vitro  study  of  vascular 
interfacial  transport  mechanics.  . 

Previous  Serial  Numbers:   NHLI-42 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.D.,  and  John  T.  Flaherty, 
M.D. 

Cooperating  Units:   Section  on  Pathology,  Cardiology  Branch 

Project  Description: 

Objective:   To  develop  methods  for  the  in  vitro  study  of  intimal  transport 
mechanics.  ■ 

Methods  Employed:   Arterial  segments  are  removed  and  placed  in  a  special 
tissue  holding  device  (described  in  previous  reports) .   The  essential 
purpose  of  the  device  is  to  permit  selected  areas  of  the  opened  vascular 
interface  to  be  studied  in  a  controlled,  thermal,  chemical  and  mechanical 
milieu.   The  surface  is  exposed  to  serum  containing  various  tagged  mole- 
cules of  interest  for  selected  periods  of  time,  following  which  the      ■ 
amount  of  tagged  material  that  has  entered  the  interface  is  estimated     m 
using  ref lectometric,  fluorescent,  or  radioactivity  measurements. 

Major  Findings:   The  major  barrier  to  protein  transport  appears  to  reside 
in  a  thin  layer  associated  with  the  endothelial-cell  surface.   This 
barrier  appears  to  remain  intact  for  periods  up  to  four  hours,  so  long    ^ 
as  the  endothelial  surface  remains  covered  with  autologous  serum. 
Barrier  function  is  decreased  by  exposure  of  the  endothelial  surface  to 
saline,  albumin  solutions,  solutions  containing  traces  of  various  polar 
solvents,  such  as  ethanol,  and  solutions  containing  various  anesthetic 
agents,  such  as  nembutal. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
These  studies  have  shown  that  it  is  feasible  to  study  protein  transport 
across  the  vascular  interface  in  a  controlled  in  vitro  situation  for 
periods  up  to  4  hours » 

Proposed  Course  of  the  Project:   This  methodology  is  to  be  applied  to  a 
wide  variety  of  questions  concerning  those  factors  which  influence  the 
permeability  of  the  arterial  vascular  interface  to  various  molecular 
species  of  interest  and  in  particular  to  proteins  and  lipids. 

Honors  and  Awards:   None. 

Publications:   None. 


li 


Serial  No.       NHLI-39 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


ras-NiH 

Individual  Project  Report 
July  I,    1970  through  June  30,  1971 

Project  Title:   Development  of  canine  and  miniature  swine  experimental  ath- 
erosclerotic animal  colonies 

Previous  Serial  Number:   NHLI-45 

Principal  Investigator:   John  T.  Flaherty,  M.D. 

Other  Investigators:   Joseph  E.  Pierce,  DVM,  Donald  L.  Fry,  MoD. 

Cooperating  Units:   Laboratory  of  Kidney  and  Electrolyte  Metabolism,  NHLI, 
and  Food  and  Drug  Administration 

Project  Description: 

Objective:   To  develop  colonies  of  dogs  and  swine  with  experimentally  in- 
duced atherosclerosis  to  be  used  as  experimental  models  of  human  athero- 
sclerosis.  To  produce  atheroma  by  various  surgical  interventions.   To 
evaluate  the  effect  of  hypothyroidism  on  the  pattern  of  atherosclerosis 
in  minipigs. 

Methods  Employed:   An  atherogenic  diet  was  used  consisting  of  cholesterol, 
cholic  acid,  6-propylthiouracil  and  cottonseed  oil.  A  group  of  32  dogs 
was  thyroidectomized  and  then  placed  on  the  diet.   Serum  cholesterol, 
triglycerides,  lipoprotein  electrophoretic  patterns  and  thyroid  function 
were  monitored  at  monthly  intervals.  Arteriovenous  shunts  were  placed  in 
the  femoral  artery  in  7  dogs  and  in  the  carotid  artery  in  8  dogs.   Thor- 
acic aorta  coarctation  was  performed  on  8  dogs.  Minipigs  were  a) 
thyroidectomized  and  placed  on  an  atherogenic  diet  (3),  b)  placed  on  the 
atherogenic  diet  only  (2)  and  c)  placed  on  a  standard  swine  diet  for  con- 
trol purposes. 

Major  Findings:   The  thyroidectomized  dogs  showed  a  marked  rise  in  serum 
cholesterol,  triglycerides  and  p-lipoproteins.   Swine  also  developed 
elevations  of  p-lipoproteins  on  the  atherogenic  diet  both  with  and  with- 
out thyroidectomy.   Arteriovenous  shunts  remained  patent  for  3-4  months. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  establishment  of  canine  and  miniature  swine  colonies  with  experi- 
mentally produced  atherosclerosis  is  essential  to  studies  of  the  patho- 
genesis of  arteriosclerotic  cardiovascular  disease.  The  evaluation  of 
the  role  of  hypothyroidism  in  the  localization  of  atheroma  in  minipigs 
is  important  in  the  interpretation  of  the  data  that  can  be  obtained  from 
hypothyroid  atherosclerotic  dogs,  as  well  as  for  evaluation  of  the  role 
of  other  tissue  factors  involved  in  the  localization  of  lipid  deposition. 

Proposed  Course  of  the  Project:   Further  comparative  studies  of  the  detailed 
topography  of  lipid  deposition,  collagen  deposition  and  smooth  muscle 


77 


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Serial  No.       NHLI-39 


PHS-NIH 

Individual  Project  Report 

July  I,    1970  through  June  30,  1971 

proliferation  will  be  carried  out  to  elucidate  the  role  of  tissue  fac- 
tors.  The  interrelations  of  these  processes  as  well  as  their  relation 
to  mechanical  factors  will  be  studied.   Based  on  our  initial  observations 
of  femoral  artery  shunt  compared  to  carotid  artery  shunts,  there  is  a 
relative  resistance  seen  in  the  common  carotid  artery  to  the  build-up  of 
lipid  deposits  under  similar  hemodynamic  conditions  which  produce  massive 
atheroma  in  the  femoral  artery. 

Honors  and  Awards:   None 

Publications: 

Flaherty,  J.T.,  Ferrans,  V.J.,  Pierce,  J.E.,  Carew,  T.E.,  and  Fry,  D.L. : 
Localizing  factors  in  experimental  atherosclerosis.   Chapter  in  Coronary 
Heart  Disease  and  Atherosclerosis.   Ed.  W.  Likoff.   Grune  and  Stratton, 
New  York,  1971. 


I 


1 
70 


Serial  No.     NHLI-40 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda^  Maryland 


Project  Title: 


PHS-NIH 

Individual  Project  Report 

July  1,    1970  through  June  30,  1971 

The  topography  of  experimental  atherosclerotic  lesions  in 
the  dog. 


Previous  Serial  Number:   NHLI-43 

Principal  Investigator:   John  T.  Flaherty,  M.D. 

Other  Investigators:   Donald  L.  Fry,  M.D.,  Joseph  E.  Pierce,  DVM,  and 
Victor  J.  Ferrans,  M.D.,  Ph.D. 


Cooperating  Units: 


Laboratory  of  Kidney  and  Electrolyte  Metabolism,  NHLI, 
Section  on  Pathology,  Cardiology  Branch,  NHLI 


Project  Description: 

Objective:   1.  To  study  the  topography  of  atheroma,  in  hyperlipemic  dogs. 

2.  To  produce  atheroma  by  altering  flow  conditions  surgically. 

Methods  Employed:  The  atherosclerotic  dogs  were  given  Evans  blue  dye  as  a 
visual  tag  for  albumin  one  hour  prior  to  sacrificing.  The  entire  ar- 
terial tree  was  removed  and  mounted  for  examination  of  the  intimal  sur- 
face following  Sudan  IV  staining.  Photographic  records  were  made  of  the 
Sudan-stained  lipid  accumulations  and  the  blue  stained-albumin  concen- 
trations.  Histologic  sections  were  obtained  of  the  principal  lesions. 
Arteriovenous  shunts  or  coarctations  were  surgically  induced  prior  to  the 
starting  of  the  atherogenic  diet  in  a  second  group  of  dogs.   The  albumin 
and  lipid  staining  patterns  were  recorded  and  flow  and  pressure  distribu- 
tion measurements  obtained  prior  to  sacrifice. 

Major  Findings:   Discrete  lipid  infiltrations  were  found  principally  at 
branch  points  and  entrance  regions.   In  early  disease  Evans  blue  patterns 
were  similar.  Histologic  examination  of  the  early  lesions  revealed  lo- 
calization of  lipid  in  the  intima  often  localized  to  endothelial  cells  or 
to  the  region  of  the  internal  elastic  lamina.   Special  connective  tissue 
stains  revealed  the  presence  of  focal  fibromuscular  proliferations  at 
flow  dividers.  The  presence  of  collagen  in  apical  regions  and  within 
intimal  pads  appeared  to  alter  the  patterns  of  lipid  infiltration  and 
deposition  in  the  vessel  wall.   Arteriovenous  shunts  in  the  femoral 
artery  produced  extensive  but  characteristic  atheroma,  compared  to  com- 
parable shunts  in  the  carotid  artery,  which  produced  only  minimal  athero- 
mas.  Carotid  shunts  did  produce  fibromuscular  intimal  thickening  in  the 
region  of  the  shunted  artery  upstream  from  the  shunt.   Coarctations  pro- 
duced atheroma  on  the  leading  slope  of  the  narrowed  segment. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute:   The 
relation  of  the  topography  of  albumin  and  lipid  accumulations  as  well  as 


lUi 


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If 


Serial   No.      NHLI-40 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

connective  tissue  changes  to  areas  in  the  vascular  system  where  mechanical 
stresses  would  be  high  suggests  a  causal  relationship- 
Proposed  Course  of  the  Project:   Detailed  topography  of  the  atheroma  will 
point  to  areas  in  the  vascular  tree  where  shear  stresses  and  flow 
patterns  should  be  further  studied  j^  vivo  and  in  models.   Experimentally 
induced  atheroma  will  show  the  effect  of  controlled  alteration  of  hemo- 
dynamics on  lipid  accumulation  and  fibromuscular  proliferation  in  vessel 
wa 1 1 s  . 

Honors  and  Awards:   None 

Publications : 

Flaherty,  J.T.,  Ferrans,  V.J.,  Pierce,  J.E.,  Carew,  T.E.,  and  Fry,  D.L.: 

Localizing  factors  in  experimental  atherosclerosis.  Chapter  in  Coronary 

Heart  Disease  and  Atherosclerosis.   Ed.  W.  Likoff.  Grune  and  Stratton, 
New  York,  1971. 


eo 


Serial  No.        NHLI-41 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,    1970  through  June  30,  1971 

Project  Title:   Endothelial  nuclear  orientation  and  morphology  and  its 
relation  to  hemodynamic  factors  • 

Previous  Serial  Number:   NHLI-44 


Principal  Investigator:   John  T.  Flaherty,  M.D. 


Other  Investigators: 


Cooperating  Units; 


Dali  J.  Patel,  M.D.,  Ph.D.,  Joseph  E.  Pierce,  DVM, 
Victor  J.  Ferrans,  M.D.,  Ph.D. 

Laboratory  of  Kidney  and  Electrolyte  Metabolism,  NHLI, 
and  Section  on  Pathology,  Cardiology  Branch,  NHLI 


Project  Description: 

Objective:   To  study  the  variations  in  nuclear  orientation,  and  morphology 
of  canine  arterial  endothelivmi  and  to  relate  these  to  shear  stresses  and 
secondary  flow  patterns.  To  evaluate  the  role  of  physiologic  shear 
stresses  in  orienting  and  molding  the  nuclei.   Surgical  transposition  of 
segments  of  arteries  to  study  the  effect  of  physiologic  shear  stresses 
in  reorienting  and  reshaping  endothelial  nuclei  in  a  chronic  _in  vivo 
preparation. 

Methods  Employed:   Arterial  trees  from  normal  dogs  were  removed,  opened 
longitudinally  and  stretched  to  in  vivo  dimensions o   The  surface  was 
then  washed  with  isopropyl  alcohol  and  the  nuclei  stained  with  Evans 
blue  dye.   Photomicrography  was  employed  to  make  a  permanent  record  of 
the  endothelial  cells  at  many  selected  locations  along  the  arterial 
tree.   Major  axis  orientation  angles  were  measured  with  a  protractor 
and  minor  axes  lengths  and  cell-population  density  were  recorded.   Sur- 
gical transposition  of  segment  of  thoracic  aorta  was  carried  out  and  the 
endothelial  patterns  studied  at  3,  10,  21  and  70  days. 

Major  Findings:   Study  of  the  variations  in  orientation,  shape,  and  densi- 
ty of  the  endothelial  nuclei  at  various  locations  along  the  arterial 
tree  suggest  a  correlation  between  hemodynamic  factors,  and  "normal" 
endothelial  cell  patterns.   Markedly  non-axial  nuclear  orientations  are 
found  in  the  aortic  arch  and  in  the  upper  abdominal  aorta  which  could 
logically  correlate  with  secondary  flow  patterns  in  these  regions. 
Marked,  but  systematic  variations  of  nuclear  shape  and  density  are  also 
found,  which  are  suggestive  of  a  relationship  to  local  hemodynamic 
factors.   The  orientation  of  endothelial  nuclei  on  the  surgically  trans- 
posed segment  showed  complete  alignment  to  the  new  flow  direction  in  10 
days . 


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Serial   No.       NHTJ-41 


1.  Cardiology  Branch 

2.  Section  on  Clinical  Biophysics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  establishment  of  a  relationship  between  endothelial  morphology  and 
orientation  to  the  blood  flow  patterns  to  which  it  is  subjected  would 
provide  a  valuable  tool  in  the  study  of  vascular  hemodynamics  in  animals 
as  well  as  in  human  beings  by  in  vitro  observation. 

Proposed  Course  of  the  Project:   Hot-film  anemometry  will  be  employed  to 
study  the  associated  blood  velocity  profiles  and  shear  distributions. 
Endothelial  nuclear  strain  measured  by  nuclear  deformation  will  be 
quantitated  and  related  to  hemodynamic  factors  in  an  rn  vitro  prepara- 
tion. 

Honors  and  Awards:   None 

Publications:   None 


01 


Serial  No.  NHLI-42(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 


Project  Title:   Healed  Valvular  Infective  Endocarditis 

Principal  Investigator:   Neil  A.  Buchbinder,  M.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 

Cooperating  Units:   None 

Project  Description:   Clinical  and  anatomic  features  are  described  in  29 
necropsy  patients  with  healed  valvular  infective  endocarditis.   Active  infective 
endocarditis  (AIE)  occurred  on  anatomically  abnormal  valves  in  24  patients,  9 
of  whom  had  congenitally  bicuspid  aortic  valves.   Six  patients  had  congestive 
heart  failure  before  AIE.   All  29  patients  had  evidence  of  valvular  dysfunction 
during  and  after  AIE,  27  of  whom  had  overt  signs  of  heart  failure  AIE  was  the 
sole  or  contributary  cause  of  valvular  dysfunction  in  at  least  23  patients. 
Aortic  valve  perforation  was  discemable  in  10  patients.   5  patients  had 
ruptured  mitral  chordae  tendineae.   The  average  time  from  AIE  to  necropsy  was 
4.3  years.   The  average  longevity  of  patients  with  aortic  valve  perforations 
was  7.8  years  and  4.1  years  for  those  with  ruptured  chordae  tendineae.   The 
absence  of  a  "myocardial  factor"  specific  to  IE  is  further  substantiated  by 
the  fact  that  only  4  of  17  patients  with  adequate  surgical  correction  of 
valvular  dysfunction  died  as  a  result  of  persistent  congestive  heart  failure. 
Death  in  the  other  13  postoperative  patients  was  caused  by  a  surgical  compli- 
cation and  not  myocardial  failure.   The  presence  of  fibrosis  of  the  papillary 
muscles  in  83%  of  patients  supports  the  view  that  this  lesion  is  the  result 
of  healed  papillary  muscle  necrosis  which  has  been  found  in  75%  of  necropsy 
patients  with  AIE. 

Honors  and  Awards :   None 

Publications:   None 


W' 


ds 


Serial  No.     NHLI-43(c) 

1.  Cardiology   Branch 

2.  Section  of  Pathology 

3.  Bethesda,    Man'land 

PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Project  Title:   Active  Non-infective  Thrombotic  Endocarditis 

Principal  Investigator:   Neil  A.  Buchbinder,  M.D. 

Other  Investigators:   William  C.  Roberts 

Cooperating  Units:   None 

Project  Description:   The  clinical  and  necropsy  features  of  45  patients  with 
active  non-infective  thrombotic  endocarditis  (ANITE)  were  studied.   Vegetations 
occurred  on  anatomically  normal  valves  in  40  patients  and  functionally  normal 
valves  in  44  patients.   Malignancy  was  present  in  39  patients,  31  of  whom  were 
being  treated  with  chemotherapy.   A  precordial  murmur  was  present  in  55%  of 
patients.   Twenty-eight  patients  were  febrile,  9  of  whom  had  positive  blood 
cultures.   The  diagnosis  was  not  made  premortem  in  any  patients.   An  embolic 
infarct  was  suspected  clinically  in  only  3  patients  but  was  found  at  necropsy 
in  31  patients,  14  of  whom  had  multiple  infarcts.   Death  in  6  patients  was 
caused  by  a  CNS  embolus .   In  no  patient  were  organisms  demonstrable  in  histo- 
logic sections  of  the  vegetation.   Thus,  bacterial  implantation  in  vegetations 
of  ANITE  is  not  felt  to  be  the  underlying  mechanism  for  the  development  of  AXE. 
The  possible  relationship  of  ANITE  to  a  coagulopathy  was  studied.   Thrombo- 
cytopenia (75,000)  was  present  in  25  of  31  patients.   Thirteen  of  the  25  had 
a  possible  etiology  (hematopoietic  malignancy  or  cytotoxic  drug)  whereas  12 
had  no  apparent  explanation.   In  only  3  of  these  patients  was  a  complete 
coagulation  profile  available  for  study.   2  of  the  3  had  criteria  disseminated 
intravascular  coagulation. 

Honors  and  Awards:   None 

Publications:   None 


I 


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Serial  No.   NHLI-44(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Project  Title:   Active  Valvular  Infective  Endocarditis 

Principal  Investigator:   Neil  A  Buchbinder,  M.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 

Cooperating  Units:   None 

Project  Description:   Clinical  and  anatomic  features  are  described  in  55 
patients  with  active  valvular  infective  endocarditis.   Fifty-eight  percent  had 
vegetations  on  previously  anatomically  normal  valves.   Predisposing  factors 
allowing  entrance  of  virulent  or  unusual  organisms,  or  alterations  of  host 
defense  mechanisms,  are  believed  to  account  for  the  frequency  of  IE  on  normal 
valves.   Valvular  dysfunction  occurred  in  70%  of  the  55  patients,  causing  con- 
gestive heart  failure  in  all.   Myocardial  lesions  were  present  in  92%  of  the 
39  patients  in  whom  multiple  histologic  sections  were  examined.   Papillary 
muscle  necrosis  was  present  in  75%  but  contributed  to  mitral  regurgitation 
in  only  one.   Myocardial  inflammation  was  focal  in  all  but  2  patients.   Myo- 
cardial lesions  are  not  believed  to  be  a  primary  cause  of  congestive  heart 
failure.   No  correlation  was  found  between  congestive  heart  failure  and  any 
or  all  myocardial  lesions.   Pericarditis  was  found  in  9  patients  (18%),  and 
in  8  a  site  of  direct  extension  of  the  inflammation  into  the  pericardium  was 
apparent.   Ring  abscesses  were  found  in  10  of  19  patients  with  aortic 
regurgitation.   The  most  common  renal  lesion  was  infective  (abscess  or 
pyelonephritis) . 

Honors  and  Awards:   None 

Publications:   None 


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Serial  No.        NHLI-45(c) 


1.  Cardiology   Branch 

2.  Section   of  Pathology 

3.  Bethesda,    Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Project  Title:   Active  Infective  Endocarditis  Confined  to  Mural  Endocardium 

Principal  Investigator:   Neil  A.  Buchbinder,  M.D. 

Other  Investigators:     William  C.  Roberts,  M.D. 

Cooperating  Units:    None 

Project  Description:   The  clinical  and  necropsy  features  of  5  patients  with 
active  infective  endocarditis  confined  to  non -valvular  (mural)  endocardium 
were  studied.   Each  patient  had  an  underlying  malignant  disease;  4  had  leukemia 
and  1  lymphoma.   The  causative  organism  was  fungal  in  each;   Candida  in  3, 
Phycomycetes  in  1  and  Aspergillus  in  1.   The  vegetations  were  located  on  the 
right  ventricular  endocardium  in  3  patients  and  left  atrial  endocardium  in  2. 
Only  one  patient  had  a  precordial  murmur  and  this  was  thought  to  be  functional. 
The  diagnosis  of  fungal  infection  was  made  in  each  patient  but  the  diagnosis 
of  endocarditis  was  not  made  in  any.   The  pathogenesis  of  this  unusual  form 
of  infective  endocarditis  was  studied.   In  each  patient  the  endocardial  lesion 
was  not  primary  but  rather  the  result  of  extension  of  an  infective  process 
originating  at  another  site.   Each  of  the  3  patients  with  vegetations  on  the 
right  ventricular  endocardium  had  numerous  myocardial  abscesses  as  part  of 
systemic  fungal  septicemia.   The  endocardial  vegetations  in  these  patients 
resulted  from  extension  of  a  myocardial  abscess  to  involve  the  endocardium. 
The  2  patients  with  IE  of  left  atrial  mural  endocardium  had  extensive  fungal 
infection  of  the  lungs  with  fungal  thrombi  in  pulmonary  vein  vegetations  into 
the  left  atrium.   No  definite  evidence  of  cardiac  dysfunction  was  found  in 
any  patient  although  the  left  atrial  vegetation  in  one  patient  was  extensive 
enough  to  have  decreased  pulmonary  venous  return. 

Honors  and  Awards :   None 

Publications:   None 


8^ 


Serial  No.   NHLT-46(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PRS-NIH 

Individual  Project   Report 

July   1,    1970    through   June    30,    19  71 

Project  Title:      The   Spleen  in  Type   I   Hyperlipoproteinemia:      Histochemical, 
Biochemical,    Microfluorimetric   and  Electron  Microscopic 
Observations 

Previous    Serial  Number:      NHLI-74(c) 

Principal   Investigator:      Victor  J.    Ferrans,   M.D. 

Other   Investigators:  L.    Maximilian  Buja,    M.D. 

William  C.    Roberts,   M.D. 
Donald   S.    Fredrickson,   M.D. 

Cooperating  Units:      Molecular  Disease  Branch,    National  Heart   and  Lung 
Institute 

Project   Description:      Histochemical,    biochemical,    microfluorimetric   and 
electron  microscopic  studies   were  made   of   the  spleen  of  a  patient  with    type  I 
hyperlipoproteinemia.      Foam  cells  were   observed   that    contained   a  material 
identified   as    ceroid  on   the  basis    of  its    autofluorescence ,    acid-fastness, 
sudanophilia,    PAS-positivity   and  insolubility   in  organic  solvents.      Electron 
microscopy   showed   that    the   ceroid  was    organized   in   the   form  of   granules  with 
concentric   lamellae   of  irregular  periodicity.      The  process   of   formation  of 
these   granules  was    described  in  detail.      The   ceroid  was    considered   to   repre- 
sent non-digestible   end-products   of    the  metabolism  of   chylomicrons   taken   up 
by  macrophages   in  splenic   sinusoids. 


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Honors  and  Awards ; 


None 


Pub'lications :  Ferrans,  V.  J.,  Buja,  L.  M. ,  Roberts,  W.  C.  ,  and  Fredrickson, 
D.  S.:   The  spleen  in  type  I  hyperlipoproteinemia:   Histo- 
chemical, biochemical,  microfluorimetric  and  electron 
microscopic  observations.   Am.  J.  Path.  (In  press). 


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Serial  No.    NHLI-47(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Ultrastructural  Studies  of  Cardiac  Biopsies  in  Primary 

Myocardial  Disease 

Previous  Serial  Number:   NHLI-7&(c) 

Principal  Investigator:   Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:     William  C.  Roberts,  M.D. 

Rashid  Massumi,  M.D.* 
Nayab  Ali,  M.D.- 
Gerald Shugoll,  M.D.** 


Cooperating  Units; 


*Cardiology  Division,  D.  C.  General  Hospital 
^*Cardiology  Service,  Veterans  Administration  Hospital. 
Washington,  D.C. 


Project  Description:   Electron  microscopic  studies  are  being  made  of  cardiac 
biopsies  obtained  from  patients  with  primary  myocardial  disease  at  D.C. 
General  Hospital  and  V.  A.  Hospital,  Washington,  D.C.   A  Konno  catheter  is 
being  used  to  biopsy  the  septal  wall  of  the  right  ventricle.   Observations 
made  indicate  that  mitochondrial  alterations,  swelling  of  the  sarcoplasmic 
reticulum  and  dilatation  of  the  transverse  tubular  system  are  the  main 
alterations  present. 

Honors  and  Awards:   None 

Publications:   Data  to  be  presented  at  Sept.,  1971,  International  Symposium 
on  Cardiomyopathies,  will  be  published  in  a  book  which  will  contain  the 
proceedings  of  this  meeting. 


ee 


Serial  No.   NHLI-48(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Electron  Microscopic  and  Histochemical  Studies  of  the  Liver 

in  GM-,  Gangliosidosis 

Previous  Serial  Number:   NHLI-73(c) 

Principal  Investigator:   Victor  J.  Ferrans ,  M.D.,  Ph.D. 

Other  Investigators:     Donald  S.  Fredrickson,  M.D. 

Howard  Sloan,  M.D. 

Cooperating  Units:   Laboratory  of  Molecular  Diseases,  National  Heart  &  Lung 
Institute 

Project  Description:   Ultrastructural  and  histochemical  studies  have  been 
made  on  liver  tissue  obtained  from  2  patients  with  biochemically  proven  GM, 
gangliosidosis.   Results  obtained  indicate  that  two  different  compounds  are 
stored  in  the  liver:   GM  ganglioside,  which  is  localized  in  the  hepatocytes, 
where  it  forms  membranous  cytoplasmic  bodies  that  are  probably  of  lysosomal 
origin,  and  an  acid  mucopolysaccharide- like  material,  which  is  present  within 
the  lysosomes  of  the  Kupfer  cells  in  the  form  of  thin-walled,  straight  tubules 
that  measure  from  170  to  200  A  in  diameter.   The  material  from  which  the 
tubules  are  derived  accumulates  at  first  in  the  endoplasmic  reticulum  of  the 
Kupfer  cells  and  is  subsequently  transferred  to  the  lysosomes,  where  the 
tubules  develop. 

The  storage  of  two  different  compounds  in  two  different  cell  types  of  the 
same   organ  is  of  great  interest  in  view  of  the  currently  held  concept  of  a 
single  enzyme  deficiency  in  this  disorder.   It  is  possible  that  both  com- 
pounds share  a  common  path  of  degradation. 

Honors  and  Awards:   None 

Publications:   To  be  prepared  for  publication. 


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Serial  No.  NHLI-49(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  19  71 

Project  Title:   A  Histochemical  and  Electron  Microscopic  Study  of  Cardiac 

Myxomas 

Previous  Serial  Number:   NHLI-75(c) 

Principal  Investigator:   Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 

Cooperating  Units:   None 

Project  Description:   Histochemical  studies  are  being  made  on  8  cardiac 
myxomas,  and  electron  microscopic  observations  have  been  made  on  2.   The 
electron  microscopic  data  support  the  concept  that  the  tumor  cells  are  derived 
from  endocardial  endothelial  cells.   The  myxoma  cells  show  a  marked  tendency 
to  form  capillary-  or  duct-like  structures.   Their  cytoplasm  contains  numerous 
filaments  that  measure  50-70  A  in  diameter  and  are  similar  to  those  described 
in  normal  endothelial  cells.   The  myxoma  cells  also  contain  variable  amounts 
of  rough-surfaced  endoplasmic  reticulum  and  iron  particles.   The  stroma  of 
the  tumor  is  composed  of  a  material  the  structure  of  which  varies  from 
amorphous  to  finely  fibrillar.   The  histochemical  studies  are  primarily 
concerned  with  the  histochemical  characterization  of  the  carbohydrate 
components  of  the  myxomatous  stroma. 

Honors  and  Awards:   None 

Publications:   None 


« 


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Project  Title; 


Serial  No.     NHLI-50 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project   Report 

July   1,    19  70    through   J'jne    30,    19  71 

Effects    of  Hyperosmotic  Perfusate   on  Ultrastructure   and 
Function  of   the  Isolated  Canine  Heart 


Previous    Serial  Number:      None 

Principal   Investigator:      Victor  J.    Ferrans ,   M.D.,    Ph.D. 

Other   Investigators:  L.    Maxi.milian   Buja,   M.D. 

Sidney  Levitsky,    M.D. 
William  C.    Roberts,   M.D. 

Cooperating  Units:      Surgery   Branch,    National  Heart   and  Lung  Institute 

Project  Description:      Ultras tructural   and    functional   observations    are   de- 
scribed on  isolated   canine  hearts   preserved   for   18  hours  with  either    filtered 
plasma  perfusate    (5    dogs)    or  with    filtered  plasma-dextran  perfusate    (6   dogs) . 
Interstitial   edema,    swelling  of  sarcoplasmic   reticulum,    and  mitochondrial 
damage  were   observed  in  each   of   the  5  hearts   perfused  by    filtered  plasma. 
In   contrast,    interstitial   edema  was    absent   in  each   of   the   6  hearts   perfused 
by   filtered  plasma-dextran,    and  swelling  of  sarcoplasmic   reticulum  and  mito- 
chondrial  damage   occurred  in  only  2.      Myocardial   compliance   also   appeared 
to  be  better  in  hearts   perfused  by   filtered  plasma-dextran   than   in  hearts 
perfused  with    filtered  plasma.      In   conclusion,    the   osmolarity  of   the  per- 
fusate is   important   in  preventing  edema   in  preserved  hearts. 


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Honors    and  Awards 
Publications 


None 


Ferrans,  V.J.,  Buja,  L.M. ,  Levitsky,  S.,  and  Roberts,  W.C. : 
Effects  of  hyperosmotic  perfusate  on  ultrastructure  and 
function  of  the  isolated  canine  heart.   Laboratory  Investi- 
gation.  In  press. 


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Serial  No.      NHLI-51(c) 

1.  Cardiology   Branch 

2.  Section  of  Pathology 

3.  Bethesda,    Maryland 

PHS-NIH 

Individual   Project   Report 

July  1,  19  70  through  June  30,  19  71 

Project  Title:   Papillary  Muscle  Dysfunction  Before  and  After  Auscultatory 

Mitral  Regurgitation.   Hemodynamic  and  Morphologic  Documentation 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:     M.  Wayne  Falcone,  M.D. 

James  A.  Ronan,  Jr.,  M.D. 

Cooperating  Units:  Department  of  Medicine,  Georgetown  University  School  of 
Medicine,  Division  of  Cardiology,  Georgetown  University 
Hospital,  Washington,  D.C. 

Project  Description:   Clinical,  hemodynamic  and  anatomic  findings  are  described 
in  a  71-year-old  man  with  "silent"  acute  myocardial  infarction.   Attention  is 
called  to  the  hemodynamic  documentation  of  mitral  regurgitation  resulting  from 
papillary  muscle  necrosis  both  shortly  before  and  shortly  after  clinical 
appearance  of  a  murmur  of  mitral  regurgitation.   The  interest  of  this  case  is 
the  fact  that  the  patient  underwent  2  cardiac  catheterizations  during  the  time 
that  the  myocardium  was  being  infarcted.   It  was  thus  possible  to  demonstrate 
the  hemodynamic  consequences  of  papillary  muscle  necrosis.   It  was  shown  that 
hemodynamic  evidence  of  mitral  regurgitation  was  present  before  auscultatory 
evidence  of  mitral  regurgitation  appeared. 

Honors  and  Awards:   None 

Publications:   None 


<?i 


Serial  No.      NHLI-5  2{c) 

1.  Cardiology   Branch 

2.  Section  of  Pathology 

3.  Bethesda,   Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Project  Title:   Aneurysm  of  the  Non-patent  Ductus  Arteriosus, 

Previous  Serial  No.   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators: 


Cooperating  Units : 


M.  Wayne  Falcone,  M.D. 
Joseph  K.  Perloff,  M.D. 

Department  of  Medicine,  Georgetown  University 
School  of  Medicine,  Division  of  Cardiology, 
Georgetown  University  Hospital,  Washington,  D.C. 


Project  Description:   Clinical  and  necropsy  findings  are  described  in  a  6-week- 
old  infant  with  aneurysm  of  the  ductus  arteriosus .   It  was  obliterated  at  the 
pulmonary  arterial  end  and  patent  at  the  aortic  end.   Observations  in  60 
previously  described  patients  with  ductal  aneurysm  disclosed  that  46  were  in 
infants  less  than  2  months  old,  4  in  children,  and  11  in  adults.   The  aortic 
end  of  the  ductal  aneurysm  is  always  patent.   The  pulmonary  arterial  end  may 
or  may  not  be  patent.   Since  nearly  half  of  the  ductal  aneurysms  tend  to 
develop  complications  (rupture,  embolism  or  infection)  operative  resection 
appears  indicated. 

Honors  and  Awards:   None 

Publications:  Falcone,  M.  W.  ,  Perloff,  J.  K. ,  and  Roberts,  W.  C.  :   Aneurysm 
of  the  non-patent  ductus  arteriosus.   Am.  J.  Cardiol.   In 
press . 


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Project  Title: 


Serial  No.      NHLI-5  3(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Congenital  Aortic  Stenosis  Resulting  from  a  Unicommissural 
Valve.   Clinical  and  Anatomic  Features  in  21  Adult  Patients 


Principal  Investigator:   William  C.  Roberts,  M.D. 
Other  Investigators: 


Cooperating  Units 


M.  Wayne  Falcone,  M.D. 
Andrew  G.  Morrow,  M.D. 
Joseph  K.  Perloff,  M.D. 

Surgery  Branch,  National  Heart  and  Lung  Institute 
and  Department  of  Medicine,  Georgetown  University 
School  of  Medicine,  Division  of  Cardiology,  George- 
town University  Hospital,  Washington,  D.C. 


Project  Description:   Clinical,  electrocardiographic,  phonocardiographic, 
radiographic,  hemodynamic  and  anatomic  findings  are  presented  in  21  adult 
patients  with  stenotic  unicommissural  aortic  valves.   Distinction  of  con- 
genitally  unicuspid  and  bicuspid  aortic  valves  before  operation  or  autopsy 
was  not  possible.   Although  the  basic  structure  of  the  valve  may  render  it 
inherently  stenotic,  the  age  at  which  a  murmur  was  first  noted  (avg.  19  years 
the  duration  of  a  known  murmur  (avg.  25  years) ,  and  the  age  of  onset  of  first 
symptoms  of  left  ventricular  outflow  obstruction  (avg.  41  years)  strongly 
suggest  that  stenosis  at  least  in  part  is  acquired.   The  relationship  of  the 
true  and  false  commissures  to  the  coronary  arterial  ostia  could  be  determined 
with  certainty  in  12  patients.   The  basic  division  of  the  aortic  valve  into 
left,  right,  and  non-coronary  cusps  is  maintained,  but  the  raphes  do  not 
extend  to  the  valve  orifice.   Because  the  aortic  valve  is  attached  to  the 
ascending  aorta  at  only  one  point  (the  true  commissure) ,  which  is  at  the  leve 
of  the  orifice,  valvotomy  is  hazardous,  and  valve  replacement  appears  indi- 
cated when  operative  treatment  becomes  necessary  in  the  adult  patient  with  a 
stenotic  unicommissural  aortic  valve. 


), 


Honors  and  Awards :   None 

Publications:   Falcone,  M.  W. ,  Roberts,  W.  C. ,  Morrow,  A.  G. ,  and  Perloff,  J, 
Congenital  aortic  stenosis  resulting  from  a  unicommissural 
valve:   Clinical  and  anatomic  features  in  21  adult  patients. 
Circulation.   In  press. 


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Project  Title: 


Serial  No.      NHLI-54 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 

Indi\ridual  Project  Report 

July  1,  19  70  through  June  30,  19  71 

Acute  and  Chronic  Effects  of  Normothermic  Anoxia  on 
Canine  Hearts:   Light  and  Electron  Microscopic  Evaluation 


Previous  Serial  Number: 

Principal  Investigator:   L.  Maximilian  Buja,  M.D. 

Other  Investigators:     Sidney  Levitsky,  M.D. 

Victor  J.  Ferrans,  M.D. ,  Ph.D. 
Sherman  G.  Souther,  M.D. 
William  C.  Roberts,  M.D. 
Andrew  G.  Morrow,  M.D. 

Cooperating  Units:   Surgery  Branch,  National  Heart  and  Lung  Institute 

Project  Description:   To  evaluate  the  acute  and  chronic  effects  of  normo- 
thermic cardiac  anoxia,  structural  and  functional  observations  were  made  on 
the  hearts  of  29  dogs  subjected  to  total  cardiopulmonary  bypass.   Elective, 
normothermic  cardiac  arrest  was  induced  by  aortic  cross-clamping  for  30 
minutes  in  7  dogs,  and  for  45  minutes  in  16  dogs.   The  remaining  6  dogs,  which 
served  as  controls,  were  placed  on  bypass  without  aortic  cross-clamping.   The 
6  control  dogs  and  6  of  7  dogs  subjected  to  30  minutes  of  cardiac  anoxic 
showed  excellent  cardiac  function  and  minimal  myocardial  damage;  one  dog  in 
the  latter  group  that  died  6-12  hours  after  bypass  showed  no  myocardial  damage, 
All  16  dogs  subjected  to  45  minutes  of  cardiac  anoxia  showed  extensive  myo- 
cardial damage.   Depressed  cardiac  function  was  demonstrated  in  3  of  5  dogs 
that  survived  for  8  days  or  longer  after  bypass .   The  acute  cardiac  damage  was 
of  the  myofibrillar  degeneration  type  and  progressed  to  replacement  fibrosis; 
this  damage  was  selectively  localized  in  the  left  ventricular  papillary 
muscles  and  subendocardium. 

Honors  and  Awards :   None 


Publications:   Buja,  L.  M. ,  Levitsky,  S.  ,  Ferrans,  V.  J.,  Souther,  S.  G., 

Roberts,  W.  C,  and  Morrow,  A.  G.  :   Acute  and  chronic  effects 
of  normothermic  anoxia  on  canine  hearts:   Light  and  electron 
microscopic  evaluation.   Suppl.  to  Circulation  43  and  44: 
In  press. 


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Serial  No.  NHLI-55(c) 

1.  Cardiology  Branch 

2.  Section  of  Pathology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual   Project    Report 

July    1,    1970    through   June    30,    1971 

Project   Title:      Causes   of   Death    and  Other  Anatomic   Observations   after 

Cardiac  Valve   Replacement    . 

Principal   Investigator:      William  C.    Roberts,    M.D. 

Other    Investigators:      Andrew   G.    Morrow,    M.D. 

Cooperating   Units:      Surgery   Branch,    National   Heart    and  Lung   Institute 

Project   Description:      From   1963   through    1970,  181   patients    died   following  re- 
placement   of    1  or  more    cardiac   valves  with   prostheses.      Each   patient  was 
studied  at  necropsy.      The  patients  were   divided   into    two   groups:      1)    107 
patients  who   died  within   2   months   of   operation    (early    deaths),    and  2)    74 
patients  who    died   at    later  periods    up    to    80   months    (late   deaths)  .      Of   the   220 
cardiac   valves    replaced   in   these  181   patients,    Starr-Edwards    prostheses  were 
used   in   200.      Among    the   causes   of   death   early   prosthetic   dysfunction  was 
responsible   in    30%,    no   anatomic    cause  was    found   in   28%,   bleeding   and   technical 
mishaps    19%,    central   nervous    system   catas trophies    7%,    associated  undiagnosed 
or   uncorrected   valve    disease    7%,    infection   4%,    and  miscellaneous    causes   5%. 
Among   the    74  patients   who   died    late,    prosthetic  dysfunction   accounted    for 
death   in    37%   and   factors    secondarily   related   to   the  prosthesis   in   24%.      These 
latter    factors    included  systemic  embolism,    CNS  hemorrhage    from  hypopro- 
thrombinemia,    secondary   endocardial    fibrosis   of   the   left   ventricle,    secondary 
intimal    fibrosis   of   the   ascending  aorta,    and  hepatitis.      In    18%   of    the    late 
deaths    the   cause  was    undetermined,    in   16%   death  was    related   to   the   underlying 
cardiac   disease   and   in   5%    the   cause   of   death  was    unrelated   to   the  heart.      Al- 
though  additional  years    of   life  have   been   provided   to  many    critically   ill 
patients    from  severe   valvular  heart    disease  by   cardiac  prostheses    it    is 
apparent    10   years    after   valve    replacement    that    the   ideal    cardiac   valve   is   not 
presently    available.      It   would   appear    from  this   study    and   others    that    the 
rigid    frame    prostheses    are   simply  not    capable   of    functioning  properly  in   a 
few   selected  hearts  with   either  small    left   ventricular   cavities   or   small    aortas. 
^^/hether  or  not    intimal   proliferation  which   occurs    in   the   aortic    root    late 
following  aortic   valve    replacement    and   also    in    the  area   of   the   coronary 
arterial   ostia  will   lead   to   chronic   myocardial    ischemia    remains    to  be   seen. 
The  degree   of   intimal   proliferation   in   the   ascending  aorta  in   the   late   deaths        _ 
corresponded   to    the   presence    or   absence   and   degree   of    renal  hemosiderosis  H 

indicating  that    the  aortic    lesion   is    due    to    turbulence   of  blood    transversing  ■ 

the  prosthetic   valve   and   that    this    turbulence    causes    intravascular  hemolysis 
which    is   apparent    in   the  kidney  by   deposition   of   iron    in    cytoplasm  of    renal 
tubules.      V^ether  or  not    the  metallic  hollow  ball    used   in  Starr-Edwards 
prostheses   will  hold   up   over   many   years    still    remains    to   be  seen,    but    there 
is    evidence    that    the   cloth-covered  struts    do   show   evidence   of  wearing   after 
about    three   years . 

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Serial  No.    NHLI-55(c) 


Honors  and  Awards:   None 

Publications:   Roberts,  W.  C.  and  Morrow,  A.  G. :  in  Vogel,  J.  (Ed.),  Long- 
term  prognosis  following  valve  replacement.   Second 
Conference  on  Cardiovascular  Disease.   Basel,  Switzerland, 
S.  Karger.   Publication  date  1971. 


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1. 


ANNUAL  REPORT  OF  THE 
CLINIC  OF  SURGERY 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  19  70  through  June  30,  19  71 

The  clinical  and  laboratory  programs  of  the  Surgery  Branch  have,  as  in 
past  years,  largely  centered  upon  the  study  of  operative  methods  for  the 
correction  of  congenital  and  acquired  heart  and  lung  diseases,  assessment  of 
the  results  of  such  operations,  and  laboratory  studies  related  to  cardio- 
vascular physiology  and  pharmacology. 

Radioi-sotope-powered  Pacemakers :   A  previous  report  has  described  the 
initial  research  effort  to  develop  a  cardiac  pacemaker  powered  by  an  isotopic 
heat  source.   This  work  has  been  carried  out  with  the  cooperation  and  support 
of  the  Atomic  Energy  Commission.   The  pacemakers,  fueled  with  metallic 
238piutonium,  were  designed  to  have  a  useful  life  of  more  than  ten  years,  to 
obviate  the  frequent  power  pack  changes  which  are  necessary  with  pacemakers 
powered  by  conventional  batteries.   After  initial  laboratory  and  bench  testing 
eight  fueled  pacemakers  were  inserted  in  normal  dogs .   Serial  observations  in 
these  animals  indicated  that  after  an  initial  stabilization  period  the  pace- 
maker rate  and  the  amplitude  and  configuration  of  the  pacing  artifact  remained 
unchanged.   Over  a  period  of  18  months,  however,  pacing  failed  in  every  animal 
after  an  average  time  of  eight  months.   In  each  case  the  electrode  was  shown 
to  be  physically  and  functionally  intact  after  the  pacemaker  was  disconnected 
from  it.   Examination  of  the  pacemakers  which  were  removed  has  revealed  the 
cause  of  failure  in  seven  of  the  eight  units  to  be  a  defective  output  tran- 
sistor in  the  pacemaker  circuit.   In  the  single  unit  in  which  the  nuclear 
power  supply  was  defective,  failure  resulted  from  detachment  of  one  of  the 
thermocouple  tapes.   Needless  to  say,  this  experience  was  discouraging,  but  re- 
inforced the  research  plan  which  emphasizes  extensive  animal  evaluation  before 
the  units  are  utilized  in  man.   A  new  series  of  pacemakers  incorporating 
an  improved  pacemaker  circuit  will  be  made  available  for  animal  implantation 
early  this  year.   The  experience  with  the  modified  pacemakers  will  determine 
the  likelihood  of  suitability  for  use  in  patients  with  complete  heart  block. 

Prosthetic  Heart  Valves;   A  continuing  interest  of  the  Surgery  Branch  has 
been  the  evaluation,  operative  treatment,  and  subsequent  followup  of  patients 
in  vrhcm  replacement  of  one  or  more  cardiac  valves  is  necessary.   The  original 
St jrr --Edwards  prostheses,  both  mitral  and  aortic,  were  constructed  in  such  a 
manner  that  much  bare  metal  was  exposed  to  blood,  and  there  was  a  large  area 
of  interface  between  metal  and  the  fabric  sewing  ring.   During  1964-66  experi- 
mental studies  revealed  that  if  all  the  metallic  surfaces  were  covered  with  a 
thin  layer  of  fabric,  the  fabric  would  become  convered  with  host  neointima, 
and  no  foreign  material  except  the  ball  was  exposed  to  blood.   This  principal 
was  incorporated  into  valves  produced  commercially  during  3  966-68,  and  aortic 
prostheses  (series  2300)  and  mitral  prostheses  (series  6300)  were  implanted 
in  approximately  100  patients.   Subsequent  evaluation  of  these  patients  by 
both  clinical  examination  and  serial  cardiac  catheterizations  revealed  that 
the  valves  had  poor  hemodynamic  characteristics.   Peak  systolic  pressure 
gradients  in  excess  of  50  mm.  Hg  were  common  after  aortic  valve  replacement, 
and  gradients  as  high  as  75  mm.  Hg  were  sometimes  recorded.   Similarly,  in  the 
mitral  position  the  6300  valves  almost  never  permitted  the  left  atrial  pres- 


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sure   to   fall   to   a  normal   level,    and  significant   diastolic  gradients  were 
invariably   seen.      Also,    in   the   aortic  position   these   valves   caused   severe 
hemolytic   anemia   in   a  number   of   patients,    even  when  perivalvular   regurgitation 
was   not   present.      This   experience   led   to   the   development   of  an   improved   series 
of   cloth-covered   prostheses   in  which    the   ratio   of  orifice   area   to   ball   di- 
a-neter  was    increased  by  nearly   20  percent.      Over   the   past    two   years    these 
valves    (aortic    -  2310,    mitral    -6310)    have  been  evaluated   in  another  100 
patients.      Postoperative  hemodynamic   assessments  have    indicated   significant 
improvement    in   comparison   to    the   2300   -   6300   series.      In    the   aortic   position 
peak   pressure    gradients   of  0   to   20   mm.    Hg  are    usual,    and   in  the  mitral 
position    the  hemodynamic   picture    is   essentially   identical    to   that   observed 
with    the   original   model  6120    (silastic   ball)    valves. 

It  has   been  hoped   that    the    fabric   covering   utilized   in  both   the   2300-6300 
and   2310-6310   valves  would   result   in   a  significant    reduction  in   thrombo- 
embolism.     To   date   this  hope  has   been  borne   out  when   these  valves  have  been 
utilized   in   the   aortic  position.      Anticoagulants   have  been   administered    for 
the    first   six  months   after  operation   and   discontinued   thereafter.      Only   two 
patients   are  known  to  have   sustained  systemic   emboli   after   cessation  of   anti- 
coagulation.     Such   encouraging   results  have  not   been  observed  with   fabric- 
covered  valves    utilized   in   the  mitral   position.      Thus,    systemic  emboli 
occurred   in  each   of   the    first    three   patients   with   6310  mitral   prostheses  with- 
in  days   after   anticoagulants  were    stopped,    and   in  one   patient    the  embolus 
proved    fatal.      At    the   present    anticoagulation   is   stopped  six  months    after 
aortic  valve    replacement,    but    continued   indefinitely   in   all  patients  with 
mitral   prostheses. 


Tissue   Heart   Valves:      In   an   attempt   to   obviate   some    of   the   disadvantages 
of   ball  or   disc   mechanical   prostheses   an   initial    laboratory   and   clinical  ef- 
fort   to    construct   satisfactory  heart   valves   of  homologous   or  heterologous 
tissue   have   been   made.      At   other   institutions   porcine   aortic   valves  have  been 
used   for  valve    replacement    in  man   after   they  were   sterilized   and   fixed   in 
formalin   and  mounted  on   a  rigid  metallic   or  plastic   frame.      The  hemodynamic 
function  of  such   valves  has   been   generally   good,    and    they  have  not  been   the 
source  of  emboli  even   though   anticoagulants  were  not   administered.    Frequently, 
however,    such   valves   have    failed  when   the   tissue   tore   away    from  its    attachment 
to   the   rigid    frame.      A  new  valve    frame  has   been   designed   in   a   cooperative 
project  with    the   Hancock   Laboratories.      This    frame    is   presently    constructed   of 
polypropylene,    and    the    legs    to  which    the   commissures   of   the   tissue  valve   are 
attached  are   made   semi-flexible.      Tissue   valves  mounted   on   rigid   and  semi- 
flexible    frames  were   studied   in   a  pulse   duplicator  while    the   stress   on   the 
sutures    anchoring  the  valve   to   the   frame  was    constantly   recorded  with   a   force 
transducer.      With   a   rigid   frame    the   stress   on   the   attachment    rose   strikingly 
as    the  valve  was    subjected   to   increased   closing  pressure.      With    the    semi-rigid 
frame,    however,    the  slight   centripital   movement   of   the   struts   prevented    this 
tension  increase   and,    in   fact,    stress    at    maximum  closing  pressure  was    some- 
times  observed   to  be   less    than    that    at    a   lower  pressure. 

Heterologous    (porcine)    valves,    preserved   and   sterilized  with   glutaralde- 
hyde,    and   constructed  on  such   semi-flexible    frames   have   been   utilized   for 
mitral   and/or    tricuspid  valve    replacement    in  20   patients.      It   has   become 
obvious    that   a  distinct    advantage   of   such   valves    is    that    they    can  be    inserted 


into  a  diminutive  left  ventricular  chamber  such  as  is  encountered  in  patients 
with  severe  calcific  mitral  stenosis  or  those  with  mitral  disease  and 
associated  aortic  stenosis.   Postoperative  hemodynamic  studies  have  been 
carried  out  at  4-6  months  and  the  function  of  the  tissue  valves  is  comparable 
tc  those  of  the  6310  ball  valves.   There  have  been  no  instances  of  thrombo- 
pmbolism  or  infection.   Anticoagulants  have  not  been  administered.   Only  con- 
tinuing clinical  followup  will  indicate  whether  tissue  valves  of  this  type 
are  sufficiently  durable  to  recommend  their  general  use. 

Research  in  Organ  Transplantation:   A  program  of  research  in  trans- 
plantation biology,  with  special  emphasis  on  cardiac  transplantation,  has  been 
instituted.   Initial  efforts  have  been  directed  toward  definition  of  selected 
fundamental  problems  utilizing  a  heterotopic  (abdominal)  heart  transplant 
model  in  inbred  rats. 

Immunologic  enhancement  (i.e.  prolongation  of  graft  survival  by  antibody) 
of  Brown  Norway  (BN)  hearts  transplanted  into  Lewis  (L)  hosts  has  been  demon- 
strated.  Control  BN  hearts  in  L  hosts  were  rejected  in  6.4  "^  0.7  (S.D.)  days. 
Pretreatment  of  L  recipients  with  107  BN  spleen  cells  given  intravenously  one 
week  pretransplant  caused  prolongation  of  graft  survival  to  11.7  "^  3.3  days 
(P  <.01).   Serum  obtained  from  L  rats  injected  with  10^  BN  spleen  cells,  when 
injected  into  normal  L  rats  at  the  time  of  transplantation  and  for  a  short 
time  thereafter,  caused  prolongation  of  graft  survival  to  11.0  'i  2.4  days. 
Thus,  immunologic  enhancement  of  cardiac  grafts  has  been  demonstrated,  al- 
though the  duration  of  graft  prolongation  is  less  than  that  previously  shown 
for  renal  grafts  in  a  qualitatively  similar  system. 

Some  in -vitro  properties  of  the  serum  factor  (S)  responsible  for  graft 
enhancement  in  this  system  have  been  examined.   No  cytotoxicity  can  be  de- 
tected in  micro cytotoxicity  tests.   However,  serum  from  BN  spleen  cell-treated 
L  rats  does  cause  significant,  reproducible,  and  immunologically  specific  de- 
pression of  unidirectional  mixed  leukocyte  reactions  (MLR)  between  L  and  irra- 
diated BN  spleen  cells .   Similar  results  were  observed  even  when  presensitized 
L  spleen  cells  were  used.   The  depression  of  MLR  was  dose-dependent.   These 
observations  support  the  concept  of  "peripheral  masking"  of  antigen,  rather 
than  central  tolerance,  as  the  mechanism  of  enhancement.   Furthermore,  the 
results  suggest  that  this  assay  (i.e.  effect  of  serum  from  immunized  subjects 
on  MLR)  may  be  used  as  a  semi -quantitative  assay  for  the  presence  of  enhancing 
antibody. 

In  another  series  of  L  rats  receiving  BN  heart  grafts,  recipients  were 
sacrificed  at  daily  intervals  on  postoperative  days  one  through  six.   Spleen 
cells  from  the  transplanted  L  hosts  were  examined  for  DNA  synthesis,  uti- 
lizing a  two  hour  incubation,  and  measuring  the  uptake  of  tritiated  thymidine. 
Significantly  increased  ^h-  thymidine  uptake,  as  compared  to  controls  and 
sham-operated  animals,  was  noted  on  postoperative  day  three,  increasing  pro- 
gressively through  day  six.   Changes  in  graft  status,  as  determined  by 
palpation  and  EKG  activity,  were  inconsistent  and  were  invariably  preceded  by 
increased  in-vitro  leukocyte  DNA  synthesis  by  at  least  48  hours.   This  study 
suggests  a  rapid  convenient,  and  probably  specific  assay  for  graft  rejections 
which  is  qualitatively  different  from  the  usual  clinical  tests. 


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Also   under  examination  are    the   anatomic  and  physiologic   consequences   of 
orthotopic   cardiac   graft   rejection.      At    the    time   of   transplantation   a  left 
ventricular  pressure   transducer,    ascending  aortic    flow  probe,    and   small 
piezoelectric   crystals   on    the   left   ventricular  endocardial   surface,    across 
the  maximum  transverse   diameter,    are    implanted.      Postoperatively,    EKG,   heart 
rate,    LV  pressure,    and   dp/dt,    cardiac   output,    stroke   volume,    LV  diameter, 
circumferential   fiber  velocity,    and   diastolic  pressure-volume    relationships 
are   examined  serially   at    rest   and   during   treadmill   exercise.      In  this  way, 
the   pathophysiology   of   graft    rejection   and    the  effects   of   immunosuppressive 
treatment   may  be   defined.      These  studies   are    in   progress. 


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I 


Serial  No.  NHTJ-56(c) 
1.  Clinic  of  Surgery 
3.   Bethesda„  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Development  of  a  percutaneous  transthoracic  pacing  wire 
for  both  routine  and  emergency  use. 

Previous  Serial  No.:   None 

Principal  Investigators:   Thomas  Q.  Winter,  M.  D. 

Robert  L.  Reis,  M.  D. 

Project  Description:   The  two  customary  methods  of  delivering  electrical 
impulses  from  a  battery  pack  to  the  heart  are  (1)  transvenous  electrodes  and 
(2)  epi.cardial  electrodes.  The  former  has  the  advantage  of  being  inserted 
under  local  anesthesia,  but  suffers  from  a  failure  rate  of  from  107o  to  257o 
due  to  the  tip  of  the  electrode  being  ejected  or  dislodged  from  the  apex  of 
the  right  ventricle.   This  necessitates  re-manipulation  of  the  electrode  to 
restore  pacing.   The  epicardial  electrode  system  has  proved  much  more  reliable, 
but  requires  a  thoracotomy  under  general  anesthesia  for  installation.  We 
have  tried  to  design  a  system  that  would  combine  the  reliability  of  the 
epicardial  system  with  the  ease  of  insertion  of  the  transvenous  system. 
This  system  employs  a  17  gauge  needle  to  make  a  left  or  right  ventricular 
puncture.   A  wire  with  a  preformed  tip  is  then  introduced  through  the  needle 
into  the  ventricular  chamber.   The  needle  is  withdrawn,  and  the  wire  is 
gently  snugged  into  the  myocardium.   For  emergency  use  the  wire  would  be 
directly  connected  to  the  pulse  generator;  for  permanent  use  the  wire  would  be 
tunneled  subcutaneously  to  the  pulse  generator,  which  would  also  lie  sub- 
cutaneously.   One  theoretical  advantage  of  this  system  is  that  pacing  thresh- 
olds from  wires  actually  lodged  in  the  myocardium  are  less  than  endocardial 
thresholds,  thus  allowing  a  lower  setting  on  the  pulse  generator  and  longer 
battery  life.  Working  with  the  Elgiloy  Company  of  Elgin,  111.  several  different 
wire  sizes  were  evaluated  (.006,  .007,  .008,  .0085,  .009,  and  .010).   The 
latter  proved  to  be  the  most  desirable.  Wires  were  coated  with  a  polyurethane 
elastomer  (Lycra)  by  the  Plastics  Unit  of  the  Instrument  Fabrication  Section, 
a. yd  were  introduced  into  dog  hearts  in  a  variety  of  ways.   It  was  possible  to 
pace  the  dog  hearts  at  a  low  threshold  (<3.5  ma).  Wires  have  been  left  in 
the  dogs  for  a  period  of  up  to  4  months.   Recent  x-rays  reveal  no  wire  fracture, 
This  system  was  also  used  to  permanently  pace  one  patient,  a  17  year  old  girl 
with  complete  heart  block  following  repair  of  a  partial  A-V  canal.  After  nine 
weeks  of  satisfactory  functioning,  the  wire  fractured  at  a  spot  where  it 
crossed  a  rib  (and  thus  was  subject  to  repeated  flexion  through  a  narrow 
radius) „   The  wire  was  removed  after  it  fractured  and  a  transvenous  electrode 
was  inserted.   The  wire  has  been  used  in  one  emergency  situation  where  it 
was  necessary  to  pace  the  heart  immediately.   The  wire  was  inserted  quickly 
and  triggered  ventricular  contractions. 


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Serial  No.  NHLI-56(c) 
1.  Clinic  of  Surgery 
3.   Bethesda,  Md. 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30, 


1971 


Current  Status:   We  have  a  wire  that  is  superior  to  any  wire  commercially 
available  for  emergency  pacing  of  the  heart o   Only  economic  considerations 
may  possibly  prevent  its  manufacture  and  widespread  use.   Its  use  as  a 
substitute  for  either  the  transvenous  or  epicardial  electrode  systems  will 
depend  on  how  reliable  the  electrode  will  prove  to  be. 


Proposed  course: 
wire . 


An  attempt  should  be  made  to  find  a  manufacturer  for  the 


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Project  Title: 


Serial  No.   NHLI-57(c) 
1.  Clinic  of  Surgery 
3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Preoperative  assessment  of  aortic  and  mitral  valve  sizes 
to  facilitate  valve  replacement  with  autogenous 
tissue  valves. 


I. 


Previous  Serial  Number:   None 


Principal  Investigators: 


Thomas  Q.  Winter,  M.  D. 
D.  Luke  Glancy,  M.  D. 
Robert  L.  Reis,  M.  D. 
Andrew  G.  Morrow,  M 


D. 


Cooperating  Unit:   Cardiology  Branch,   NHLI 


Project  Description:   To  reduce  the  time  of  aortic  or  mitral  valve  replace- 
ment when  autogenous  tissue  valves  are  utilized  it  is  essential  that  a 
preoperative  assessement  of  approximately  what  size  valve  will  be  required, 
so  that  one  surgeon  can  construct  the  valve  while  another  opens  the  chest 
and  prepares  the  operative  site.   To  gain  experience  in  predicting  the 
valve  size,  all  preoperative  left-ventricular  and  aortic  root  cines  which 
had  accompanying  measuring  grids  were  reviewed. 

Measurements  were  made  at  the  area  of  the  annulus,  at  the  sinus  of 
Valsalva  area,  and  at  the  narrowest  part  of  the  ascending  aorta.  Measure- 
ments were  corrected  for  magnification  error,  and  the  results  were 
correlated  with  the  actual  valve  size  subsequently  employed  at  operation. 
The  measurement  made  at  the  annulus  was  the  most  valuable  in  predicting 
the  size  of  the  valve  actually  used.   Using  this  measurement,  the  correct 
valve  size  was  predictable  in  the  majority  of  cases  analyzed,  and  the 
predicted  valve  size  never  differed  from  that  of  the  valve  utilized  by  more 
than  one  size,  well  within  the  margin  of  error  allowed. 

To  date,  we  have  only  analyzed  two  cines  in  patients  who  have  had 
mitral  valve  replacement.   It  is  too  early  to  judge  the  usefulness  of 
that  procedure. 

Proposed  Course:   Continue  to  accumulate  experience  and  publish  the 
results  if  the  data  for  estimating  the  size  of  the  mitral  valve  proves 
accurate,  as  this  has  not  yet  been  reported. 


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Serial  No.  NHLI-58(c) 
1,  Clinic  of  Surgery 
3.   Betbesda,  Md. 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   A  new  cause  for  diastolic  murmur  following  caged-ball 
replacement  of  the  aortic  valve 

Previous  Serial  Number:   None 


Principal  Investigators: 


Thomas  Q.  Winter,  M.  D. 
Robert  L.  Reis,  M.  D. 
William  C.  Roberts,  M.  D, 


Cooperating  Unit:   Section  of  Pathology,  Cardiology  Branch,  NHLI 

Project  Description:   A  diastolic  decrescendo  murmur  along  the  left  sternal 
border  following  aortic  valve  replacement  usually  signifies  a  peri-basilar 
leak  around  the  prosthetic  valve.  Recently  a  patient  with  a  cloth-covered 
aortic  valve  was  seen  who  manifested  a  diastolic  murmur  but  at  autopsy 
no  peri-basilar  leak  was  found.  Fibrous  tissue  had  grown  into  the  valve 
seating  ring  making  the  valve  relatively  stenotic  and  at  the  same  time 
providing  an  uneven  seating  surface  for  the  metal  ball,  allowing  a  modest 
amount  of  aortic  regurgitation.  This  has  not  been  previously  reported „ 

Proposed  Course:   Project  completed.  Manuscript  in  process  of  being 
submitted  for  publication. 


/U 


Project  Title: 


Serial  No,  NHLI-59(c) 
1.  Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Four  Years'  experience  with  fabric-covered  Starr-Edwards 
ball  valves 


Previous  Serial  Number:   None 


Principal  Investigators:   Thomas  Q.  Winter,  M.  D. 

Robert  L.  Reis,  M.  D. 
A.  G.  Morrow,  M.  D. 

Project  Description:   Fabric-covered  Starr-Edwards  ball  valves  have  been 
used  for  both  aortic  and  mitral  valve  replacements  at  this  Institution  for 
over  four  years.   Approximately  100  patients  with  the  2300/6300  series 
valves  have  been  followed  for  up  to  four  years.  The  followup  period  for 
the  later  series,  the  2310/6310  series,  has  been  approximately  two  years. 
For  each  group  determinations  of  mean  valve  area,  peak  systolic  gradient, 
and  left  atrial  pressure  are  being  made  from  the  postoperative  catheteriza- 
tion data.   The  incidence  of  emboli  in  terras  of  patient  months  will  be 
given  for  each  group.   These  data  will  be  stored  on  a  Wylbur  Data  Set  and 
be  available  for  future  reference,  as  well  as  serving  as  the  initial 
batch  of  data  in  the  Retrospective  Study  of  Cardiac  Valve  Replacement 
which  was  begun  approximately  one  year  ago.   (The  data  from  the  230  patients 
are  presently  being  transferred  to  key  punch  cards;  for  this  reason 
specific  numbers  are  not  available  at  this  time.) 

Proposed  Course:   The  information  gathered  in  the  above  study  will  be 
submitted  for  publication.   The  title  of  the  project  should  be  changed 
from  "Retrospective"  to  "Continuing"  and  data  should  be  accumulated  on 
all  the  prosthetic  valve  replacements  performed  at  this  Institution  and 
stored  in  a  system  such  as  the  Wylbur  Data  System  so  that  it  will  be 
available  for  easy  reference  in  the  future. 


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Proiect  Title: 


Serial  No.  NHLI-60 
1.   Clinic  of  Surgery 
3o   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

An  exploration  of  the  usefulness  of  secondary  parameters  in 
improving  accuracy  of  stroke  volume  estimation  by  computer 
analysis  of  aortic  pulse  contour. 


t 

4 


Previous  Serial  No.:   None 


Principal  Investigators: 


David  B.  Melvin,  M.  D. 
Kenneth  Kempner,  M.  A, 
Robert  L.  Reis,  M.  D. 


I 


Cooperating  Unit:   Computer  Systems  Laboratory,  DCRT 

Project  Description:   Many  formulas  have  been  proposed  for  the  estimation 
of  cardiac  stroke  volume,  and  thus  cardiac  output,  by  computer  analysis  of 
aortic  pulse  contour.   Several  of  these  have  been  of  great  clinical  useful- 
ness. A  critical  limitation  experimentally  and  clinically  has  been  a 
decreasing  degree  of  accuracy  with  abrupt  changes  in  peripheral  vascular 
resistance.  While  changes  of  this  degree  are  not  common,  it  is  precisely 
during  such  periods  that  a  rapid  and  reliable  evaluation  of  cardiac  output 
is  most  needed.   It  was  felt  that  by  monitoring  other  parameters  simultaneously, 
a  pattern  change  suggestive  of,  and  quantitatively  related  to,  these  systematic 
errors  in  current  methods  might  be  detected,, 

Eight  dogs  were  studied.   Continuous  records  were  made  of  1)  aortic  root  flow 
by  electromagnetic  flol^7meter,  2)  aortic  root  instantaneous  pressure,  3)  left 
femoral  arterial  pressure,  4)  right  femoral  flow  by  a  Doppler  ultrasonic 
flow  probe,  and  5)  right  femoral  flow  by  an  electromagnetic  flow  probe. 
Recordings  were  made  on  electromagnetic  tape.  The  dogs  were  given  a  series 
of  drugs  to  alter  cardiac  and  peripheral  vascular  function.   Partial  venous 
inflow  occlusion  was  effected  briefly  during  each  pharmacologic  intervention. 

Proposed  Course:   Experimental  data  will  be  digitized  for  analysis,   Tiiitial 
computations  will  be  1)  an  estimated  stroke  volume  for  each  beat  by  several 
currently  used  formulas,  2)  the  actual  stroke  volume  by  aortic  electro- 
magnetic flow  probe,  and  3)  the  errors  incurred  in  estimation.   The  percentage 
of  error  (positive  or  negative)  will  be  plotted  against  time  for  each  entire 
experiment.   Then,  on  the  same  axis  can  be  plotted  multiple  derivations  of 
the  parameters  measured  --  (for  example,  the  Doppler  flow,  the  aorto- femoral 
pulse  accentuation,  the  femoral  pulse  pressure,  etc.).   Any  visually  suggested 
correlation  will  be  tested  mathematically.   A  new  formula  with  a  factor 
incorporating  the  value  which  correlates  with  error  will  be  written.   The  new 
formula  will  then  be  applied  to  the  original  curves,  and  error  assessed 
again.   Completion  of  data  analysis  and  preparation  of  manuscript  for 
publication. 


/vd 


Project  Title: 


Serial  No.  NHLI-61 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970   through  June  30,  1971 

The  direct  and  reflex  effects  of  isolated  changes  in  pH, 
pOo)  ^i^d  pC02  upon  systemic  and  pulmonary  vascular 
resistance 


Previous  Serial  No.:   None 

Principal  Investigators:   Jefferson  F.  Holl ingsworth,  M.  D. 

Robert  L.  Reis,  M.  D. 

Other  Investigators:      Bradley  M.  Rodgers,  M.  D. 

John  W.  Yarbrough,  M.  D. 
Frederick  H.  Levine,  M.  D. 
David  B.  Melvin,  M.  D. 
David  M.  Conkle,  M.  D. 

Project  Description:   The  isolated  effects  of  pH,  p02.  arid  pC02  upon  systemic 
and  pulmonary  vascular  resistance  are  incompletely  defined  because 
compensatory  mechanisms  tend  to  obscure  individual  changes.   A  canine 
preparation  utilizing  cardiopulmonary  bypass  and  employing  isolated  perfusion 
and  oxygenation  of  the  systemic  and  of  the  pulmonary  circulations  has  been 
developed.   Twenty-two  NIH  foxhounds  were  studied.   This  preparation 
facilitates  the  investigation  of  isolated  pH,  p02.  ^^id  pC02  changes  upon 
systemic  and  pulmonary  circulations.   Utilizing  this  preparation,  neurological 
pathways  remain  intact,  but  there  is  complete  humoral  isolation  of  the 
systemic  and  pulmonary  circulations. 

Results:   Neither  airway  hypoxia,  pulmonary  arterial  hypoxia,  nor  a  combination 
of  these  factors  significantly  altered  pulmonary  vascular  resistance.   Systemic 
hypoxia,  however,  significantly  increased  pulmonary  vascular  resistance. 
Systemic  hypercapnic  acidosis  with  a  normal  p02  and  a  physiologic  pulmonary 
circulation  also  significantly  increased  pulmonary  Tascular  resistance.   A 
combination  of  systemic  hypercapnic  acidosis,  and  hypoxia  produced  a  synergistic 
increase  in  pulmonary  vascular  resistance.   It  is  proposed  that  these  changes 
in  pulmonary  vascular  resistance  are  reflexly  mediated  since  humoral  isolation 
of  the  circulations  was  confirmed.   Neither  systemic  hypercapnia  with  a 
physiologic  pH,  nor  systemic  acidosis  with  a  physiologic  pC02  produced  a 
significant  increase  in  pulmonary  vascular  resistance. 

The  direct  effects  of  selective  changes  in  the  systemic  pH,  p02,  and  pC02  on 
systemic  vascular  resistance  were  also  evaluated.   Systemic  hypoxia  produced 
no  change  in  systemic  vascular  resistance,  however,  one  minute  folbwing 
reoxygenaticn  systemic  vascular  resistance  significantly  decreased.   Systemic 
hypercapnia  with  a  physiologic  pH  also  increased,  systemic  vascular  resistance. 
Lactic  acidosis  with  a  constant  pC02  did  not  alter  systemic  vascular  resistance. 


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Serial  No.   NHLI-61 
1.    Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-MHI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Metabolic  alkalosis  significantly  decreased  systemic  vascular  resistance. 

Proposed  Course:   An  investigation  of  the  direct  effect  of  airway  and 
pulmonary  vascular  hypercapnia,  acidosis,  and  hypercapnic  acidosis  upon 
pulmonary  vascular  resistance  is  proposed. 


1 


« 


Serial  No.     NHLI-62 


1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH  j^ 

Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Measurement  of  left  ventricular  diameter  and  contraction 
velocity  by  ultrasound 

Previous  Serial  No.:   None 

Principal  Investigators:   Edward  B.  Stinson,  M.  D. 

Glenn  Rohmoeller 
Andrew  G.  Morrow,  M.  D., 

Cooperating  Unit:   Biomedical  Engineering  and  Instrumentation  Branch 

Project  Description:   Internal  left  ventricular  transverse  diameter  in  dogs 
is  measured  continuously  and  instantaneously  by  ultrasound  methods.  Lead 
titanate-zirconate  piezoelectric  crystals,  which  oscillate  at  eight  megahertz 
in  the  thickness  mode  when  stimulated  electrically,  are  cut  into  6  mm. 
diameter  disks  and  mounted  in  lucite  housings.  Two  crystals  are  implanted 
on  the  endocardial  surface  of  the  left  ventricle  across  the  maximum  internal 
transverse  diameter  and  the  electrical  leads  are  exteriorized.   One  crystal 
is  pulsed  and  the  receiving  crystal  monitoredo   Sound  travels  through  blood 
at  a  speed  of  1.5  millimeters  per  microsecond.   By  processing  the  receiving 
amplifier  output  with  a  tracking  gate,  similar  to  the  type  of  electronics 
system  used  by  the  military  to  track  rockets,  an  analog  output  proportional 
to  left  ventricular  diameter  is  displayed  on  a  scope  or  chart  recorder o 

Results:   Preliminary  results  indicate  that  with  the  tracking  gate  the 
system  is  practical .   Several  dogs  have  been  successfully  monitored  during 
exercise. 

Proposed  Course:   The  piezoelectric  crystals  are  being  evaluated  as  to  their 
antenna  properties  and  sonic  techniques  are  being  applied  to  maximize  beam 
wtdili  and  range. 


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Serial   No.     NHTJ-63 

1,   Clinic  of  Surgery 
3.   Bethesda,  Md. 


Project  Title: 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Effects  of  chronic  cardiac  dener^/ation  on  the  cardiac 
resDonse  to  exercise 


Previous  Serial  No< 


None 


Principal  Investigators:   Edward  B.  Stinson.  M.  D. 

Andrew  G.  Morrow,  M.  D. 

Project  description:   An  intact  dog  model  has  been  developed  to  evaluate 
instantaneous  cardiac  responses  to  treadmill  exercise.   After  preoperative 
treadmill  training,  animals  are  chronically  instrumented  by  implantation 
of  miniature,  solid-state  pressure  transducers  in  the  left  ventricle  and 
aorta,  a  flow  transducer  around  the  ascending  aorta,  piezoelectric  crystals 
on  the  endocardial  surface  of  the  left  ventricle  across  the  maximum  trans- 
verse diameter,  and  pacing  wires.   Experimental  subjects  are  cardiac-denervated 
by  regional  neural  ablation.   After  recovery  from  operation,  the  dogs  are 
studied  at  rest  and  during  exercise  and  the  following  parameters  are 
measured  directly  or  derived  offline  from  magnetic  tape  recordings:   heart 
rate,  cardiac  output,  stroke  volume  ,  peak  ejection  velocity,  acceleration  of 
ejection,  left  ventricular  peak  systolic  and  end-diastolic  pressure,  left 
ventricular  dp/dt,  aortic  phasic  and  mean  pressure,  stroke  work  and  power, 
left  ventncular  diameter,  volume,  and  rate  of  circumferential  fiber  shorten- 
ing. The  effects  of  heart  rate  control  and  pharmacologic  autonomic 
blockade  on  exercise  response  in  both  normal  and  cardiac-denervated  animals 
are  also  being  evaluated. 

Results:   Preliminary  results  in  two  cardiac-denervated  and  three  control 
dogs  indicate  that  profound  alterations  from  normal  adaptive  mechanisms 
are  induced  by  cardiac  denervation.   In  particular,  they  illustrate  a  major 
role  for  the  Frank-Starling  mechanism  in  the  response  of  the  denervated 
(and  by  inference,  the  transplanted)  heart  to  physical  stress.   At  present 
the  collection  and  analysis  of  data  have  not  been  completed. 

Proposed  course.   To  be  continued. 


**X 


Serial  No.    NHLI-64 
1 .      Clinic  of   Surgery 
3.      Bethesda.  Md. 


3. 


Project  Title: 


PHS-NIH 
Individual  Project  Reports 
July  1,  1970  through  June  30,  1971 

The  effects  of  chronic  right  heart  failure  on  left 
ventricular  function 


Previous  Serial  No. 


None 


Principal    Investigators:      John  W.  Yarbrough,   M.   D. 

Robert  L.  Reis,   M.   D. 


Other  Investigators: 


David  Melvin,  M.  D. 

Jefferson  Hollingsworth,  M.  D, 

David  Conkle,  M.  D. 


Project  Description:   Left  ventricular  function  curves  were  inscribed  by 
means  of  a  right  heart  bypass  preparation  at  constant  heart  rate  and 
aortic  pressure  in  17  American  foxhounds.   In  7  dogs,  severe  right  heart 
failure  had  been  produced  by  creation  of  tricuspid  regurgitation  and 
pulmonary  arterial  constriction  2-3  months  previously.   The  normal  foxhounds 
served  as  controls.  Left  ventricular  function  curves  were  depressed  in  all 
7  dogs  with  chronic  right  heart  failure  compared  to  the  curves  inscribed 
in  the  control  dogs.  Dilatation  and  hypertrophy  of  the  right  ventricle 
was  evident  in  addition  to  hepatosplenomegaly,  severe  malnutrition,  and 
ascites  in  each  of  the  7  dogs  with  right  heart  failure. 

The  mechanism  by  which  chronic  right  heart  failure  produces  left 
ventricular  dysfunction  has  not  been  defined.  Possibly,  septal  hypertrophy 
alters  left  ventricular  compliance  and  reduces  left  ventricular  function. 
Metabolic  defects  from  malnutrition  may  play  a  mare  direct  role. 


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Serial  No.  NHLI-65 
1 .   Clinic  of  Surgery 
3.   Bethesda,  Md. 


Project  Title: 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Evaluation  of  glutaraldehyde- fixed  fascia  lata  as  a  material 
for  tissue  valve  leaflets 


Previous  Serial  No, 


None 


Principal  Investigators:   John  W.  Yarbrough,  M„  D. 

Robert  L.  Reis,  M.  D. 


Other  Investigators: 


Jefferson  Hollingsworth,  M.  D, 
David  Melvin,  M.  D. 
David  Conkle,  M,  D. 


Project  description:   Fascia  lata  was  obtained  from  cadavers  without  the 
use  of  sterile  technique.   The  fascia  was  immediately  used  to  construct 
valves  on  the  Reis-Hancock  strut  and  then  placed  in  0.25%  glutaraldehyde. 
A  portion  of  the  fascia  was  sent  for  culture  at  the  time  of  construction 
of  the  valve  and  again  at  the  time  of  implantation  of  the  fixed  valve  in 
the  recipient. 

Five  Holstein  calves  and  one  sheep  were  used  for  implantation  of  the  valves 
in  the  mitral  position.   At  the  time  of  implantation  all  valves  were  sterile 
to  culture.  The  valves  were  noted  to  be  very  stiff  and  nonpliable.  Two 
calves  survived  for  two  months.   The  other  animals  died  within  24  hours 
from  pulmonary  edema.   Postmortem  examination  showed  the  valves  to  be  rigid, 
but  not  incompetent.  The  two  calves  that  survived  for  two  months  also  died 
from  pulmonary  edema.  The  valves  were  rigid  and  partially  overgrown  with 
neo-intima „ 

In  vitro  flow  determinations  across  the  valves  showed  them  to  be  stenotic 
and  nonpliable.  The  rigidity  of  the  valves  in  the  two  month  survivors 
demonstrated  that  the  fixed  fascia  did  not  become  more  pliable  after 
implantation.   It  was,  therefore,  concluded  that  glutaraldehyde- fixed  facia 
lata  is  not  a  suitable  material  for  use  in  constructing  tissue  valve 
prostheses. 

Proposed  Course:   Paper  being  prepared  for  publication. 


n*/: 


Project  Title: 


Serial  No.  NHLI-66 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Effect  of  positive  pressure  ventilation  on  pulmonary 
vascular  resistance 


5 


Previous  Serial  No.:   None 

Principal  Investigators:   Edward  M.  Mullin,  M.  D. 

Robert  E.  Sloane,  M.  D. 

Thomas  Q.  Winter,  M.  D. 

Robert  L.  Reis,  M.  D. 

Project- Description:   It  has  been  a  clinical  impression  that  positive  pressure 
ventilation  with  a  volume  controlled  respirator  can  augment  cardiac  output 
and  stabilize  the  hemodynamic  state  in  the  immediate  postoperative  period. 
This  effect  seems  most  pronounced  in  patients  with  elevated  pulmonary- 
vascular  resistance  secondary  to  long  standing  mitral  valvular  disease.  To 
document  and  quantitate  this  effect,  patients  undergoing  mitral  valve 
replacement  or  open  commissurotomy  have  been  studied  in  the  early  postoperative 
period.  Hemodynamic  assessements  with  PVR  measurements  were  recorded  using 
first  a  volume  respirator  (Engstrom) ,  and  then  during  spontaneous  ventilation 
via  the  Briggs  adapter.   Arterial  blood  gases  were  obtained  with  every 
intervention,  and  the  patient's  clinical  status  noted.  Measurements  were 
repeated  during  isuprel  infusion. 

Results:   There  is  a  statistically  significant  change  in  PVR  (+607o)  in 
switching  from  the  Engstrom  to  Briggs  adaptor,  which  was  slightly  modified 
by  isuprel o   The  pH,  pCO^,  p02  were  relatively  constant  but  cardiac  output 
appears  to  decrease.   Statistical  analysis  of  these  parameters  is  in  progress. 
Preliminary  data  indicate  that  there  is  a  beneficial  effect  from  the  use  of 
positive  pressure  ventilation  in  the  postoperative  period. 

Proposed  Course:   The  manuscript  is  in  process  of  preparation  and  will  be 
s.ioi.dtted  for  publication. 


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Serial  No,      NHTJ-67rp\ 
1,      Clinic  of   Surgery 
3.      Bethesda,   Md, 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Evaluation  of  the  operative  treatment  of  mitral  stenosis 
Previous  Serial  No „ :   None 

Principal  Investigators:   Edward  M.  Mullin,  Jr.,  M,  D„ 

D.  Luke  Clancy,  M.  D. 
Andrew  C.  Morrow,  M.  D. 


Other  Investigators: 


Lawrence  Higgs,  M.  D. 
Stephen  Epstein,  M.  D, 


Cooperating  Unit:   Cardiology  Branch,  NHLI 

Project  description:   A  study  of  closed  mitral  commissurotomies  (CMC),  open 
mitral  commissurotomies  (OMC) ,  and  valve  replacements  (MVR)  for  mitral  stenosis 
performed  from  1964  through  1969  was  undertaken,  to  compare  clinical  status 
and  catheterization  findings.   Records  were  reviewed  for  age,  sex,  rhythm, 
clinical  classification,  evidence  of  emboli,  mitral  calcification,  previous 
and  subsequent  operations,  and  associated  heart  murmurs.   Operative  notes 
vjere  reviewed  for  presence  of  LA  thrombus,  attempts  at  CMC  or  OMC,  and  mitral 
regurgitation  incurred.   Postoperative  followup  involved  recording  emboli, 
cause  of  death  if  any,  and  calls  or  letters  to  all  patients  not  seen  within 
the  past  year.  Pre-  and  postoperative  catheterization  data  were  analyzed 
for  LA,  RV  systolic,  LA-^LV  gradient,  CI,  and  MVA. 

Results:   The  patients  included  53  CMC,  20  OMC  and  51  MVR.   As  expected  the 
MVR  patients  represented  an  older  group  with  more  advanced  valvular  disease, 
a  higher  percentage  of  atrial  fibrillation,  equal  sex  distribution,  more  LA 
thrombi,  and  a  higher  operative  mortality.   There  was  no  mortality  in  the 
CMC  group,  though  four  patients  required  subsequent  operations.   Two  patients 
undergoing  OMC  succumbed  (10%) .   Comparison  of  the  catheterization  data 
reveals  that  the  best  results  were  obtained  in  CMC  and  indicates  that  thi?  is 
a  satisfactory  and  safe  operation  in  selected  patients. 


Proposed  Course: 
publication. 


A  manuscript  is  in  process  and  will  be  submitted  for 


//S 


I 


Serial  No.   NHLI-68(c) 
1.  Clinic  of  Surgery 
3,  Bethesda,  Md„ 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Closure  of  atrial  septal  defect  with  a  perforated  patch 

Previous  Serial  No „ :   None 


Principal  Investigators:   Edward  M.  Mullin,  M.  D. 

Robert  L.  Reis,  M.  D. 

Project  Description:   The  hemodynamic  effects  of  patch  closure  of  atrial 
septal  defect  have  been  well  described,  and  the  operation  itself  has  become 
a  relatively  routine  procedure »   In  a  small  group  of  adults,  however, 
preoperative  cardiac  catheterization  has  suggested  the  possibility  of  left- 
heart  decompensation  with  complete  closure  of  an  ASD.   Two  such  patients 
have  recently  undergone  operative  repair  of  ASD  using  a  perforated  Teflon 
patch,  to  allow  residual  left- to-right  shunting  and  to  prevent  sudden 
cardiac  failure.  The  pre-  and  postoperative  course,  the  catheterization 
data,  and  operative  techniques  have  been  reviewed  and  form  the  content  of 
this  project. 

Results:    In  the  first  patient  (K.D.) ,  careful  intraoperative  pressure 
recordings  as  the  ASD  was  closed  are  complimented  by  pre-and  postoperative 
catheterizations.   In  the  second  (M.W„)  postoperative  data  are  pending 
though  preoperative  assessment  was  thorough. 

Both  patients  have  had  long  and  complicated  postoperative  courses  with 
evidence  of  LV  failure.  The  successful  use  of  a  perforated  interatrial 
patch  to  allow  left  heart  decompression  is  a  reportable  technique  and  bears 
further  investigation. 


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Proposed  course: 
publication. 


A  manuscript  is  in  progress  and  will  be  submitted  for 


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Serial  No.  NHLI-69<c) 
1.  Clinic  of  Surgery 
3.   Bethesda,  Md. 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Augmentation  of  myocardial  contractility  with  coronary 
bypass  grafts 

Previous  Serial  No.:   None 


Principal  Investigators: 


Cooperating  Unite: 


Edward  M.  Mull in,  M.  D. 
Robert  E.  Sloane,  M.  D. 
Robert  L.  Reis,  M.  D. 
Kenneth  Kempner,  M.  A. 

Computer  Systems  Laboratory,  DCRT 


Project  Description:   Coronary  bypass  vein  grafts  are  presently  being 
performed  in  patients  with  coronary  atherosclerosis  and  angina  with  the 
rationale  that  increased  coronary  blood  flow  beyond  the  point  of  obstruction 
will  result  in  improved  coronary  perfusion.  This  improved  perfusion  sho'jld 
reflect  itself  in  augmented  contractility  of  the  ischemic  myocardium.   The 
present  study  was  designed  as  an  intraoperative  assessment  of  LV  contractility 
by  means  of  computer  analysis  of  high  fidelity  LV  pressure  recordings.  The 
contractile  state  was  quantified  by  using  V^gj^  based  on  a  graph  plot  of 
dp/dt/P  against  P  (either  total  or  derived)  . 

Recordings  of  LV  and  Ao  pressure  were  made  after  the  vein  grafts  were 
constructed,  and  the  patient  successfully  separated  from  bypass.   Contractility 
was  then  measured  with  the  graft  (or  grafts)  open  or  occluded  for  3--5 
minutes.  Where  possible,  this  sequence  was  performed  in  duplicate.   Changes 
in  contractile  state  were  correlated  with  vein  graft  flow  as  measured  by 
individual  flow  probes. 


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graft  flow,  there  was  a  significant  (30%)  augmentation  of  the  contractile 
state.  This  method  of  analysis  of  the  effect  of  coronary  vein  grafts  has 
merit  and  warrants  further  investigation. 

Proposed  course:   Continued  application  of  intraoperative  contractile 
assessment  of  the  effects  of  coronary  bypass  grafts. 


//« 


Serial  No.     NHLI-70 


1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title;   The  effect  of  protamine  sulfate  on  myocardial  contractility 
in  the  intact  dog 

Previous  Serial  No.:   None 

Principal  Investigators:   Gordon  N.  dinger,  M.  D. 

Robert  E.  Sloane,  M.  D. 
Edward  B.  Stinson,  M.  D. 
Andrew  G.  Morrow,  M.  Do 

Project  description:   Normal  adult  dogs  were  instrumented  with  aortic  flow 
probes  and  solid  state  aortic  and  left  ventricular  pressure  transducers 
and  were  studied  in  the  intact  state  several  days  after  recovery  from 
operation.  Two  dogs  also  had  regional  cardiac  denervation.   Animals  were 
studied  while  sedated  and  determinations  made  on-line  (utilizing  analog 
computation  of  left  ventricular  dp/dt/P  plotted  against  P,  i.e.,  contractile 
element  velocity)  with  use  of  V^^  as  an  index  of  myocardial  contractility. 
The  effect  of  therapeutic  dosage  of  protamine  sulfate  given  intravenously 
was  evaluated  in  each  dog  both  in  the  normal  resting  state,  and,  during 
another  study,  after  beta-adrenergic  blockade  with  propranolol.  Preliminary 
data  have  shown  no  effects  on  contractility  in  these  preparations.   A  small 
number  of  dogs  similarly  instrumented  were  subjected  to  standard  periods  of 
cardiopulmonary  bypass  and  protamine  sulfate  evaluated  following  cessation 
of  bypass.   Small  decrements  in  contractility  have  been  observed  under  these 
circumstances.  Protamine  sulfate  has  shown  no  effect  in  the  normal  dog 
made  acutely  hypertensive  with  neosynephrine. 

These  preliminary  data  indicate  that  protamine  sulfate  does  not  have 
a  direct  negative  inotropic  effect  on  the  normal  canine  myocardium.   Its 
action  on  the  nyocardium  following  cardiopulmonary  bypass  indicates  some 
vasoactive  influence  which  remains  to  be  elucidated.   The  mechanisms  of  this 
action  are  currently  under  evaluation. 

Proposed  Course:   Further  elucidation  of  the  mechanism  of  action  of 
protamine  sulfate  on  the  peripheral  vascular  system.   To  be  submitted  for 
publication. 


■B3«   - 


fff 


Serial  No.  NHLI-71 
1.  Clinic  of  Surgery 
3.   Bethesda,  Md. 


Project  Title: 


PHS-NIH 
Individual  Proiect  Report 
July  1,  1970  through  June  30,  1971 

Evaluation  of  the  effects  of  corticosteroids  on  myocardial 
contractility 


Previous  Serial  No. 


None 


PrinciDal  Investigators: 


Paul  L.  Teclclenberg,  M.  D„ 
Edward  B.  Stinson,  M.  D. 
Andrew  G.  Morrow,  M.  D. 


Project  Description:   Eleven  normal,  nine  chronic  cardiac  denervated  (four 
after  beta-blockade  with  propranolol),  and  five  acutely  adrenalectomized 
and  cardiac  denervated  dogs  were  studied  on  cardiopulmonary  bypass,  at 
constant  coronary  blood  flow,  by  means  of  an  isovolumic  left  heart  preparation. 
Methyl-prednisolone  (MP),  15-30  mg./Kg.,  was  given  IV  and  changes  in  myo- 
cardial contractility  were  assessed  by  measurement  of  left  ventricular  peak 
pressure  (LVP)  ,  LV  dp/dt,  and  LV  contractile  element  velocity  (niax  V). 

Results:   In  normal  dogs  MP  caused  significant  enhancement  of  myocardial 
contractility  (LVP  +  217.,  LV  dp/dt  +  27%,  max  V  +  22%,  P  <  .01  in  all). 
Chronic  cardiac  denervated  animals  showed  similar  effects,  but  with 
significantly  greater  responses  than  normal  dogs  in  LVP  and  LV  dp/dt  (P  <  .01)  . 
These  changes  were  greatly  attenuated  or  abolished  in  beta-blocked-denervated, 
and  in  acutely  adrenalectomized  and  denervated  dogs. 

The  results  of  this  study  confirm  an  acute  positive  inotropic  effect  of  MP 
in  vivo,  but  suggest  that  this  influence  is  mediated  indirectly  by  the 
release  and/or  potentiation  of  endogenous  catecholamines. 

Proposed  Course:   Manuscript  to  be  submitted  for  publication. 


tic 


Serial  No.  NHLI-72 
1.  Clinic  of  Surgery 
3.  Bethesda,  Md, 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Mechanisms  of  the  pulmonary  vascular  response  to  hypovolemic 
shock 


s- 


Previous  Serial  No.:   None 


Principal  Investigators: 


Other  Investigators: 


Frederick  H.  Levine,  M.  D. 
Robert  L.  Reis,  M.  D. 

Jefferson  F.  Hollingsworth,  M.  D. 
David  M.  Conkle,  M.  D„ 


Project  Description;   Increased  pulmonary  vascular  resistance  (PVR)  which 
occurs  with  hypovolemic  shock  (S)  is  thought  to  be  related  to  both  neural 
and  humoral  factors »  The  pulmonary  and  systemic  circulations  were  separately 
perfused  by  two  pump  oxygenator  systems.  Neural  pathways  were  intact  but 
humoral  isolation  of  the  circulations  was  confirmed.  A  pulsatile  pump  was 
employed  in  the  systemic  circulation  and  stroke  volume  was  adjusted  so  that 
mean  arterial  pressure  was  100  mm.  Hg.   Systemic  and  pulmonary  arterial 
p02  and  pC02  were  maintained  physiologic.   Pulmonary  blood  flow,  systemic 
blood  flow,  pulmonary  arterial,  left  atrial,  and  systemic  arterial  pressures 
were  continuously  recorded.  PVR  was  assessed  before  and  after  (S)  by 
resistance- flow  curves  constructed  by  increasing  pulmonary  flow  from  50-200 
cc./Kg./min.   (S)  was  produced  by  decreasing  stroke  volume  until  mean 
arterial  pressure  was  30  mm.  Hg .  Twenty  experiments  were  performed  on  ten 
dogs.  PVR  was  unchanged  when  assessed  5  minutes  after  (S)  was  induced. 
One  liter  of  blood  (physiologic  pH)  obtained  from  the  systemic  circulation 
10  minutes  after  (S)  was  added  to  the  pulmonary  circulation;  PVR  increased 
from561  +  70  (dynes-sed-cm"^)  to  748  +  90  (p^.05).   Systemic  acidosis 
9pH  7.14  +  .06)  developed  after  30-45  minutes  of  (S)  but  the  PVR  remained 
unchanged.  When  the  pH  of  the  pulmonary  blood  was  decreased  (7.08  +  .08) 
by  lactic  acid  infusion  PVR  increased  from  521  +  104  to  794  +  130  (p  <.05). 

Thus  neither  systemic  hypotension  (baroreceptors)  nor  metabolic  acidosis 

in  the  systemic  circulation  (chemoreceptors)  produced  reflex  changes  in  PVR. 

Increased  PVR  in  (S)  results  from  the  effects  of  humoral  factors  on  the  lung. 

Proposed  Course:   Project  com.pleted  -  abstract  submitted  for  publication. 


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Serial  No.   NHLI-73 


1.   Clinic  of  Surgery 
3.   Bethesda,  Md . 

PKS-NII-I 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Hemodynamic  effects  of  bretylium  tosylate  in  the  intact  dog 

Previous  Serial  No.:   None 

Principal  Investigators:   Robert  E.  Sloane,  M,  D. 

Gordon  N.  dinger,  M.  D. 
Edward  B.  Stinson,  M.  D. 
Andrew  G.  Morrow,  M.  D. 

Project  Description:   Controlled  studies  of  the  inotropic  effect  of  bretylium 
tosylate  have  been  done  in  vitro .   However,  data  obtained  from  intact  mammals 
is  inconclusive.  Miniature  solid  state  pressure  transducers  were  implanted 
in  the  left  ventricle  and  proximal  aorta  of  dogs.   Aortic  flow  probes  and 
atrial  pacing  wires  were  placed  as  well.  From  3  to  7  days  postoperatively, 
the  animals  were  studied  in  intact  state.  Myocardial  contractility  was 
determined  by  extrapolating  to  V^ax  the  linear  isovolumic  portion  of  the 
vector  loop  produced  by  dp/dt/P-LVED  displayed  against  P-LVED  on  an 
oscilloscope.   Control  Vniax>  dp/dt,  LVP,  LVEDP,  AoP,  and  cardiac  output 
were  compared  before  and  after  administration  of  bretylium  tosylate  5  mg./Kg. 

Results:   The  study  is  at  present  in  progress  so  that  results  are  preliminary'. 
A  biphasic  rise  in  V^ax  was  noted  in  the  order  of  lO-ACX;  dp/dt  also  rose 
10-1007„.  LVP  rose,  LVED  fell,  mean  AoP  increased  and  C  .0 .  did  not  change 
significantly.  The  inotropic  effect  of  the  drug  was  confirmed.   Prior 
administration  of  propranolol  0.5  mg./Kg.  completely  abolished  the  biphasic 
inotropic  action  of  bretylium.  Further  studies  of  the  mechanism  of  action 
are  in  progress. 


/xa- 


Serial  No.       NHLI-74 
1.      Clinic   of   Surgery 
3.      Bethesda,   Md. 


Project  Title: 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

The  use  of  isobutyl  cyanoacrylate  in  microvascular 
anastomosis 


Previous  Serial  No.:   NHLI-87 

Principal  Investigators:   Sherman  G.  Souther,  M.  D. 

Sidney  Levitsky,  M.  D„ 
Andrew  G.  Morrow,  M.  D. 


Other  Investigator: 
Cooperating  Unit: 


William  C.  Roberts,  M.  D. 


Pathology  Section,  Cardiology  Branch,  NllLI 


Project  Description:   The  infrarenal  abdominal  aorta  in  albino  rats  weighing 
120  to  480  grams  was  divided  between  occluding  clamps,  and  stay  sutures 
of  8-0  and  9-0  nylon  were  placed.   Isobutyl  cyanoacrylate  was  applied  to 
the  approximated  edges  of  the  divided  vessel,  and  the  clamps  were  removed. 
The  animals  were  sacrificed  at  intervals  varying  from  24  hours  to  6  weeks, 
and  sectioiB  of  the  anastomoses  were  obtained  for  histologic  study.   This 
group  of  rats  was  compared  to  a  control  group  in  which  anastomoses  performed 
with  a  continuous  suture  of  8-0  or  9-0  nylon. 

Results:   Five  of  28  anastomoses  in  the  isobutyl  cyanoacrylate  group  became 
occluded,  and  1  of  24  anastomoses  in  the  suture  group  became  occluded. 
Comparison  of  sections  through  patent  anastomoses  revealed  a  more  marked 
and  prolonged  acute  and  chronic  inflammatory  response  in  the  vessels  in 
the  isobutyl  cyanoacrylate  group  than  in  the  suture  group.   In  addition, 
degeneration  of  the  media  and  deposition  of  calcium  occurred  in  the  vessels 
in  the  isobutyl  cyanoacrylate  group. 

Propoyed  Course:   A  manuscript  has  been  prepared  from  these  data  and  has 
e2n  submitted  for  publication.   The  project  is  completed. 


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Serial  No.      NKLI-75 
1.      Clinic   of   Surgery 
3.      Bethesda,   Md. 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Prolongation  of  cardiac  allografts  in  the  rat  by  alloantisera 

Previous  Serial  No.:   None 


Principal  Investigators: 


Sherman  G.  Souther,  M.  D, 
Edward  B.  Stinson,  M.  D. 
Andrew  G.  Morrow,  M.  D. 


Project  description:   Survival  of  heterotopic  (abdominat)  heart  allografts 
was  studied  in  inbred  rats.  Nine  control  Lewis  rats  (L)  rejected  (loss 
of  EKG  activity)  Brown  Norvjay  (BN)  grafts  in  6,4  +  0.7  (S.D.)  days. 
Thirteen  L  rejected  hybrid  (LBN)  grafts  in  14  +  3.4  days.   IV  administration 
of  10^  BN  or  LBN  bone  marrow  cells  to  seven  L  and  spleen  cells  to  five  L 
seven  days  preoperatively  caused  significant  prolongation  of  BN  grafts  to 
11.7  +  3.3  and  9.6  +  2.1  days  respectively  (p  ^  .05)  .  Administration  of  BN 
bone  marrow  or  spleen  cells  to  10  L  did  not  cause  significant  prolongation 
of  LBN  graft  survival.   Serum  obtained  from  L  seven  days  after  injection 
with  either  10 'BN  or  LBN  spleen  cells  was  given  IV  intraoperatively  and  for 
nine  days  postoperatively  (total  8  ml.)  to  each  of  four  L  with  BN  grafts, 
and  prolonged  survival  to  11.0  +2.4  days  (p  <.05).   Control  sera  did  not 
prolong  graft  survival. 

Alloantiserum  prolongs  survival  of  cardiac  allografts  in  the  rat,  although 
less  dramatically  than  previously  shown  for  renal  grafts. 

Proposed  course:   The  project  is  completed  and  a  manuscript  will  be  prepared. 


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Project  Title: 


Serial  No.  NHLI-7  6 
1 .   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Mixed  leucocyte  reaction  (MLR)  as  an  assay  for  the  presence 
of  enhancing  alloantiserum 


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Previous  Serial  No.:   None 


Principal  Investigators: 


Sherman  G.  Souther,  M.  D. 
Edward  B.  Stinson,  M,  D. 
Robert  0.  Gordon,  M.  D. 
Andrew  G.  Morrow,  M.  D. 


Other  Investigator:       Joost  J.  Oppenheim,  M.  D. 

Cooperating  Unit:    Laboratory  of  Microbiology,  NIDR 

Project  description:   Alloantisera  that  enhance  survival  of  heterotopic 
(abdominal)  heart  allografts  in  inbred  rats  were  tested  in  MLR.  Lewis  (L) 
rats  administered  10'  Brown  Norway  (BN)  rat  bone  marrow  or  spleen  cells 
intravenously  (IV)  7  days  prior  to  transplantation  exhibited  significant 
prolongation  of  BN  graft  survival.  Passive  administration  to  normal  L  rats 
of  serum  obtained  from  L  rats  7  days  after  such  IV  immunization  similarly 
prdonged  BN  graft  survival.  The  unidirectional  MLR  of  normal  or  presensitized 
L  with  irradiated  BN  spleen  cells  was  also  suppressed  by  this  immune  serum 
as  compared  to  MLR's  containing  normal  L  serum.  However,  washed  splenic 
leucocytes  from  these  presensitized  L  rats  when  incubated  in  normal  L  serum 
showed  increased  proliferative  activity  to  irradiated  BN  spleen  cells. 
Since  these  L  cells  after  removal  of  "enhancing"  serum  were  very  active  in 
the  MLR,  this  suggests  that  enhancement  rather  than  central  tolerance  was 
the  mechanism  of  graft  prolongation.  Twenty  percent  alloantiserum  decreased 
the  MLR  of  unsensitized  L  with  irradiated  BN  as  much  as  6- fold.  Decreasing 
degrees  of  suppression  were  demonstrated  with  10%  and  5%  antiserum,  and  none 
with  2.57o.  Alloantiserum  suppression  of  MLR  remained  demonstrable  in  the 
absence  of  hemolytic  complement.   These  suppressive  antisera  have  no  cytotoxic 
effects  in  microcytotoxicity  assays.   Immunologic  specificity  was  demonstrated 
because   "enhancing"  serum  failed  to  suppress  the  MLR  of  L  and  irradiated 
ACI  rat  leucocytes.  Thus,  inhibition  of  MLR  can  be  used  as  an  assay  for 
the  presence  of  "enhancing"  alloantiserum. 

Proposed  course:   The  immune  serum  is  being  evaluated  for  specific  antibody 
and  the  importance  of  the  complement  fixing  fragment. 


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Serial  No,   NHLI-77 
1,   Clinic  of  Surgery 
3.   Bethesda,  Md. 


Project  Title: 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Immediate  in  vitro  leukocyte  DNA  synthesis: 
indicator  of  heart  allograft  rejection 


An  early 


Previous  Serial  No, 


None 


Principal  Investigators: 


Shemian  G.  Souther,  M.  D, 
Edward  Bo  Stinson,  M.  D. 
Robert  0.  Gordon,  M.  D. 
Andrew  G.  MorroxN^,  M.  D. 


Other  Investigator: 


Joost  J.  Oppenheim 


Cooperating  Unit:   Laboratory  of  Microbiology,  NIDR 

Project  description:   The  in   vitro  DNA  synthesis  by  splenic  leukocytes  from 
Lewis  (L)  rats  bearing  heterotopic  (abdominal)  Brown  Norway  (BN)  rat  heart 
allografts  was  studied.   Allografts  in  nine  L  rats  were  rejected  (loss  of 
EKC  activity)  in  6.4  +  0.7  (S.D.)  days.  Twelve  L  rats  were  sacrificed  at 
days  1-6  (2  each  day)  after  grafting;   the  allografts  were  excised  for 
pathologic  study,  and  the  spleens  were  removed  for  study  of  leukocyte  DNA 
synthesis.   One  tici  of  H-thymidine  was  added  to  a  suspension  of  3  x  10 
L  leukocytes  in  1.5  ml.  RPMI  1640  culture  medium  supplemented  with  107=  fetal 
calf  serum.  After  two  hours  of  incubation  at  37°  C,  the  cells  \<iere   washed 
with  isotonic  saline  and  treated  with  57=,  TCA  at  4°  C .   The  acid  precipitable 
radioactivity  was  determined.  Mean  counts  per  minute  (cpm)  of  12  normal  L 
rat  spleen  cell  suspensions  was  527  +  165  (S.D.)  (range  204-734).   Mean  cpm 
of  animals  with  allografts  were  increased  above  normal  L  as  follows:   day  2, 
1.3x;  day  3,  2. Ox;  day  4,  2.4x;  day  5,  7.2x,  and  day  6,  7.5x.   By  day  3 
the  increase  in  DNA  synthesis  was  significantly  elevated  above  normal,  and 
there  was  no  overlap  with  the  range  of  normal  L  rats.   Sham  operated  animals 
showed  no  increase  in  lymphocyte  activation.   Changes  in  the  status  of  the 
grafts  determined  by  palpation  and  EKG  were  inconsistent  and  were  invariably 
preceded  by  increased  _in  vitro  leukocyte  DNA  synthesis  by  at  least  48  hours. 

Proposed  Course:   These  studies  will  be  continued  utilizing  peripheral 
blood  leukocytes  in  dogs  bearing  orthotopic  cardiac  grafts. 


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Serial  No.      NHLI-78 


3. 


Clinic  of  Surgery 
Be the s da,  Md. 


Project  Title: 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Creation  of  aorto-pulmonary  shunts  with  cyanoacrylate 
tissue  adhesive 


Previous  Serial  No. :   None 

Princpal  Investigators:   Sherman  G.  Souther,  M.  D. 

Edward  B.  Stinson,  M.  D. 
Andrew  G.  Morrow,  M.  D. 

Project  description:   A  rapid  method  of  creating  aorto-pulmonary  shunts  was 
devised.   Through  a  left  thoracotomy  in  dogs,  the  common  adventitia  between 
the  aorta  and  main  pulmonary  artery  was  opened,  and  the  aorta  and  pulmonary 
artery  were  approximated  with  isobutyl  cyanoacrylate  or  fluoroeikyl  cyano- 
acrylate.  Through  a  purse-string  suture  on  the  opposite  wall  of  the 
pulmonary  artery,  a  large  needle  was  inserted  into  the  pulmonary  artery 
and  then  into  the  aorta.   The  needle  was  removed,  and  a  Fonkalsrud  septectomy 
punch  was  inserted  into  the  aorta  through  the  tract  made  by  the  needle.   The 
wall  of  the  aorta  and  pulmonary  artery  in  the  area  approximated  with  the 
tissue  adhesive  was  engaged  with  the  punch  and  a  portion  was  removed.   The 
purse-string  suture  was  tied.   A  good  thrill  in  the  pulmonary  artery   indicated 
a  satisfactory  shunt. 

Results:   Thirty-two  dogs,  18  adults  and  14  puppies,  had  shunts  performed. 
A  5  mm.  diameter  punch  was  used  in  adults,  and  a  3  mm.  punch  was  used  in 
puppies.   Four  adults  and  one  puppy  died  from  intraoperative  hemorrhage; 
one  adult  and  one  puppy  died  from  late  postoperative  hemorrhage.   In  five 
adult  dogs,  the  operation  was  done  without  dissection  of  the  aorta  and 
pulmonary  artery  and  without  application  of  the  glue.    These  animals  were 
considered  a  control  group.   Three  months  after  operation,  two  adult  dogs 
with  continuous  murmurs  were  reoperated,  and  2  mm.  and  3  mm.  shunts  were 
closed  using  total  cardiopulmonary  bypass.   These  dogs  survived  without 
difficulty.   The  remainder  of  the  11  adults  were  sacrificed  three  months  after 
operation.   Two  dogs  had  continuous  murmurs,  and  each  had  a  2  mm.  shunt  at 
autopsy.   The  remaining  nine  dogs  had  no  murmur,  and  at  autopsy  the  shunt 
was  completely  closed. 

Proposed  course:   The  puppies  will  be  followed  for  one  year  to  evaluate  the 
late  results  of  operation. 


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ANNUAL  REPORT  OF  THE 

EXPERIMENTAL  THERAPEUTICS  BRANCH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1970  through  June  30,  1971 


BIOCHEMICAL  PHARMACOLOGY 


I.   Biochemistry  of  Neurohumoral  Amines 

Enzymes  of  the  Pineal  Gland.   A  protein  fraction  from  rat  liver  was  found 
to  stimulate  tryptophan  hydroxylase  isolated  from  beef  pineal.   Since  the 
fraction  appears  identical  to  liver  phenylalanine  hydroxylase,  we  believe 
that  these  two  hydroxylating  enzymes  are  similar  and  that  substrate  specific- 
ity may  be  determined  by  various  subunit  combinations.   Hydroxyindole  0- 
methyltransf erase  (HOMT)  has  been  purified  from  beef  pineals  and  shown  to 
have  a  molecular  weight  of  about  78,000,  consisting  of  2  identical  subunits 
of  39,000.   This  enzyme  is  a  major  protein  of  the  pineal  representing  4% 
of  the  total  soluble  proteins  in  this  gland.   HOMT  exists  in  2  higher  molec- 
ular weight  forms  and  in  2  differently  charged  species.   It  is  possible  that 
conversion  of  one  form  to  another  may  result  in  changes  in  enzyme  activity 
noted  during  diurnal  variations.   Protein  kinases  may  play  a  key  role  in 
control  of  such  diurnal  rhythms  acting  either  at  a  transcriptional  level 
(histone  phosphorylation)  or  by  modifying  enzymes  directly.   Bovine  pineals 
were  found  to  contain  active  protein  kinases  which  are  strongly  stimulated 
by  cyclic  AMP.   At  least  two  of  these  kinases  have  been  partially  purified. 

Serotonin:   Chronic  transection  (5  weeks)  of  spinal  cord  in  cats  results 
in  an  80%  loss  of  tryptophan  hydroxylase  and  serotonin  content.   Since  all 
the  descending  serotonergic  fibers  degenerate,  residual  serotonin  and 
synthetic  enzyme  suggest  the  presence  of  serotonergic  interneurones.   Other 
studies  in  cats  demonstrate  that  administration  of  5-hydroxytryptophan 
enhances  monosynaptic  reflexes  and  that  tricyclic  antidepressant  drugs 
act  synergistically  to  greatly  enhance  the  effect.   This  finding  has  rele- 
vance to  clinical  situations  such  as  hypotonia  in  Down's  syndrome. 

Other  studies  reveal  the  following:   1)  serotonin  synthetic  rates  and 
tryptophan  hydroxylase  levels  are  not  altered  in  morphine  addicted  mice, 
contrary  to  reports  from  other  laboratories;  2)  there  is  no  change  in  the 
level  of  tryptophan  hydroxylase  during  REM  sleep  deprivation  in  rats, 
although  others  report  an  increase  in  serotonin  turnover;  and  3)  using  a 
system  for  continuous  monitoring  of  gastric  secretion  in  the  rat,  an 
inhibitory  effect  of  serotonin  (i.v.)  on  histamine  stimulated  secretion  was 
observed  while  5-hydroxytryptophan  had  no  effect. 

Histamine:   The  development  of  new  methods  of  assay  for  histamine  and 
its  metabolizing  enzyme,  histaminase,  has  permitted  more  definitive  studies 
on  this  compound.   The  gastrointestinal  tract  contains  the  highest  amount  of 
histaminase  in  the  rat,  although  appreciable  amounts  exist  in  the  adrenals, 
pancreas  and  thymus.   Histaminase  activity  is  increased  in  the  thymus  of 
spontaneously  hypertensive  (SH)  rats  compared  to  controls.   The  rise  in 
plasma  histaminase  following  dosing  with  heparin  was  accompanied  by  a  severe 


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depletion  in  the  intestinal  content  of  the  enzyme.   The  plasma  level  of 
histaminase  returned  to  normal  within  5  hours  and  the  intestinal  level  vas 
restored  within  10  hours. 

The  possible  role  of  histamine  in  inflammation  and  sv;elling  was  examined 
using  the  heat  injured  paw  of  the  rat.   Heat  injury  caused  a  70%  depletion 
of  histamine  in  paw  skin,  substantial  swelling  and  a  5  to  10-fold  increase 
in  the  histamine  content  of  paw  exudate.   Prior  depletion  with  the  histamine 
liberator  48/80  partially  blocked  the  effects.   Compound  48/80  causes  the 
release  of  histamine  from  mast  cells  by  an  energy  requiring  reaction.   The 
release  is  strongly  inhibited  in  vitro  by  dibutyryl  cyclic  AMP  and  a  variety 
of  compounds  which  inhibit  phosphodiesterase.   It  appears  that  cyclic  AMP 
is  involved  in  release  of  histamine  from  mast  cells. 

Catecholamines:   The  brainstem  levels  of  norepinephrine  in  spontaneously 
hypertensive  (SH)  rats  are  20  to  30%  less  than  those  of  normal  rats.   It  was 
found  that  the  aromatic  L  amino  acid  decarboxylase  in  the  brainstem  was 
decreased  by  50%  possibly  accounting  for  the  lower  norepinephrine  content. 
A  number  of  studies  were  done  using  catecholamine  precursors  to  replete  the 
brainstem  levels.   A  significant  inverse  correlation  between  blood  pressure 
and  brainstem  norepinephrine  was  observed.   Peripheral  administration  of 
6-hydroxydopamine  which  destroys  a  large  percentage  of  the  peripheral 
sympathetic  nerves  was  not  effective  in  reducing  the  blood  pressure  in  the 
hypertensive  animals.   The  plasma  renin  activity  of  SH  rats  was  found  to  be 
distinctly  elevated.   This  renin  activity  appeared  to  be  of  extrarenal  origin, 
but  no  direct  correlation  between  this  enzyme  and  catecholamines  has  been 
established. 

II.   Metalloproteins 

The  primary  amino  acid  sequence  of  the  iron-sulfur  protein,  rubredoxin, 
has  been  largely  completed.   The  points  of  major  interest  are  the  finding 
of  N-formylmethionine  at  the  N  terminus  and  the  positioning  of  the 
cysteinyl  residues.   The  sulfhydryl  groups  of  these  4  cysteinyl  residues  form 
the  ligands  for  the  iron  atom  and  are  arranged  in  a  tetrahedron.   Evidence 
for  this  type  of  binding  has  been  obtained  by  near  infrared  absorption  and 
circular  dichroism  and  by  Laser-Raman  spectroscopy.   There  does  appear  to  be 
a  distortion  in  the  tetrahedral  symmetry  as  evidenced  by  highly  dichroic 
single  crystal  absorption  spectra  and  splitting  of  the  d  ->  d  transition  bands 
in  the  near  infrared.   These  observations  have  been  confirmed  by  x-ray 
crystallographic  analysis. 

The  techniques  for  measuring  absorption  and  circular  dichroism  of  protein 
solutions  in  the  near  infrared  have  been  applied  to  plant  ferredoxin,  and 
adrenodoxin.   A.t  least  one  of  the  two  iron  atoms  in  each  of  these  proteins 
is  bound  in  a  tetrahedral  configuration.   Spectral  analysis  also  indicates 
that  there  is  significant  interaction  between  the  two  iron  atoms  in  each 
molecule.   Based  on  these  observations  and  other  reported  physical  studies, 
a  model  for  the  active  center  of  these  two  important  iron-sulfur  proteins 
has  been  proposed. 


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PHYSIOLOGICAL  CHEMISTRY 

I.  Factor  XIII  and  Fibrin  Crosslinking 

Fibrin  clots  from  normal  plasma  contain  approximately  6  moles  e-(Y~ 
glutamyl) lysine  crosslink  per  mole  fibrin,  whereas  clots  from  individuals 
with  Factor  XIII  deficiency  contain  little  or  none  of  this  crosslink 
(0.02-0.64  mole/mole).   Thus,  recurrent  bruising  and  bleeding,  and  abnormal 
wound  healing  in  these  cases  may  be  due  to  the  lack  of  e-Cy-glutamyl) lysine 
formation. 

II.  Kinin  System 

The  purification  of  human  and  rat  urinary  kallikreins  has  permitted  the 
development  of  an  improved  assay  for  urinary  kallikrein,  which  has  been 
applied  clinically.   In  SH  rats  urinary  kallikrein  was  significantly  elevated 
(at  11  and  16  weeks)  above  that  in  control  animals.   Significant  progress 
has  been  made  in  the  purification  of  what  may  be  a  specific  activator  of 
prekallikrein  which  the  data  indicate  arises  from  inactive  Hageman  factor. 
Antibodies  to  hunan  and  rat  urinary  kallikrein  have  been  prepared.   Both 
block  action  of  the  enzyme  on  kininogen  but  ester  hydrolysis  is  not  affected. 
The  principle  of  affinity  chromatography  has  been  used  in  the  purification 
of  human  prekallikrein  activator,  human  kallikrein  and  human  kininogens. 

III.  New  Biologically  Active  Peptides 

The  peptide  nature  of  villikinin  has  been  more  firmly  established  with 
studies  on  material  of  bovine  origin.   As  with  canine  material,  bovine 
villikinin  is  a  low  molecular  weight  substance  which  can  be  purified  on 
cation  exchange  resins.   It  is  also  inactivated  by  Pronase,  papain  and 
chymotrypsin  and  not  by  trypsin.   In  confirmation  of  work  elsewhere  a  pressor 
substance  was  found  in  the  plasma  of  dogs  following  bilateral  renal  artery 
ligation.   The  biologic  spectrum  of  activity  is  similar  to  that  of  angioten- 
sin I,  but  not  angiotensin  II.   Boiling  the  plasma  abolishes  activity  sug- 
gesting an  angiotensin  I-protein  complex.   The  bald-faced  hornet  venom  sac 
was  shown  to  contain  1-5  mg  of  serotonin  and  histamine,  as  well  as  hypotensive 
substance  which  may  also  be  a  peptide.   Eight  synthetic  analogues  of  ranaten- 
sin  (undecapeptide)  were  tested  for  hypotensive  and  uterine  stimulant 
activity.   The  c-terminal  nona  and  deca  peptides  were  fully  active,  while 
Val^-Ala^  ranatensin  was  inactive  on  blood  pressure  and  the  uterus. 

IV.  Biochemical  Analysis 

Droplet  countercurrent  chromatography  (DCCC,  earlier  described  by  us)  has 
been  further  developed  with  the  construction  of  an  even  simpler  unit  con- 
sisting of  only  teflon  tubing.   DCCC  has  been  used  for  the  separation  of 
peptides,  proteins,  and  ribonucleic  acids.   Reproducibility  is  excellent, 
elution  volumes  are  accurately  predicted  from  the  partition  coefficient  and 
recovery  is  quantitative  with  as  little  as  1  pg  of  material.   Another  form 
of  countercurrent  chromatography,  gyration  locular  countercurrent  chromatog- 
raphy (invented  in  LTD,  NHLI) ,  has  been  evaluated  and  found  to  have  higher 
resolving  power  and  speed  of  analysis  than  DCCC,  but  lower  capacity  and  more 


i^l't 


'i!i;!l" 


*»«„ 


^ 


i. 


/3/ 


complexity  in  execution. 

The  gas  chromatographic  (GC)  method  for  the  analysis  of  phenylthiohydan- 
toin  derivative  of  amino  acids  has  been  improved  by:   development  of  a  new 
blend  of  stationary  phases  that  gives  better  isolation  and  faster  analysis; 
the  use  of  helium  as  carrier  gas;  and  high  temperature  conditioning  of  the 
columns.   Methylthiohydantoins  were  also  studied  and  separated  with  greater 
resolution  than  hitherto  obtained.   Chemical  ionization  mass  spectrometry 
(Ci;iS,  performed  by  LC,  NHLI)  was  compared  to  GC  for  assay  of  phenylthiohy- 
dantoins;  while  GC  is  simpler,  the  CIMS  method  has  the  potential  of  greater 
sensitivity  and  speed  of  analysis  when  used  with  a  computer  and  automatic 
sample  applicator. 

EXPERIMENTAL  MEDICINE 

I .  Histamine 

A  specific  radioassay  for  histamine  in  urine  has  been  developed.   Normal 
histamine  excretion  averaged  11  yg/24  hr  (n=16)  with  a  range  of  3  to  40; 
these  values  are  lower  than  that  found  with  less  specific  (e.g.  fluorescence) 
assays.   S-^H-histamine  has  been  prepared  enzymatically  and  its  fate  studied 
in  6  patients.   The  3--^H  label  permits  distinguishing  between  the  activity    1 
of  histaminase  (diamine  oxidase)  which  releases  tritiated  water  and  the       < 
activity  of  monoamine  oxidase  which  leads  to  formation  of  tritiated  acidic 
metabolites.   Histaminase  activity  accounted  for  the  metabolism  of  only  5 
to  8%  of  the  injected  histamine.   Histamine-N-methyl-transf erase  and  monoamine 
oxidase  are  the  more  important  enzymes  in  histamine  metabolism,  and  not 
histaminase  as  aad  been  thought  previously.   We  have  found  abnormally  high 
levels  of  serum  histaminase  in  15  of  23  patients  with  medullary  carcinoma  of 
the  thyroid.   High  levels  of  histaminase  activity  are  also  present  invariably 
in  tumor  tissue.   Thus,  serum  histaminase  activity  serves  as  a  useful 
diagnostic  tool  and  tissue  histaminase  activity  serves  as  a  tumor  marker. 
Plasma  histaminase  activity  increases  markedly  after  small  doses  of  intra- 
venous heparin,  the  response  being  similar  to  that  of  plasma  lipoprotein 
lipase  activity.   Four  patients  with  Type  I  hyperlipoproteinemia  and  one 
with  Type  IV  had  subnormal  responses  of  serum  histaminase  activity  to  both 
low  (10  units  heparin/kg)  and  high  (75  units/kg)  doses  of  heparin.   The 
relationship  of  histaminase  to  lipolysis,  if  any,  is  unknown. 

II.  Kallikrein 

Kallikrein  is  an  enzyme  which  controls  the  production  of  bradykinin,  the 
most  potent  vasodilator  known.   An  improved  esterolytic  assay  has  been  used 
to  study  extensively  the  excretion  of  urinary  kallikrein  in  man.   Significant 
differences  from  normal  were  found  in  hypertensive  subjects,  e.g.  a  mean  of 
3.8  enzyme  units  (per  24  hr)  in  male  hypertensives  compared  to  9.0  in  normal 
men.   Two  hypertensive  subjects  had  no  detectable  urinary  kallikrein;  a  possi- 
ble genetic  relationship  is  being  studied. 


/J> 


d 


III.   Cardiovascular  Drugs 

>G<-266  (a  benzyl  ether  of  metaraminol)  has  been  administered  to  7  patients 
with  essential  hypertension.   At  doses  of  30  to  800  mg/day  there  were  slight 
to  moderate  decreases  in  blood  pressure,  most  marked  usually  on  the  early 
morning  standing  pressure.   Three  patients  had  significant  changes  in  SCOT 
and  SGPT  levels  which  were  probably  drug  related.   The  drug  is  not  a  good 
source  of  metaraminol  since  only  1%  of  the  administered  drug  could  be  re- 
covered as  metaraminol  in  the  urine. 


i. 


A  technique  has  been  developed  for  studying  the  reactivity  of  small  strips 
of  human  temporal  artery  _in  vitro.   Tissue  has  been  obtained  from  seven  normo- 
tensive  neurosurgical  patients.   Dose-response  curves,  and  the  agonist- 
antagonist  values  for  alpha  adrenergic  receptors  were  found  to  be  comparable 
to  those  of  animal  blood  vessels.   This  technique  holds  great  promise  for  pre- 
cise evaluation  of  vascular  responsiveness  in  patients  (hypertension,  etc.). 

IV.   Collagen  Metabolism 

Seventy  per  cent  of  54  patients  with  hepatoma  were  found  to  have  abnormally 
high  values  of  serum  protocollagen  proline  hydroxylase  (PPH)  activity.   This 
is  comparable  to  the  occurrence  of  alpha-f etoprotein  in  hepatoma.   Thus, 
serum  PPH  activity  serves  as  a  useful  diagnostic  test  for  hepatoma  and  we 
are  now  studying  it  serially  to  evaluate  the  effects  of  therapy.   Elevated 
serum  PPH  activity  also  seems  to  indicate  liver  cell  injury  and  has  been 
found  to  rise  acutely  in  both  man  and  rabbits  24  hours  after  exposure  to 
fluorinated  hydrocarbon  anesthetics.   In  tissues  PPH  activity  is  a  good  in- 
dicator of  the  rate  of  collagen  synthesis.   We  have  found  that  PPH  activity 
is  markedly  elevated  in  the  skin  of  patients  with  scleroderma  and  in  keloid 
and  hypertrophic  scars.   These  findings  help  to  explain  the  excessive  collagen 
found  in  these  conditions  and  provide  more  insight  into  pathogenesis  and 
treatment. 


NEUROENDOCRINOLOGY 


I.   Taste 

We  have  hypothesized  that  some  form  of  chemical  sieving  controls  the  non- 
specific portion  of  pre-neural  taste  events.   Experiments  demonstrating  in- 
hibitory effects  of  thiol-containing  drugs  and  amino  acids  support  this  con- 
cept.  Locally  applied  proteases  affect  all  taste  qualities  and  suggest  that 
these  enzymes  are  effective  on  protein  of  the  taste  bud  exposed  to  the  oral 
environment.   We  suppose  this  protein  is  part  of  the  membrane  of  the  pre- 
dominant cells  of  the  taste  bud,  the  type  I  receptor  cell.   There  is  also 
a  specific  portion  of  the  pre-neural  events  of  taste  which  relate  to  each 
taste  quality.   The  specific  taste  events  are  most  probably  involved  with 
the  binding  of  tastant  to  the  receptor  membrane. 


E" 


We  have  described  an  equation  by  which  taste  phenomena  occur  in  normal  man 
and  in  patients  with  various  abnormalities  of  adrenal  cortical  function.   This 
equation  is: 

K  (T)" 


■'- '  -"-max   K  (T)^+  1 


^33 


WB^ 


where  I  is  the  intensity  of  tastant  at  concentration  (T)  ,  Ipiax'  ^^^^   maximum 
intensity  at  highest  (T)  and  K  is  a  constant.   The  model  which  is  described 
by  this  equation  would  explain  why  intensity  functions  in  taste  reach  a 
maximum  at  high  tastant  concentrations;  taste  molecules  would  complex  with 
all  available  receptor  molecules  and  introduction  of  more  tastant  could  not 
form  more  complexes.   Deficiency  and  excesses  of  carbohydrate-active  steroids 
in  man  may  affect  the  pre-neural  events  of  taste  by  directly  affecting  the 
binding  constants  by  which  tastant-receptor  molecule  complexes  occur  or  by 
affecting  the  stoichiometry  of  binding.   Our  data  demonstrate  that  excessive 
and  inadequate  amounts  of  these  steroids  decrease  the  binding  constants  for 
all  taste  qualities  because  the  intensity  curves  are  all  shifted  to  higher 
concentrations.   Apparently  there  is  a  concentration  of  steroid  at  which 
intensity  is  maximal  for  a  given  tastant  concentration  and  either  an  excess 
or  a  deficiency  of  steroid  lowers  the  intensity. 

We  have  placed  the  entire  problem  of  taste  acuity  within  the  framework  of 
medical  practice.   The  prevalence  of  the  disease.  Idiopathic  Hypogeusia, 
which  we  have  recently  described,  indicates  the  need  for  an  awareness  and 
an  understanding  of  the  loss  of  taste.   Results  of  a  single  blind  study  in- 
dicate that  oral  administration  of  zinc  is  beneficial  in  patients  with  this 
disorder  although  the  mechanism  is  not  yet  known.   Injection  of  Zn"-*  in  rats 
demonstrated  that  the  tongue  was  one  of  the  most  active  tissues  in  accumula- 
ting label,  behind  bone  and  liver.   Correlating  these  studies  with  those  pre- 
viously noted  in  which  zinc  was  found  in  the  epithelial  layer  of  papillae  by 
laser  microprobe  spectroscopy  suggest  that  taste  bud  bearing  papillae  and 
perhaps  even  taste  buds  themselves  may  avidly  take  up  zinc. 

II.  Olfaction 

We  have  limited  the  role  of  vitam.in  A  in  olfaction  to  one  which  involves 
retinol  binding  protein  (RBP)  and  vitamin  A  alcohol,  separated  abnormalities 
of  vision  from  olfaction  in  hepatitis  and  clarified  the  interrelationship 
between  olfactory  function  and  gonadal  function  in  lower  mammals.  An  ex- 
haustive report  (5,000  citations)  on  the  molecular  basis  of  olfaction  has 
been  prepared  for  the  Office  of  Naval  Research  during  this  past  year;  the 
requirements  which  an  adequate  molecular  theory  of  olfaction  must  satisfy 
have  been  established. 

III.  Trace  Metal  Metabolism 

1.   Metals  and  Hormones.   We  have  found  that  the  A  -3  hydroxysteroid- 
dehydrogenase-isomerase  step  in  steroidogenesis  in  rat  and  cat  requires 
copper.   Tissue  metal  concentrations  are  dependent  on  endogenous  adrenal 
steroids.   The  liver  appears  to  be  an  important  source  of  metals  which  are 
mobilized  by  increased  amounts  of  circulating  adrenocorticosteroids.   Serum 
concentrations  of  copper  and  zinc  are  influenced  by  both  estrogen  and 
progesterone,  but  not  by  LH  or  FSH.   It  is  interesting  that  intrauterine 
devices  coated  with  copper  are  more  effective  in  controlling  fertility  than 
the  same  devices  coated  with  Teflon.   We  have  established  that  serum  con- 
centrations of  copper  (also  ceruloplasmin)  and  zinc  exhibit  circadian  changes 
parallel  in  time  to  secretion  of  Cortisol;  similar  changes  were  observed  for 
urinary  copper  but  not  zinc.   Interestingly,  these  changes  are  not  abolished 

6  /J/ 


by  blocking  endogenous  adrenocortical  or  ACTH  secretion  for  3  days. 

2.  Metal-Protein  Interactions.   Metals  in  urine  are  not  "free"  but  rather 
appear  as  ligands  with  relatively  small  molecular  weight  peptides  or  proteins. 
Two  distinct  metal  containing  proteins  have  been  identified  in  dog  kidney. 
One  (MW  34,000)  contains  equal  amounts  of  copper  and  zinc  and  is  probably 
cytocuprein.   The  other  (MW  10,000)  corresponds  to  metallothionein  in  size 
but  contains  only  copper,  without  zinc  or  cadmium.   Kidney  metallothionein 
may  differ  significantly  in  different  animal  species. 

3.  Metal-Neurotransmitter  Interactions .   Metals  are  generally  inhibitory 
of  the  uptake  of  norepinephrine  (NE)  and  choline  (Ch)  by  brain  synaptosomes 
and  for  the  activity  of  both  Na-K  activated  and  Mg  activated  ATPase  activity. 
The  specific  nature  of  this  inhibition  has  been  systematically  investigated 
and  related  to  the  nature  of  the  metal-neurotransmitter  complex.   However,  two 
metals,  Mn  and  Sn,  appear  to  enhance  NE  uptake.   Sn  acts  to  enhance  Na-K 
ATPase  activity  specifically  (without  an  effect  on  Mg-ATPase  activity)  and 
thereby  the  uptake  of  NE  by  brain  synaptosomes.   These  studies  suggest  a 
physiological  role  for  Sn. 

IV.   Steroid  Hormones  and  Neural  Function 


3. 


Steroid  hormones  are  known  to  influence  sensory  and  central  nervous 
function.   NE  and  Ch  uptake  by  brain  synaptosomes  is  markedly  inhibited  by 
carbohydrate-active  steroids,  apparently  via  inhibition  of  Na-K  activated 
ATPase.   No  effect  of  these  steroids  was  observed  on  Na  or  K  currents  of 
the  isolated  squid  nerve  indicating  their  effect  on  neural  function  is  on 
metabolism  of  nerve  (i.e.  maintaining  membrane  potential);  they  also  appear 
to  affect  synaptic  delay.   In  addition,  these  steroids  appear  to  operate 
through  effects  on  myelin. 


/SS' 


1   1 

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I 


Serial  No.   NHLI-79(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland  E. 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Urinary  Excretion  of  Kallikrein 

Principal  Investigator:   Harry  S.  Margolius,  Ph.  D. ,  M.D. 

Other  Investigators:   Ronald  Geller,  Ph.  D.,  Wybren  DeJong,  M.D. ,  Vida 
Beaven,  Ph.  D.  and  John  Pisano,  Ph.  D. 

Cooperating  Units:   Normal  Volunteer  Office  ,, 

Project  Description:  Ijll"' 


Objectives:   To  identify  the  magnitude  of  urinary  excretion  of  kallikrein  in 
normal  volunteers,  patients  with  various  diseases,  control  and  spontaneously 
hypertensive  rats  (SHR) ,  and  anesthetized  dogs  subjected  to  various  mani- 
pulations and  drugs. 

Methods :   Patients:   24  hour  urine  collections  were  obtained  from  50  normal 
volunteers  and  a  like  number  of  subjects  with  various  diseases.   The  amount 
of  kallikrein  excreted  was  measured  in  two  ways:   1)  using  the  method  of  Beaven, 
et  al  (Clin.  Chim.  Acta,  In  Press)  which  measures  esterolytic  capability  of 
urine  kallikrein  and  2)  using  standard  bioassay  techniques.   Recoveries  of 
pure  standard  human  urinary  kallikrein  were  measured  in  each  urine  sample  and 
ranged  from  70-100%. 

Rats:   24  hour  urine  collections  were  obtained  from  male  Wistar  control, 
Sprague-Dawley  control  and  SHR.   Urine  was  assayed  for  kallikrein  esterase 
activity  and  kallikrein  biologic  activity.   In  addition,  urine  volume,  body 
weight,  urine  protein  excretion  and  blood  pressure  were  also  measured. 

Dogs:   The  effects  of  acute  unilateral  renal  artery  constriction  and 
angiotensin  infusion  or  ureteral  excretion  of  kallikrein  were  measured  in 
20-25  kgm  dogs  anesthetized  with  pentobarbital.   Both  bioassay  and  esterolytic 
activity  were  assessed,  as  well  as  arterial  blood  pressure,  renal  arterial 
blood  flow  .IPC-  u.rine  output. 

Major  Findings:   Patients:   The  24-hour  urinary  excretion  of  kallikrein 
ranged  from  3.76  to  19.38  enzyme  units  (I.E.U.  =  1  pmole  Tolsylarginine 
methylester  hydro lyzed/min)  with  a  mean  of  9.00  in  25  normal  males.   In  23 
normal  females  the  range  was  2,43-19.67  E.U.  with  a  mean  of  6.66  E.U.   In  11 
male  patients  with  essential  hypertension,  the  values  ranged  from  0.00-8.91 
with  a  mean  of  3.78  E.U./24  hour.   In  9  female  patients  with  essential  hyper- 
tention  excretion  ranged  from  0.00-7.52  with  a  mean  of  2.99  E.U./24  hours. 
In  8  patients  with  proven  pheochromocytoma  kallikrein  excretion  varied  from 
3.53-33.27  with  a  mean  of  14.92  E.U./24  hours.   No  consistently  abnormal 


/37 


! 


>.  fO 


Serial  No.   NHLI-79(c) 

findings  have  been  found,  as  yet,  in  patients  with  burns,  carcinoid  syndrome, 
hypoparathyroidism,  or  metastatic  carcinoma.   It  is  of  interest  that  both  the 
parents  of  a  27-year  old  white  female  with  hypertension  of  unknown  etiology, 
and  a  urine  kallikrein  excretion  of  0.00  E.U./24  hours,  also  excrete  ab- 
normally low  amounts  of  kallikrein  in  their  urine. 

Rats:   Urine  kallikrein  excretion  in  control  Wistar  and  spontaneously 
hypertensive  rats  (SHR)  was  examined  in  male  animals  at  7,11,16  and  25  weeks 
of  age.   At  seven  weeks,  levels  of  urinary  kallikrein  were  similar  in  both 
groups;  significant  increases  appeared  in  SH  rats  at  11  weeks  and  were 
greater  at  16  weeks  but  declined  by  35  weeks.   In  all  groups  the  SHR  exhibit 
higher  urinary  excretion  rates  and  higher  blood  pressures  -  while  body  weight, 
urinary  protein  excretion  and  urine  volume  are  similar. 

Dogs:   We  have  been  unable  to  demonstrate  acute  changes  in  kallikrein 
excretion  rates  to  date.   Neither  unilateral  renal  artery  constriction  nor 
angiotensin  infusion  have  produced  significant  or  consistent  changes  in 
urinary  kallikrein  excretion  rate  or  concentration. 

Significance:   The  data  indicate  that  urinary  excretion  of  kallikrein,  an 
enzyme  which  controls  production  of  the  most  potent  vasodilator  substance 
known,  bradykinin,  is  altered  in  hypertension  in  animals  and  man.   This 
finding  clearly  separates  a  special  group  of  patients  with  hypertension. 
This  may  provide  a  starting  point  for  understanding  the  role  of  kinins  in 
pathogenesis  of,  or  compensatory  responses  to  hypertension. 

Proposed  Course  of  Project:   Man:   Enlarge  and  clarify  patient  categories  in 
which  urinary  kallikrein  excretion  is  abnormal,  and  correlate  plasma  kalli- 
krein levels  and  assess  kinin  metabolism  in  hypertension. 

Rats:   Parallel  studies  in  SHR,  renal  and  DOCA-salt  hypertensive  and 
control  animals. 

Publications:   None 


/3e 


A 


Serial  No.   NHLI-80(c) 

1.  Experimental  Therapeutics  Branch 

2 .  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  of  the  Biological  Role  of  Histamine:   Histaminase 
Activity  in  Various  Physiological  and  Pathological  States. 

Previous  Serial  Number:   NHLI-162(c) 

Principal  Investigator:   Stephen  B.  Baylin,  M.D. 

Other  Investigators:   Michael  A.  Beaven,  Ph.D.,  Zdenka  Horakova,  Ph.D.,  n 

Harry  R.  Reiser,  M.D.,  and  Albert  Sjoerdsma,  M.D.,  Ph.D.         «pl''» 

Cooperating  Units:   None  i«i,]|.ir 


Project  Description: 

Objectives:   This  project  is  a  continuation  of  the  clinical  studies  of 
histaminase  activity  in  normal  and  disease  states.   The  studies  included: 
1)  further  investigation  of  the  elevated  histaminase  activity  in  serum  and 
tissues  of  patients  with  medullary  carcinoma  of  the  thyroid;  2)  study  of  the 
appearance  of  histaminase  in  plasma  after  heparin  in  normal  individuals  and 
in  patients  with  hyperlipoproteinemia;  and  3)  evaluation  of  the  effect  of  the 
histaminase  inhibitor,  aminoguanidine,  on  food  consumption. 

There  are  two  conditions  in  normal  individuals  in  which  plasma  or  serum 
histaminase  activity  may  be  high;  pregnancy  and  after  the  administration  of 
heparin.   Previous  studies  from  this  laboratory  have  shown  that  high  serum 
activity  may  be  present  in  some  patients  with  medullary  carcinoma  of  the 
thyroid,  and  that  high  histaminase  activity  is  present  in  this  tumor  tissue. 
The  relationship  between  histaminase  and  this  malignancy  has  been  further 
explored.   Additional  work  is  now  being  done  concerning  the  dynamics  of  plasma 
histaminase  activity  increase  after  parenteral  heparin.   Since  this 
phenomenon  is  not  specific  to  histaminase,  and  other  enzyme  activities  like 
lipoprotein  lipase  also  increase,  studies  of  plasma  histaminase  activity  are 
being  done  in  a  disease  state  involving  a  deficiency  of  lipoprotein  lipase. 
Type  I  hyperlipoproteinemia  (Fredrickson  classification). 

Methods:  The  assay  of  histaminase  activity,  by  the  measurement  of  tritiated 
water  formation  upon  deamination  of  3-  H-histamine,  was  described  in  Project 
Report  NHLI-162(c),  1969-70. 

All  subjects  who  participated  in  the  studies  were  either  patients  or  normal 
volunteers  hospitalized  in  the  Clinical  Center,  NIH.   Serum  samples  from' 
patients  with  medullary  carcinoma  of  the  thyroid  have  been  donated  by  several 
investigators  throughout  the  country,  in  particular,  Dr.  Kenneth  Melvin,  New 

1  nf 


\f 


^ 


Serial  No.   NHLI-80(c) 

England  Medical  Center,  Tufts  University,  Boston,  Mass. 

Major  Findings:   1)  Histaminase  activity  in  medullary  carcinoma  of  the  thyroid. 
The  previous  observations  of  elevated  serum  histaminase  activity  in  patients 
with  medullary  carcinoma  of  the  thyroid  have  been  extended.   In  50  normal 
individuals  the  mean  serum  histaminase  activity  was  1.5  units/ml  serum  (1  unit 
=  1  uumole  histamine  deaminated/hr) .   Of  the  23  patients  with  medullary 
carcinoma  of  the  thyroid,  21  (91%)  had  serum  histaminase  activity  greater 
than  2. A  units/ml  (>  1  standard  deviation  from  the  normal  mean)  and  15  patients 
had  serum  histaminase  activity  greater  than  3.5  units/ml  (>  2  standard  devia- 
tions from  normal).   In  these  15  patients,  serum  histaminase  activity  ranged 
from  3.6  to  90  units/ml. 

The  histaminase  activity  of  normal  human  tissues  and  of  tumor  tissues  from 
patients  with  widely  disseminated  medullary  carcinoma  of  the  thyroid  was 
examined.   Only  kidney  and  intestine  had  histaminase  activity  greater  than 
50  units/g  tissue.   In  contrast,  the  presence  of  microscopic  foci  of  medullary 
carcinoma  tissue  within  an  organ  was  associated  with  much  higher  levels  of 
histaminase  activity  in  that  organ  than  would  normally  be  present.   For 
example,  690  units  of  histaminase/g  was  found  in  ovarian  tissue  containing 
foci  of  medullary  carcinoma  tissue  compared  to  13  units/g  in  normal  ovary. 
In  patients  affected  with  both  medullary  carcinoma  and  pheochromocytoma,  the 
medullary  carcinoma  tissue  could  be  differentiated  from  the  pheochromocytoma 
tissue  by  the  high  histaminase  activity  in  the  medullary  carcinoma  tissue  and 
the  low  activity  (<  20  units/g)  in  the  pheochromocytoma  tissue. 

2.  Rise  in  plasma  histaminase  activity  in  response  to  intravenous  heparin 
administration.   Plasma  histaminase  activity  of  man  has  been  shown  by  other 
investigators  to  increase  after  parenteral  administration  of  heparin.   This 
response  to  heparin  has  been  studied  in  detail  with  the  sensitive  radioassay 
for  histaminase  activity.   At  a  dosage  of  10  units/kg  heparin  i.v. ,  at  10 
minutes  post  injection  plasma  histaminase  activity  increased  1  to  2-fold; 
with  20  units/kg  2.5  to  6-fold;  and  with  75  units/kg    10  to  40-fold.   Peak 
activity  was  reached  on  the  average  at  10  minutes  following  injection  with 
all  doses  used.   Clearance  of  the  enzyme  activity  from  plasma  was  exponential 
after  all  doses,  and  return  to  baseline  levels  was  reached  within  two  hours 
with  the  20  unit/kg  dose. 

The  above  time  course  of  the  increase  in  plasma  histaminase  activity  follow- 
ing heparin  administration  resembled  that  observed  by  other  investigators  for 
lipoprotein  lipase.   The  appearance  of  histaminase  activity  in  plasma  was 
investigated  in  patients  with  Type  I  hyperlipoproteinemia  and  other  hyper- 
lipoproteinemias.  At  a  dose  of  10  units  heparin/kg  4  patients  with  this  dis- 
order showed  at  10  minutes  plasma  histaminase  activities  of  0,  0,  0.2,  and 
0.4  units/ml  plasma.   In  contrast,  the  mean  increase  in  histaminase  activity 
in  21  normal  individuals  was  1.5  +  0.5  units/ml  (+2  SEM) .   With  75  units 
heparin/kg,  3  Type  I  patients  showed  an  increase  of  0.8,  4.4,  and  4.7  units/ 
ml  plasma  whereas  the  increase  in  histaminase  activity  in  10  normals  ranged 
from  6.8  to  49.8  units/ml.   In  2  patients  with  Type  IV  hyperlipoproteinemia 
and  in  5  patients  with  Type  V  disease,  histaminase  activity  increased  from 


-A 


Serial  No. 


NHLI-80(c) 


0.9  to  6.0  units /ml  after  10  units  heparin/kg.   One  patient  with  Type  IV 
disease  failed  to  respond  to  10  units  heparin/kg  and  also  had  a  low  response 
(2.8  units/ml)  to  75  kg.   In  vitro  studies  showed  that  concentrations  of 
protamine  or  heparin  that  were  sufficient  to  inhibit  lipoprotein  lipase  had 
no  effect  on  histaminase  activity;  1  M  sodium  chloride,  inhibited  histaminase 
activity  as  well  as  lipoprotein  lipase  activity. 

3.   In  vivo  studies  with  aminoguanidine.   A  preliminary  evaluation  of  the 
therapeutic  effects  of  the  histaminase  inhibitor,  aminoguanidine,  in  a  patient 
with  widely  disseminated  medullary  thyroid  carcinoma  was  briefly  described 
in  the  previous  report,  NHLI-162(c).   Regression  of  tumor  was  not  achieved, 
but  an  increase  in  appetite,  associated  with  a  gain  in  weight,  was  observed. 
We  have  received  recently  a  report  from  another  center  indicating  that  amino- 
guanidine had  a  beneficial  effect  on  appetite  in  one  patient  with  medullary 
carcinoma  of  the  thyroid.   Thus,  we  decided  to  evaluate  the  effect  of  amino- 
guanidine on  appetite  and  growth  in  animals. 

In  two  separate  experiments  (18  and  34  days  duration)  involving  a  total  of 
16  rats  in  each  of  the  control  and  treatment  groups,  a  statistically  significant 
increase  in  food  consumption  was  seen  with  a  daily  dose  of  50  mg/kg  amino- 
guanidine sulphate  given  perorally  by  stomach  intubation.   A  10  mg/kg  dose  also 
produced  an  average  increase  over  control  values  in  each  study  but  failed  to 
reach  statistically  significant  levels.  Weight  gain  was  significantly  greater 
in  the  50  mg/kg  group  in  the  second  experiment  and  all  treatment  groups  showed 
an  average  weight  increase  greater  than  controls.   In  a  third  experiment, 
involving  15  animals  each  in  a  control  group  and  a  group  receiving  50  mg/kg  of 
aminoguanidine,  food  consumption  and  weight  gain  were  slightly  greater  in  the 
treatment  group  but  the  difference  was  not  statistically  significant.   The 
concentration  of  aminoguanidine  in  tissues  of  chronically  treated  animals  was 
highest  in  intestine  and  kidney  and  was  present  in  lower  amounts  in  salivary 
gland,  liver  and  thymus.   The  drug  was  not  detected  in  brain.   Histaminase 
activity  was  inhibited  by  more  than  90%  in  the  intestine  and  thymus  of  amino- 
guanidine treated  animals. 

Significance:   1)  The  recent  studies  have  confirmed  that  histaminase  activity 
is  an  excellent  biochemical  marker  for  medullary  thyroid  carcinoma  tissue.   A 
relationship  between  detection  of  high  histaminase  activity  and  the  presence 
of  tumor  cells  has  been  further  suggested.   Use  of  measurements  of  histaminase 
activity  in  serum  and  tissues  should  continue  to  prove  useful  as  a  diagnostic 
and  prognostic  tool • in  this  disease. 

2)  The  apparent  abnormality  of  plasma  histaminase  response  to  heparin  in 
patients  with  Type  I  hyperlipoproteinemia  and  possibly  in  the  patients  with 
Type  IV  disease  was  a  surprising  finding.   Such  measurements  may  prove  a  use- 
ful adjunct  to  the  measurements  of  lipoprotein  lipase  in  detection  of  Type  I 
disease. 

3)  The  aminoguanidine  studies  in  rats  have  in  general  paralleled  the  initial 
observations  made  in  patients  receiving  aminoguanidine.   The  apparent  direct 
effect  of  this  drug  on  appetite  might  suggest  utility  for  this  drug  in  treatment 


iifi/ 


"Iffl" 


Serial  No.   NHLI-80(c) 

of  anorectic  patients  with  malignancies  and  other  chronic  disorders.   There 
were  no  apparent  side-effects  from  any  dose  of  aminoguanidine  given. 

Proposed  Course  oi  Project:   1)   Studies  on  histaminase  activity  in  medullary 
carcinoma  of  the  thyroid  will  be  continued  to  examine  further  the  exact 
nature  and  origin  of  the  enzyme.   2)  The  reason  for  the  deficiency  of 
histaminase  release  in  patients  with  Type  I  hyperlipoproteinemia  will  be  in- 
vestigated.  The  role,  if  any,  of  histaminase  in  lipolysis  and  the  possible 
use  of  histaminase  determinations  in  the  diagnosis  of  lipid  disorders  will 
be  explored.   3)  Studies  will  be  continued  in  animals  to  determine  if  there 
is  a  significant  effect  of  aminoguanidine  on  appetite. 

Honors  and  Awards:   None 

Publications: 

1.   Baylin,  S.B.,  Beaven,  M.A. ,  Engelman,  K. ,  and  Sjoerdsma,  A.:  Elevated 
histaminase  activity  in  medullary  carcinoma  of  the  thyroid  gland.   New 
Eng.  J.  Med.   283:  1239-1244,  1970. 


//> 


Serial  No.  NHLI-81(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


Project  Title: 


Studies  on  6-Azuridine  Triacetate  (Azaribine)  in  Animals  and 
Man. 


Previous  Serial  Niomber:   NHLl-160 


Principal  Investigator 
Other  Investigators: 


Harry  R.  Keiser,  M.D. 

Milan  Slavik,  M.D. ,  Ph.D.,  Walter  Lovenberg,  Ph.  D. 
Robert  I.  Henkin,  M.D. ,  Stephen  Baylin,   M.D. ,  and 
Albert  Sjoerdsma,  M.D.,  Ph.  D. 


Cooperating  Units:   None 

Project  Description: 

Objectives :   6-Azuridine  triacetate  is  a  potent  pyrimidine  antimetabolite,  for 
oral  use,  developed  in  Czechoslovakia  and  in  the  United  States.   The  drug 
inhibits  de  novo  pyrimidine  synthesis  by  blockade  of  the  enzyme  orotidine-5- 
phosphate  decarboxylase.   It  has  shown  therapeutic  efficacy  in  patients  with 
psoriasis,  mycosis  fungoides,  rheumatoid  arthritis,  chorioepithelioma,  poly- 
cythemia vera  and  viral  eye  infections.   An  abnormal  pattern  of  amino-aciduria 
was  noted  in  patients  and  animals  receiving  the  drug.   We  showed  previously  in 
animals  that  this  was  not  a  renal  effect  but  that  the  drug  produced  alterations 
in  serum  amino-acids  resembling  three  inborn  metabolic  diseases  in  man;  g-alani- 
nemia,  homocystinuria  and  cystathioninuria.   Our  animal  studies  had  also  in- 
dicated that  Azaribine  administration  produced  changes  in  zinc  metabolism  and 
in  taste  acuity.  Thus,  our  objectives  were  to  administer  Azaribine  to  patients 
with  scleroderma  to  study;   1)  amino-acids  in  blood  and  urine  to  determine  if 
similar  biochemical  alterations  occurred  in  man  as  in  animals;   2)  possible 
beneficial  effects  of  this  drug  in  patients  with  scleroderma;  3)  effects  on 
taste  acuity;  and  4)  effects  on  metabolism  of  copper  and  zinc.   We  also 
sought  information  from  animal  studies  about  the  effects  of  Azaribine  on  the 
activity  of  enzymes  essential  in  the  metabolism  of  sulfhydryl-containing 
amino-acids. 

Methods;   Seven  women,  aged  44  to  62  years,  with  scleroderma  were  given 
Azaribine  (Calbiochem)  orally  in  equal  doses  each  8  hours  for  7  day  periods, 
beginning  with  3  gm  daily  and  increasing  by  3  gm  daily  each  7  day  period  for  a 
total  of  4  periods  or  28  days.   Urine  collections  and  blood  samples  were, 
obtained  during  an  initial  control  period  and  on  the  last  2  days  of  each 
period.   Amino-acids  were  analyzed  using  standard  techniques  for  analysis  of 


k 


/fS 


Serial  No.   NHLI-8l(c) 

physiologic  fluids  on  a  Beckmaii  120C  amino-acid  analyzer.   Analyses  for  copper 
and  zinc  were  done  by  atomic  absorption  spectrophotometry  according  to  the 
method  of  Moret  and  Henkin  (Clin.  Chem. ,  In  press).   Measurements  of  taste 
acuity  were  obtained  by  use  of  techniques  reported  in  detail  previously  by 
Di.  Henkia.   Evaluation  of  the  effects  of  Azaribine  on  scleroderma  included 
measurements  of  range  of  joint  motion,  pulmonary  function  tests,  and  measure- 
ments of  proline  hydroxylase  activity  and  the  physico-chemical  properties  of 
collagen  in  punch  biopsies  of  skin  before  and  at  the  end  of  drug  therapy. 
Eight  rats  were  given  either  Azaribine,  1  gm/kg  daily,  or  water  by  gastric 
gavage  for  10  days  and  the  activity  of  cystathionin-e  synthetase  in  samples 
of  their  liver  was  determined  according  to  the  method  of  Mudd  et  al  (J. B.C. 
242.''-t382,  1965). 

Major  Findings:   After  one  week  of  Azaribine  administration  to  patients  at  a 
dose  of  3  gm  daily  only  minimal  changes  were  found  in  serum  amino-acids. 
After  2  weeks  of  therapy  two  amino-acids  usually  absent  from  human  serum, 
homocystine  and  6-alanine  appeared  and  significantly  increased  levels  of 
threonine,  glycine,  methionine  and  histidine  were  found.   The  concentrations 
of  these  amino-acids  in  serum  increased  with  the  Azaribine  dose  reaching  a 
pleateau  usually  at  9  gm  daily.   At  this  dose  the  level  of  homocystine  was 
41.4  ±  19.1  nM/ral  of  serum  (mean  ±  SEM)  and  that  of  3-alanine  was  23.7  ±  3.6. 
Analysis  of  urine  revealed  the  expected  spill  over  of  these  amino-acids  into 
the  urine.   All  of  the  patients  had  some  anorexia  and  became  slightly  anemic 
at  the  high  dose  of  Azaribine.   Three  of  the  patients  had  definite  subjective 
improvement  and  mild  objective  improvement  in  their  disease.   Median  detection 
thresholds  for  the  four  tastants  prior  to  treatment  were  not  different  from 
normal,  although  median  recognition  thresholds  for  urea  were  slightly  elevated. 
During  drug  therapy  there  was  no  change  in  median  detection  or  recognition 
thresholds  except  for  the  recognition  of  sour,  which  was  significantly  in- 
creased.  No  subjective  changes  in  taste  acuity  were  reported  by  the  patients. 
Azaribine  administration  to  patients  was  associated  with  decreases  in  serum 
copper  from  142  ±6.9  yg/100  m.l  (mean  ±  SEM)  to  121  ±8.8  and  in  serum  zinc 
from  79  ±  5.4  to  60  ±  3.9  pg/100  ml. 

Azaribine  administration  to  rats  produced  an  inhibition  of  42%  in  liver 
cystathionine  synthetase  activity  when  compared  to  controls.  There  was  no 
effect  of  Azaribine  on  this  enzyme  in  in  vitro  studies. 

Significance:   Azaribine  administration  to  man  produces  significant  alterations 
in  the  metabolism  of  a  number  of  different  amiViO -acids.   The  mechanism  for 
these  changes  seems  to  be  an  inhibition  of  enzymes  which  are  dependent  on 
pyrldoxal  phosphate.   The  finding  that  the  pyridoxal  dependent  enzyme  cysta- 
thionine synthetase  is  inhibited  by  Azaribine  in  vivo  but  not  in  vitro 
indicates  that  it  is  not  the  drug  per  se  but  most  probably  a  metabolite  or  an 
induced  metabolic  change  which  is  responsible  for  the  changes  observed.   Of 
major  interest  is  the  finding  that  Azaribine  administration  produces  reversible 
changes  in  serum  and  urine  amino-acids  which  resemble  several  inborn  metabolic 
diseases.   This  may  lead  to  increased  understanding  of  these  disorders. 


/♦'f' 


Serial  No.   NHLI-81(c) 

There  was  some  evidence  of  a  beneficial  effect  from  Azaribine  on  sclero- 
derma.  However,  the  study  was  not  long  enough  to  evaluate  this  aspect 
adequately. 

The  objective  changes  in  taste  acuity  produced  by  Azaribine  may  be  related 
to  the  changes  noted  in  serum  zinc  and  copper.   However,  there  were  no 
subjective  changes  in  taste  acuity  observed  in  any  of  the  patients  and  this 
should  not  be  a  problem  in  patients  treated  with  this  drug. 

Proposed  Course  of  Project:  1.  Administration  of  Azaribine  to  patients  with 
scleroderma  for  periods  of  6  months  or  more  to  evaluate  a  possible  beneficial 
effect. 

2.  Measurement  of  pyridoxal  phosphate  levels  to  determine  if  the  enzyme 
inhibition  produced  by  Azaribine  administration  is  due  to  a  deficiency  of 
this  necessary  cof actor - 

3.  Further  study  of  some  of  the  specific  amino-acid  abnormalities  noted 
in  patients  with  scleroderma. 

Publications:   None 


,f|/ 


!:i:ii: 


I 


//r- 


IBI" 


Serial  No.   NHLI-82(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Metabolism  of  Hydroxyproline  and  Collagen 

Previous  Serial  Number:   NHLI-161(c) 

Principal  Investigator:   Harry  R.  Keiser,  M.D. 

Other  Investigators:   I.  Kelman  Cohen,  M.D. 

Albert  Sjoerdsm.a,  M.D.  ,  Ph.D. 
Charles  Vogel,  M.D. 

Cooperating  Units:   Solid  Tumor  Centre,  Uganda  Cancer  Institute,  Kampala, 
Uganda;  NCI 

Project  Description: 

Objectives:   1.   Development  of  methods  for  studying  collagen  metabolism  in 
man  and  animals.   2.   Application  of  these  methods  to  the  study  of  collagen 
metabolism  in  normal  man,  healing  wounds,  and  the  pathogenesis  of  various 
collagen  diseases.   3.   Study  of  the  effect  of  various  agents  on  wound  healing 
and  the  selection  and  evaluation  of  agents  for  treatment  of  collagen  diseases, 
especially  scleroderma.   4.   Elucidation  of  the  biochemistry  of  keloids  and 
hypertrophic  scars,  two  examples  of  overabundant  wound  healing.   5.  Determina- 
tion of  the  origin  of  protocollagen  proline  hydroxylase  (PPH)  in  the  blood  and 
its  clinical  significance. 

Methods :   The  amino  acid  hydroxyproline  (Hyp)  is  a  unique  marker  for  collagen. 
Methods  for  assaying  Hyp  in  urine,  plasma  and  tissue  have  been  developed  and 
tested  in  this  laboratory  and  described  previously.   Methods  were  also 
developed  for  studying  the  physicochemical  properties  of  collagen  in  tissues, 
for  measurement  of  the  rate  of  collagen  synthesis  in  tissues,  and  for  bio-  - 
chemical  studies  of  standardized  healing  wounds.   These  techniques  have  been 
applied  to  studies  of  wound  healing  in  normal  man  and  laboratory  animals,  to 
studies  of  wound  healing  in  patients  with  scleroderma,  and  to  studies  of  ab- 
normal wound  healing,  i.e.  keloid  and  hypertrophic  scar. 

Recently  we  have  set  up  the  technique  for  measuring  collagenase  activity 
in  tissue  samples  according  to  methods  developed  in  NIDR.   Tissue  samples  are 
grown  for  5  days  in  tissue  culture  with  daily  changes  of  media.   The  harvested 
media  is  pooled  and  its  collagenolytic  activity  measured  by  incubating  it  with 
a  radioactively  labelled  collagen  gel  and  then  measuring  the  radioactivity 
released.   This  method  coupled  with  our  techniques  for  measuring  collagen  syn- 
thesis allows  us  to  evaluate  both  collagen  formation  and  breakdown  in  tissue. 


/¥£ 


Serial  No.   NHLI-82(c) 

We  have  devised  a  simple  technique  for  administering  controlled  concentra- 
tions of  volatile  anesthetic  agents  to  4  rabbits  at  once.   This  is  accomplished 
by  utilizing  a  Foregger  anesthesia  machine  to  vaporize  the  anesthetic,  mix  it 
with  oxygen  or  room  air  and  to  control  all  flow  rates.   The  gas  mixture  is 
delivered  into  a  large  metal  animal  cage  totally  enclosed  in  a  large  poly- 
ethylene bag.   Gas  is  exhausted  from  the  cage  by  a  one  way  valve  made  from  a 
piece  of  Penrose-drain. 

Major  Findings:   Our  studies  of  serum  PPH-activity  have  now  been  extended  to 
over  450  patients  from  both  the  U.S.A.  and  Africa.   Our  normal  level  is  up  to 
570  dpm/ml/hr.   Very  high  levels  (from  1000  to  14,362)  were  found  in  56%  and 
elevated  levels  (>  570)  were  found  in  70%  of  54  patients  with  hepatoma.   As  a 
diagnostic  test  PPH  level  ranks  with  alpha-feto-protein,  another  serum  factor 
found  in  hepatoma,  which  was  positive  in  59%  of  these  patients.   Elevated  ""i) 

levels  of  serum  PPH  were  found  also  in  34%  of  32  patients  with  neoplasms  VL 

metastasizing  to  liver,  in  31%  of  35  patients  with  hepatitis  and  in  10%  of  49  Irti 
patients  with  cirrhosis.  Serum  PPH  levels  were  elevated  in  85%  of  13  newborns  ii;ii;]| 
and  in  all  of  10  patients  after  surgery  under  anesthesia  with  the  fluorinated  ^n',i 
hydrocarbons,  methoxyf lurane  or  halothane.   Serum  PPH  activity  was  not  in-  "'"|] 

creased  in  patients  with  any  of  the  common  "connective  tissue"  diseases,  in  l„„ 

patients  with  tumors  metastasizing  to  bone  or  with  primary  bone  tumors,  Paget 's       "'"'" 
disease  or  after  anesthesia  with  nitrous  oxide.   There  was  a  general  correla-         iii'iii 
tion  between  serum  PPH  levels  and  serum  alkaline  phosphatase  levels  when  eleva- 
tions of  the  latter  were  of  liver  origin  but  not  when  they  were  from  bone.  '|| 

I  ifii  II 
We  have  found  that  rabbits  show  similar  changes  in  serum  PPH  and  other  11(11'' 

enzymes  after  methoxyf  lurane  exposure,  as  does  man.   This  occurs  in  rabbits  '*"" 

at  inspired  gas  concentrations  v/hich  are  so  low  that  the  animals  are  only 
sleepy  and  not  really  anesthetized.   Serum  PPH-activity  rose  rapidly  in  animals 
exposed  to  0.3%  methoxyf lurane  for  2  hours  from  control  levels  of  916  +  366 
dpm/ml/hr  (mean  +  SEM)  to  peak  levels  of  7612  +  1164  (p  <  .01)  at  12  to  28 
hours  after  the  start  of  the  experiment.   It  returned  to  initial  levels  within 
48  to  96  hours.   Serum  levels  of  serxom  glutamic  oxaloacetic  transaminase  (SCOT) 
rose  more  slowly  from  control  levels  of  40+4  S.F.  units  to  peaks  at  48  to  96 
hours  of  402  +  124  (p  <  .02).   A  similar  change  was  noted  in  levels  of  serum 
glutamic  pyruvate  transaminase  (SGPT)  but  there  was  no  change  in  serum  levels 
of  alkaline  phosphatase.   Rabbits  exposed  to  N2O  for  2  hours  showed  no 
significant  changes  in  serum  PPH  activity.   When  cycloheximide  (Calbiochem) , 
a  potent  inhibitor  of  protein  synthesis,  was  given  intraperitoneally  to  rabbits 
in  doses  of  1  mg/kg  B.W.,  it  alone  produced  significant  elevations  in  serum 
activities  of  PPH,  SCOT,  and  SGPT.   When  rabbits  received  both  cycloheximide 
and  methoxyf lurane  the  effects  on  serum  levels  of  PPH,  SGOT,  and  SGPT  were 
additive.   Assay  of  the  livers  of  these  animals  for  PPH-activity  showed  no 
significant  differences  between  any  of  the  4  groups,  i.e.,  controls,  cyclo- 
heximide alone,  methoxyf lurane  alone  and  cycloheximide  plus  methoxyf lurane. 

PPH-activity  in  tissues  is  a  good  indicator  of  the  rate  of  collagen  syn- 
thesis.  We  have  expanded  our  data  on  PPH-activity  in  punch  biopsies  of  skin 
to  include  10  normals,  20  patients  with  sclerodema,  one  patient  with  morphea 

2  /^7 


Serial  No.   NHLI-8  2(c) 


(localized  plaques  of  scleroderma)  and  17  patients  with  miscellaneous,  non- 
connective  tissue  diseases.   PPH-activity  is  significantly  higher  in  apparent- 
ly uninvolved  parasacral  skin  from  patients  with  scleroderma  when  compared  to 
tout  of  normals,  being  364  +  24  vs  180  +  16  dpm/mg  dry  wt/hr  (mean  +  SEM)  . 
PPH-activity  was  even  greater,  626  +  60  in  samples  of  obviously  involved  fore- 
arm skin  of  patients  with  scleroderma.   The  average  levels  of  PPH-activity  in 
both  forearm  and  parasacral  skin  of  patients  with  miscellaneous  diseases  were 
not  significantly  different  from  that  of  controls  but  were  significantly 
different  from  that  ox  patients  with  scleroderma.   PPH-activity  in  skin  biopsies 
from  a  patient  with  morphea  (localized  scleroderma)  was  216  dpm/mg/hr  in  un- 
involved skin,  518  in  the  center  of  a  plaque  of  morphea,  and  848  in  the 
advancing  edge  of  that  plaque. 

The  mean  level  of  PPH-activity  in  keloids  from  8  patients,  4015  +  626 
dpm/mg/hr  (mean  +  SEM),  is  greater  (.02  <  p  <  .05)  than  that  in  hypertrophic 
scars  fiom  6  patients,  1713  +  511.   The  mean  levels  of  PPH-activity  in  both 
are  greater  (p  <  .01)  than  those  in  skin  from  10  normal  subjects,  179  +  16, 
and  in  normal  appearing  scars  from  5  patients,  209  +  35.   There  is  no  signifi- 
cant difference  in  PPH-activity  between  skin  of  normal  subjects  and  normal 
appearing  scars.   However,  mean  PPH-activity  adjacent  to  either  keloid  or 
hypertrophic  scar  in  10  patients,  574  +  112,  is  significantly  less  than  that 
of  both  keloid  and  hypertrophic  scar  (p  <  .01)  yet  significantly  higher  than 
that  of  normal  scar  and  skin  of  normals  (p  <  .01).   While  only  very  preliminary 
results  are  available,  collagenase  activity  in  keloids  and  hypertrophic  scars 
appears  to  be  considerably  elevated  over  that  in  normal  skin. 

Significance:   All  evidence  indicates  that  elevated  PPH-activity  in  serum  is 
of  hepatic  origin.   We  believe  that  serum  PPH-activity  constitutes  a  new  and 
sensitive  indicator  of  liver  cell  injury.   We  have  shown  that  the  rabbit  serves 
as  a  good  animal  model  for  studying  effects  of  anesthetic  agents.   The  use  of 
serum  PPH  levels  adds  a  new  dimension  for  analyzing  the  hepatotoxic  effects 
of  these  agents.   The  fantastically  high  levels  of  PPH-activity  in  serum  of 
patients  with  hepatoma  makes  it  a  useful  tool  in  the  diagnosis  of  this  neoplasm. 
While  relatively  rare  in  the  U.S.,  hepatoma  is  one  of  the  most  common  malignant 
tumors  in  other  countries.   Serum  PPH  levels  may  prove  useful  in  following 
either  chemotherapy  or  surgical  extirpation  of  this  rapidly  fatal  malignancy. 

The  finding  of  elevated  levels  of  PPH-activity  in  the  skin  of  patients 
with  scleroderma  and  morphea  confirms  the  clinical  impression  of  increased 
collagen  synthesis.   This  is  an  important  finding  since  it  occurs  in  both 
obviously  involved  and  apparently  uninvolved  sclerodermatous  skin.   Whether 
this  change  is  primary  or  secondary  in  the  pathogenesis  of  this  disease  remains 
to  be  elucidated. 

The  increased  PPH-activity  in  keloids  and  hypertrophic  scars  indicates 
that  the  rate  of  collagen  synthesis  is  markedly  elevated  in  these  states.   This 
helps  explain  the  excessive  collagen  present  in  these  examples  of  overabundant 
wound  healing.   It  also  provides  a  yardstick  for  evaluating  drug  interventions 
aimed  at  controlling  this  disfiguring  complication. 


f^e 


Serial  No.   NHLI-82(c) 


Proposed  Course  of  Project:   1.   Continued  studies  of  serum  PPH-activity  in 
patients,  especially  as  a  tool  for  evaluating  therapy  in  patients  with  hepatoma. 

2.  Studies  of  other  anesthetic  agents  for  their  effects  on  serum  PPH 
levels  in  man  and  on  rabbits.   Special  attention  will  be  given  the  fluorinated 
hydrocarbons  which  have  shown  sporadic  liver  toxicity  in  man. 

3.  Further  characterization  of  the  biochemistry  of  keloids  and  hyper- 
trophic scars  with  studies  of  the  effects  of  various  therapeutic  interventions 
upon  these  states. 

Honors  and  Awards :   None 


Publications : 

1.   Stein,  H.D. 
wounds.   J. 


and  Keiser,  H.R.:   Collagen  metabolism  in  granulating 
Surg.  Res.   In  press. 


Keiser,  H.R. ,  Stein,  H.D. ,  and  Sjoerdsma,  A.:   Increased  proto- 
collagen  proline  hydroxylase  activity  in  sclerodermatous  skin. 
AMA  Arch.  Dermatol.   In  press. 


/-/f 


Serial  No.     NHLI-83 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Effects  of  Pharmacological  Agents  on  Central  Neurotransmission 
Processes  using  the  Spinal  Cord  as  a  Simple  Model  System. 

Previous  Serial  Number:   None 

Principal  Investigator:   B.  V.  Clineschmidt ,  Ph.  D. 

Other  Investigators:   Albert  Sjoerdsma,  M.D. ,  Ph.  D. 

Project  Description: : 

Objectives:   Anatomical,  biochemical  and  pharmacological  studies  support  the 
view  that  serotonin  (5-HT)  and  norepinephrine  (NE)  serve  as  neurotransmitter 
substances  in  the  spinal  cord  and  brain.   Because  of  its  relative  anatomical 
simplicity  and  the  fact  that  spinal  reflexes  are  stable  over  long  periods  of 
time  which  are  often  required  for  full  development  of  a  drug's  action,  the 
spinal  cord  would  seem  to  provide  a  good  model  for  investigating  the  effects 
of  substances  believed  to  influence  central  monoamine  neurotransmission.   The 
specific  objective  of  the  study  now  in  progress  was  to  determine  if  tricyclic 
antidepressants  affect  the  action  of  5-HT  on  spinal  reflexes. 

Methods:   Spinal  reflex  activities  from  an  Ly  ventral  root  are  amplified  and 
displayed  on  an  oscilloscope.   The  reflex  is  evoked  by  stimulating  the 
corresponding  dorsal  root  in  unanesthetized  spinal  cats.   The  5-HT  precursor 
5-hydroxytryptophan  (5-HTP)  is  injected  to  elevate  the  level  of  5-HT  in  the 
cord.   Imipramine  or  a  related  agent  is  administered  either  before  or  after 
5-HTP. 

Major  Findings:  5-HTP  (50  mg/kg)  produces  a  slowly  developing  increase  in  the 
height  of  the  spinal  monosynaptic  reflex  (MSR) .   Sixty  minutes  after  5-HTP, 
the  height  of  the  MSR  is  250%  of  the  pre-drug  size.   The  tricyclic  antidepres- 
sant imipramine  (5  mg/kg)  causes  a  decrease  of  about  25%  in  the  height  of  the 
MSR.   Following  pretreatment  with  imipramine,  5-HTP  increases  the  height  of 
the  MSR  to  nearly  750%  of  its  control  size  within  sixty  minutes  after  in- 
jection.  In  contrast  to  its  effect  in  untreated  animals,  imipramine  given 
sixty  minutes  after  5-HTP  causes  a  very  rapid  increase  of  more  than  four- 
fold in  the  height  of  the  MSR.  In  cats  with  chronic  spinal  transection  (Tg-Tg), 
5-HTP  produces  its  usual  increase  in  the  monosynaptic  spike  height.   However, 
imipramine  administered  after  5-HTP  causes  a  decrease  instead  of  an  increase 
in  the  height  of  the  MSR. 


/^ 


Serial  No.  NHLI-83 

The  enhancement  of  the  MSR  produced  by  5-HTP  plus  a  tricyclic  agent  is 
antagonized  by  the  5-HT  antagonists  cinanserin  (2.5  mg/kg)  and  methysergide 
(1-2  mg/kg) .   The  interaction  between  5-HTP  and  imipramine  does  not  occur  in 
animals  pretreated  with  RO  4-4602  to  prevent  decarboxylation  of  5-HTP  to  5-HT. 
Other  antidepressants  such  as  amitriptyline  and  desmethylimipramine  have 
qualitatively  the  same  effects  on  the  MSR  as  imipramine  both  in  untreated  and 
5-HTP  pretreated  animals. 

Significance;  The  observations  indicate  that  tricyclic  antidepressants  of  the 
imipramine  type  enhance  a  central  effect  of  5-HT,  i.e.  the  increase  in  the 
MSR  following  5-HTP  administration.   A  similar  effect  on  the  actions  of  5-HT 
at  higher  levels  in  the  C.N.S.  might  be  responsible  for  the  therapeutic  and 
some  of  the  side  effects  of  tricyclic  antidepressants.   Although  much  of  the 
current  thinking  on  the  mechanism  of  the  antidepressant  action  of  these 
compounds  involves  central  noradrenergic  processes,  our  results  show  that 
central  actions  of  5-HT  must  also  be  considered. 

5-HT  has  been  shown  to  have  a  beneficial  effect  on  hypotonia  in  Down's 
syndrome.   Its  effectiveness  here  might  depend  upon  influencing  spinal 
reflex  activity  in  which  case  the  combination  of  5-HTP  and  a  tricyclic  agent 
could  prove  superior  to  giving  5-HTP  by  itself. 

Proposed  Course  of  Project:  1)   5-HT  levels.   To  this  point,  we  have  assumed 
that  5-HTP  administration  increases  spinal  cord  5-HT.   5-HT  levels  in  lumbo- 
sacral cord  will  be  assayed  before  and  after  5-HTP.   2)   Mechanism  of 
interaction.   Tricyclic  antidepressants  inhibit  uptake  of  5-HT  into  monoamine 
nerve  terminals.   We  plan  to  determine  if  the  order  of  potency  of  tricyclic 
compounds  in  inhibiting  5-HT  uptake  corresponds  to  their  order  of  potency 
in  enhancing  the  5-HTP-induced  increase  in  the  spinal  MSR.   3)  Catecholamine 
precursors.   The  NE  and  dopamine  precursor  L^-dihydroxyphenylalanine  (DOPA) 
affects  spinal  reflexes.   The  combination  of  DOPA  with  tricyclic  agents  will 
be  studied.   Also,  the  effect  of  imipramine  on  the  spinal  actions  of  di- 
hydroxyphenylserine  (precursor  of  NE  only)  and  alpha-methyldopa  may  be 
investigated. 

Publications:   None 


/r/ 


Serial  No.   NHLI-84(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  y.edicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Clinical  Investigation  of  Cardiovascular  Drugs 

Previous  Serial  Number:   NHLI-158(c) 

Principal  Investigator:   David  Horwitz,  M.D. 

Other  Investigators:   Albert  Sjoerdsma,  M.D.,  Ph.D.,  Griff  T.  Ross,  M.D. , 

Ph.D.,  B.  Van  Clineschmidt,  Ph.D.,  Harry  S.  Margolius, 
M.D.,  Ph.D.,  and  Richard  Wyatt,  M.D. 

Cooperating  Units:   Endocrinology  Service  Unit,  Reproduction  Research  Branch, 
National  Institute  of  Child  Health  and  Human  Development; 
Adult  Psychiatry  Branch,  National  Institute  of  Mental 
Health. 

Project  Description: 

A.   Influence  of  Various  Central  Nervous  System  Drugs  on  Plasma 
Gonadotropin  Levels 

Objectives:   In  animals  it  has  been  possible  to  demonstrate  the  influence  of 
the  cenLral  nervous  system  on  gonadal  function  and  to  modify  ovulation  by  the 
use  of  drugs  which  alter  the  levels  of  amine  neuromediators  in  the  central 
nervous  system.   It  is  not  known,  however,  whether  similar  effects  can  be 
obtained  in  man  or  which  amine  mediator  plays  a  predominant  role.   Our  studies 
have  been  concerned  with  the  influence  of  drugs  which  alter  central  nervous 
system  amines  on  the  blood  levels  of  pituitary  gonadotropins  as  measured  by 
radioimmunoassay.   Initial  observations  were  made  in  postmenopausal  women  or 
in  males  because  they  have  relatively  stable  levels  of  gonadotropins.   Such 
studies,  utilizing  the  drugs  parachlorophenylalanine,  reserpine,  pargyliue, 
desipramine,  L-dopa,  methyldopa,  a-methyl-5-hydroxytryptophan,  a-methyl-par.-i- 
tyrosine  and  a-methylphenylalanine,  were  inconclusive. 

We  have  since  attempted  observations  in  cycling  premenopausal  women.   Be- 
cause the  critical  peaks  of  luteinizing  hormone  (LH)  and  follicle-stimulating 
hormone  (FSH)  which  accompany  ovulation  are  present  only  during  one  to  two 
days  per  cycle,  it  has  been  necessary  to  obtain  daily  samples  over  many  months. 

Major  Findings:   Results  are  available  from  three  women,  each  followed  through 
six  consecutive  months  during  which  methyldopa  therapy  was  alternated  with  a 
control  drug.   In  each  instance  there  was  pronounced  reduction  of  cyclic  peaks 
of  LH  during  therapy  with  methyldopa  and  lesser  effects  on  FSH  levels. 


/ra- 


Serial  No.   NHLI-84(c) 


Significance:   This  is  the  first  demonstration  that  gonadotropin  levels  can 
be  altered  in  man  by  drugs  which  are  not  hormones.   It  is  an  initial  step 
toward  the  goals  of  defining  the  role  of  the  central  nervous  system  in  the 
control  of  gonadotropins  in  man  and  of  developing  new  methods  for  altering 
fertility. 

Proposed  Course  of  Project:   Studies  will  be  pursued  in  additional  subjects 
and  with  other  agents.   Simultaneous  measurements  of  plasma  estrogens  and 
progesterone  will  be  made  to  determine  how  feedback  controls  have  been  altered. 

B.  Studies  in  Vascular  Headache 

Objectives:   A  role  of  serotonin  in  migraine  headaches  has  been  suggested  by  ""iim 

reports  of  changes  in  plasma  levels  of  serotonin  and  the  urinary  excretion  of  ll|!],i» 

its  degradation  product,  5-HIAA,  during  attacks  of  migraine;  the  clinical  h'"!'* 

usefulness  of  the  serotonin  antagonists  cyproheptadine  and  methysergide  makes  l|il!|, 

the  relationship  more  plausible.   We  therefore  undertook  short-term  observa-  "ff' 

tions  of  the  effects  of  an  inhibitor  of  serotonin  synthesis,  parachlorophenyl-  ""|I' 

alanine  (PCPA) ,  in  hospitalized  patients  with  migraine;  the  drug  was  well-  ,Lu" 
tolerated  and  appeared  to  be  beneficial,  but  results  were  inconclusive.   As  a 
consequence  a  controlled  long-term  outpatient  study  of  the  effects  of  PCPA  in 
migraine  was  begun. 

Major  Findings:   Eight  patients  with  frequent  episodes  of  typical  migraine, 
which  responded  poorly  to  conventional  medications,  have  so  far  been  entered 
into  a  6  month  double-blind,  cross-over  study  comparing  the  effects  of  PCPA 
with  those  of  a  placebo.   Analysis  of  therapeutic  effects  is  not  yet  available. 
Two  of  the  eight,  each  with  a  history  of  allergy,  developed  pronovinced  drug- 
related  reversible  eosinophilia  and  were  withdrawn  from  the  study  without 
adverse  effects. 

Proposed  Course  of  Project:   The  study  will  be  pursued  in  additional  subjects. 

C.  Role  of  Brain  Amines  in  Sleep 

Objectives:   Previous  studies  in  man  have  shown  that  the  rapid  eye  movement 
(REM)  phase  of  sleep  is  decreased  when  serotonin  synthesis  is  reduced  by  the 
drug,  parachlorophenylalanine  (PCPA).   REM  sleep  is  restored  in  such  subjects 
when  the  block  in  serotonin  synthesis  is  overcome  by  giving  the  serotonin 
precursor,  5-hydroxy tryptophan.   Similar  results  have  been  obtained  in  animals 
with  a  variety  of  pharmacologic  manipulations  which  alter  serotonin  levels. 
In  animals  it  has  been  possible  to  demonstrate  monophasic  sharp  increases  in 
the  electrical  activity  of  the  pontine  oculomotor  nuclei,  lateral  geniculate 
nuclei  and  visual  cortices  beginning  shortly  before  and  persisting  through  the 
REM  phase  of  sleep;  this  electrical  activity  designated  as  pontine-geniculate- 
occipital  (PGO)  spikes,  has  been  hypothesized  to  be  critically  necessary  for 
REM  sleep  and  is  grossly  disturbed  by  treatment  of  animals  with  PCPA.   A  possible 

2  /SS 


Serial  No.    NHLI-84(c) 

analogue  to  PGO  spikes  designated  as  phasic  integrated  potentials  (PIP)  is 
under  study  in  patients  during  normal  sleep  and  during  treatment  with  PCPA. 

Major  Findings:   Studies  have  been  completed  in  two  subjects  with  migraine 
who  received  PCPA  as  possible  therapy.   During  treatment  with  PCPA  there  was 
a  decrease  in  PIP  counts  in  REM  sleep  and  an  increase  during  non-REM  sleep, 
yielding  a  four-fold  rise  in  the  non-REM  to  REM  ratio  of  PIP  activity.   Such 
changes  resemble  those  of  PGO  spikes  in  animals  receiving  PCPA,  supporting 
the  hypothesis  that  PIP  waves  in  humans  are  analogous  to  PGO  waves  in  cats. 

Proposed  Course  of  Project:   Studies  are  being  pursued  in  additional  subjects. 

D.  Reactivity  of  Human  Temporal  Arteries 

Objectives:   Increased  vascular  reactivity  has  been  reported  in  hypertensive 
humans  by  studies  utilizing  a  variety  of  techniques.   Interpretation  has  been 
difficult  because  of  differing  levels  of  initial  tone  or  reflex  feedback  and 
differing  wall-lumen  ratios  in  hypertensive  and  normotensive  subjects.   In  an 
attempt  to  investigate  this  problem  in  a  simplified  system,  we  initiated  a 
study  of  the  _in  vitro  responses  of  helical  strips  prepared  from  biopsied  human 
temporal  arteries.   This  technique  had  previously  been  used  in  this  department 
to  study  aortic  strips  from  genetic  hypertensive  and  normotensive  rats;  there 
were  no  systematic  differences  in  responses  to  the  sympathetic  neuromediator 
norepinephrine  nor  in  the  dissociation  characteristics  of  alpha  receptors  in 
the  two  groups. 

Major  Findings:   Studies  of  temporal  arteries  from  seven  normotensive  neuro- 
surgical patients  have  been  completed.   Optimal  tension  varied  from  1200  to 
5000  mg  and  was  achieved  when  strips  were  stretched  35  to  45%  above  initial 
lengths.   Average  concentrations  eliciting  half-maximum  responses  were 
3.3  X  10~5  mg/ml  for  norepinephrine  and  2.6  x  10-4  mg/ml  for  phenylephrine. 
The  negative  log  of  the  molar  concentration  of  phentolamine  which  reduced  the 
effect  of  a  double  dose  of  phenylephrine  to  that  of  a  single  dose  (pA„)  was 
7.8;  this  is  comparable  to  values  obtained  in  studies  on  alpha  adrenergic 
receptors  in  blood  vessels  of  animals. 

Significance:   Development  of  this  technique  promises  to  permit  resolution  of 
the  question  of  hypersensitivity  of  vascular  smooth  muscle  in  hypertensive 
humans  and  will  permit  comparison  of  receptors  in  normotensive  and  hypertensive 
subjects. 

Proposed  Course  of  Project:   The  observations  on  normotensives  will  be  expanded 
and  comparison  made  with  vessels  from  hypertensive  subjects. 

E.  Cardiovascular  Effects  of  L-dopa 

Objectives:   Blood  pressure  reduction  has  been  widely  reported  as  a  side-effect 
when  patients  with  Parkinson's  disease  are  treate;d  with  large  doses  of  L-dopa. 


Serial  No.   NHLI-84(c) 


Observations  in  animals  have  suggested  that  L-dopa  and  its  analogue,  the 
antihypertensive  agent  methyldopa,  exert  their  hypotensive  effects  via  the 
central  nervous  system  through  conversion  to  amines.   The  compound,  L-a-methyl~ 
a-hydrazino-3,4-dihydroxyphenylpropionic  acid  (MK-486) ,  blocks  the  decarboxyla- 
tion of  L-dopa  and  methyldopa  to  amine  products  in  peripheral  tissues  but  does 
not  enter  the  central  nervous  system.   In  so  doing  it  has  been  reported  to 
promote  the  accumulation  of  the  L-dopa-derived  amine,  dopamine,  in  the  brain 
and  to  reduce  the  dose  requirements  for  treating  Parkinson's  disease.   Use  of 
MK-486  thus  offers  an  opportunity  to  distinguish  between  central  and  peripheral 
loci  for  the  hypotensive  effects  of  L-dopa  and  methyldopa  in  patients. 

Major  Findings:   Four  patients  with  mild  hypertension  were  studied  while  re- 
ceiving L-dopa  alone  in  daily  doses  of  2.0  gm  and  also  after  the  addition  of 
MK-486  in  a  daily  dose  of  150  mg  (one  subject)  or  500  mg  (3  subjects).   Two 
showed  low  grade  blood  pressure  reduction  while  receiving  L-dopa  alone;  there 
was  no  evidence  of  enhancement  of  hypotensive  effects  after  the  addition  of 
MK-486. 

Proposed  Course  of  Project:   Further  studies  are  planned  using  higher  doses 
of  L-dopa  and  MK-486  and  combining  MK-486  with  methyldopa. 

Honors  and  Awards:   None 

Publications:   None 


,iii  / 


m  « 


/s-r 


Serial  No.   NHLI-85(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  the  Biological  Role  of  Histamine:  Examination  of 
Histaminase  Activity  in  Rat  Tissues  and  Its  Release  by 
Heparin 

Previous  Serial  Number:   NHLI-153 

Principal  Investigator:   Michael  A.  Beaven,  Ph.D. 

Other  Investigators:   Stephen  B.  Baylin,  M.D. 

Wybren  De  Jong,  M.D.,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   As  a  part  of  the  study  of  the  metabolism  of  histamine  in  the 
body,  the  enzyme  histaminase  was  examined  in  detail.   Histaminase  is  thought 
to  be  identical  to  the  enzyme  diamine  oxidase  by  some  workers  while  the 
enzymes  are  thought  to  be  separate  entities  by  other  workers.   The  objectives 
of  this  work  were  (1)  to  determine  whether  histaminase,  as  measured  by  the 
release  of  tritium  from  6--^H-histamine  (see  Project  Report  NHLI-153,  1969-70), 
was  the  same  enzyme  as  that  which  deaminates  putrescine  (diamine  oxidase)  and 
whether  the  assays  of  the  two  enzyme  activities  were  interchangeable;  (2)  to 
establish  the  distribution  of  histaminase  in  tissues;  and  (3)  to  study  the 
release  of  histaminase  from  tissues  by  heparin.   Heparin  is  known  to  raise 
histaminase  levels  in  plasma  of  experimental  animals  and  man;  the  histaminase 
is  thought  to  be  released  from  liver  in  guinea  pig  and  from  intestine  in  rat. 
The  present  studies  in  rat  examined  the  release  of  histaminase  by  heparin  in 
a  number  of  tissues  and  the  kinetics  of  this  release.   These  studies  were  in- 
tended to  complement  the  investigation  of  the  release  of  histaminase  by 
heparin  in  man. 

Methods:   Histaminase  activity  was  determined  by  measurement  of  the  release 
of  tritium  from  e--^H-histamine  as  described  in  Project  Report  NHLI-153,  1969- 
70.   Diamine  oxidase  activity  was  determined  by  a  modification  of  the  method 
of  Okujama  and  Kobayashi  (Arch.  Biochem.  Biophys.  95:  242,  1961)  with  6--^H- 
putrescine  as  substrate. 


Major  Findings: 

1) 

Biochemical 

studies  of  histaminase. 

The 

data 

showed 

that 

histaminase 

activity 

was  associated  with  diamine  oxidase  activity 

in  all 

the 

tissues  stud 

ied. 

Th 

e  deamination  of  6-^H-histamine  and 

6-%- 

-putrescine 

i.7as 

inhibited  to 

an 

equa 
_3h- 

1  extent  by 

aminoguanidine  (Irrv  2.5 

x  10- 

-8  M) 

The 

deamination 

of  e 

histamine  was  inhibited  by  putrescine  and  the 

deamination 

1 

^» 

Serial  No.   NHLI-85(c) 


of  g-  H-putrescine  was  inhibited  by  histamine.   Kinetic  studies  showed  that 
the  inhibition  was  competitive.   The  studies  also  indicated  that  the  assay  of 
histamlnase  activity  was  interchangeable  with  the  assay  of  diamine  oxidase 
activity  although  the  histaminase  assay  was  more  sensitive  and  reproducible. 


There  were  dissimilarities  in  the  two  enzyme  activities.   The  effects  of 
pH  and  substrate  concentrations  on  the  deamination  of  g-^H-histamine  and  6- 
%-putrescine  were  different.   Also,  tritium  was  released  from  3--^H-histamine 
but  not  from  B-'^H-putrescine.   The  possibility  that  6--^H-histamine  and  B-^H- 
putrescine  were  deaminated  by  the  same  enzyme  but  by  different  mechanisms 
was  reviewed  in  Project  Report  NHLI-153,  1969-70. 

Other  studies  showed  that  histaminase  could  be  differentiated  from  plasma 
amine  oxidase  (benzylamine  oxidase)  in  human  plasma  and  from  purified  beef 
plasma  amine  oxidase  by  g-^H-histamine  since  this  amine  was  not  a  substrate 
for  either  plasma  amine  oxidase. 

2)  Distribution  of  histaminase  activity  in  tissues.   Levels  of  histaminase 
activity  in  rat  tissues  ranged  from  0  to  8000  yymoles  histamine  deaminated/g 
tissue/hr  as  follows: 


Rat  Tissue 


Histaminase  Activity* 


G.I.  tract;  appendix 

8000 

small  intestine 

3300-6200+ 

stomach  (glandular) 

250 

Thymus 

500-1100+ 

Adrenals 

150-350 

Pancreas 

41 

Lung 

38 

Bone  marrow 

28 

Spleen 

27 

Liver,  kidney,  prostate 

7 

Heart 

5 

Plasma 

2.8-4. 6+ 

Submaxillary  gland,  skin 

testicles,  brain 


0 


*ypmoles  histamine  deaminated/g  tissue  or  ml  plasma/hr  , 

mean  value  from  several  experiments. 
+These  values  indicate  the  range  of  values  from  different 

experiments. 

The  high  levels  of  histaminase  activity  in  intestinal  tract,  thymus  and 
adrenals  were  found  in  both  normal  and  germ-free  rats.   The  histaminase  levels 
in  thjmius  diminished  with  age  and  were  decreased  by  treatment  with 
dexamethasone  (see  below) .   Interesting  differences  in  histaminase  activity 
were  observed  in  thymus  of  normotensive  Wistar  and  spontaneously  hypertensive 
(SH)  Wistar  rats  as  seen  below: 


/s-7 


Serial  No.   NHLI-85(c) 

Histaminase  Activity  in  Thymus* 


Rat 


Wistar,  normal 
Wistar,  SH 
Sprague-Dawley , 

control 
Sprague-Dawley , 

with  dexamethasone"'' 


Age: 


6  wk 


497+26(5) 
1260+89(8) 


S  wk 


10  wk 


20  wk 


456+38(11)    438   (2)   307+49(6) 
1100+54(16)   1077+99(5)   299+32(6) 

960+160(5) 

263H-45(5) 


*MMmoles  histamine  deaminated/g  tissue/hr,  mean  value  + 
S.E.  (.number  of  animals). 

'^'These  animals  were  treated  with  dexamethasone,  500  ug/kg, 
for  3  days. 

In  guinea  pigs,  high  levels  of  histaminase  activity  were  found  in  lymph  nodes, 
spleen  and  thymus.   Histaminase  activity  was  also  found  in  lymphocytes  isolated 
from  spleen  and  bone  marrow  but  not  in  circulating  lymphocytes  of  guinea  pigs. 

3)  Release  of  tissue  histaminase  by  heparin.   Heparin  reduced  histaminase 
activity  in  rat  intestine  and  adrenals  but  not  in  thymus.   The  extent  of 
the  depletion  was  dependent  upon  the  dose  of  heparin.   Doses  of  4,000, 
20,000  and  40,000  units/kg,  i.v.,  reduced  histaminase  activity  in  intestine 
by  40%,  63%  and  76%,  respectively,  with  maximum  depletion  15  to  30  min  after 
the  injection  of  heparin.   Maximum  levels  of  histaminase  activity  in  plasma, 
200,  585  and  538  ypmoles  histamine  deaminated/ml/hr ,  were  seen  15  to  30  min 
after  injection  of  4,000,  20,000  and  40,000  units  heparin/kg.   Histaminase 
activity  in  plasma  declined  to  near  normal,  2-4  yymoles/ml/hr ,  by  5  hr.   At 
this  time,  the  enzyme  activity  in  the  intestine  began  to  rise  and  by  10  hr 
the  activity  was  back  to  normal,  4,200  ppmole/g/hr.   The  data  showed  that 
a)  histaminase  in  intestine  was  almost  completely  depleted  by  heparin;  b)  the 
enzyme  in  intestine  was  synthesized  and  was  turned  over  at  a  rapid  rate; 
c)  histaminase  was  removed  from  plasma  within  5  hr;  and  d)  histaminase  in 
thymus  was  impervious  to  the  depleting  action  of  heparin,  which  suggested 
that  the  enzyme  in  thymus  may  be  different  from  that  in  intestine. 

Significance:   The  studies  have  shown  that  histaminase  and  diamine  oxidase 
are  similar  or  identical  enzymes  in  various  tissues  and  that  the  assays  of 
these  two  enzyme  activities  are  interchangeable.   This  finding  has  enabled 
us  to  correlate  the  data  obtained  with  the  assay  of  histaminase  activity, 
which  is  the  more  sensitive  and  reproducible  assay,  with  the  previously 
published  findings  with  the  assay  of  diamine  oxidase  activity.   The  distribu- 
tion of  histaminase  activity  in  rat  tissues  indicated  that  high  histaminase 
activity  was  present  in  thymus,  which  has  not  been  previously  described  as  a 
source  of  histaminase,  as  well  as  in  adrenals  and  in  intestine.   The  studies 
of  the  heparin  release  of  histaminase  have  confirmed  that  in  rat  the  intestine 
is  the  primary  source  of  histaminase;  the  data  have  shown  that  the  intestine 


/'^ 


Serial  No.   NHLI-85(c) 


can  be  almost  completely  depleted  of  histaminase  and  that  the  enzyme  is 
turned  over  at  a  rapid  rate.   A  rapid  turnover  of  histaminase  has  also  been 
noted  in  medullary  carcinoma  of  the  thyroid  tissue.   The  experiments  with 
heparin  in  animals  have  provided  necessary  information  for  the  interpreta- 
tion of  studies  of  heparin  release  of  histaminase  in  man. 

Proposed  Course  of  Project:   The  significance  of  histaminase  in  th3anus  and 
lymphatic  tissue  will  be  examined,  for  example,  by  determining  the  factors 
which  influence  the  levels  of  histaminase  in  these  tissues.   The  animal 
studies  with  heparin  have  raised  a  number  of  important  questions  concerning 
the  studies  of  histaminase  release  by  heparin  in  man.   One  aspect  that  will 
be  examined  is  whether  the  defect  in  release  of  histaminase  by  heparin  in 
Type  I  hyperlipoproteinemia  is  due  to  an  absence  of  enzyme  in  the  intestine 
or  due  to  a  defect  in  release  of  the  enzyme  from  intestine.   The  mechanism 
of  the  heparin  release  of  histaminase  will  be  studied  in  animals  to  provide 
a  basis  for  the  further  study  of  the  action  of  heparin  on  histaminase  in  man. 


'  J'" 


Honors  and  Awards :   None 


Publications: 


1.  Beaven,  M.A.  and  Jacobsen,  S.:  A  new  assay  for  histaminase  activity: 
Measurement  of  tritiated  water  from  6(side  chain  label) -H^-histamine. 
J.  Pharmacol.  Exptl.  Therap.   176:  52-64,  1971. 


/Sf 


Serial  No.   NHLI-86(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  Catecholamines  in  Human  Disease. 

Previous  Serial  Number:   NHLI-163(c) 

Principal  Investigator:   Harry  R.  Keiser,  M.D. 

Other  Investigators:   Milan  Slavik,  M.D.,  Ph.D.,  Stephen  Baylin,  M.D.  , 

David  Horwitz,  M.D.,  and  Albert  Sjoerdsma,  M.D. ,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   During  the  past  year  our  research  in  the  area  of  catecholamine 
metabolism  has  centered  on  evaluation  and  study  of  the  clinical  pharmacology 
of  a  new  antihypertensive  agent,  MK-266.   This  drug  is  the  chlorobenzyl  ether 
of  metaraminol.   Upon  metabolism,  metaraminol  is  released  and  should  function 
as  a  false  neurotransmitter  in  sympathetic  nerves  and  thereby  lower  blood 
pressure. 

Methods :   Catecholamines  in  blood  and  urine  and  catecholamine  metabolites  in 
urine  were  measured  by  methods  developed  and  used  in  this  laboratory  for  years. 
The  determination  of  metaraminol  in  urine  of  patients  receiving  MK-266  was 
performed  by  a  modification  of  the  method  of  Pugsley  and  Johnson  (J.  Pharm. 
Pharmacol.,  1968)  as  we  have  reported  previously. 

Major  Findings:   MK-266  has  now  been  administered  to  7  patients  with  essential 
hypertension.   In  the  first  3  patients  single  oral  doses  as  large  as  400  mg 
had  no  effect  on  blood  pressure.   Six  of  the  patients  have  received  doses  of 
300  to  800  mg/day  for  periods  of  4  to  8  days.   Three  of  these  patients  had 
slight  to  moderate  decreases  in  blood  pressure,  most  marked  usually  in  the 
standing  position.   Three  patients  developed  significant  changes  in  SCOT  and 
SGPT  levels  during  drug  administration  and  one  of  these  patients  had  con- 
comitant nausea  and  emesis.   These  symptoms  disappeared  and  abnormal  laboratory 
tests  returned  to  normal  when  the  drug  was  stopped.   One  other  patient  had  a 
maculcpapular  skin  eruption  on  the  second  day  of  MK~266  administration.   This 
disappeared  within  hours  when  the  drug  was  stopped  and  did  not  return  when  the 
drug  was  restarted.   No  central  nervous  system  symptoms  were  observed  in  any 
patients.   We  have  been  able  to  recover  only  about  1%  of  the  administered  drug 
as  metaraminol  in  the  urine  of  5  patients  tested. 


fte 


^ 


Serial  No. 


NHLI-86(c) 


Significance:   MK-266  is  an  ether  analog  of  metaraminol  which  is  released 
slowly  on  metabolism.   Metaraminol  has  no  direct  pressor  activity  of  its  own 
bat  is  capable  of  releasing  norepinephrine.   Previous  studies  have  indicated 
that  continuous  intravenous  infusions  of  metaraminol  lead  to  a  lowering  of 
blood  pressure.   MK-266  holds  promise  of  permitting  the  oral  administration 
of  metaraminol.   An  antihypertensive  effect  would  provide  evidence  in  favor 
of  the  "false  neurotransmitter  concept".   We  have  seen  some  blood  pressure 
effect  from  MK-266  but  because  of  unexpected  liver  .toxicity  we  have  not  been 
able  to  administer  large  doses  of  drug.   Larger  doses  of  drug  are  desirable 
since  only  1%  can  be  recovered  as  the  presumably  active  metabolite  metaraminol. 
Thus,  the  clinical  usefulness  of  the  drug  is  still  in  doubt,  and  the  validity 
of  the  false  neurotransmitter  concept  is  unaffected  by  these  studies. 

Proposed  Course  of  Project:   MK-266  will  be  administered  carefully  to  a  few 
more  hypertensive  patients  to  allow  more  complete  elucidation  of  its  potential 
usefulness. 

2.   A  careful  search  will  be  made  for  other  metabolites  of  this  drug  in 
urine  of  patients. 

Honors  and  Awards:   None 

Publications:   None 


45/ 


Serial  No.  NHLI-87(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  19  71 


Project  Title:   Studies  on  the  Biological  Role  of  Histamine:  Metabolism  of 
6-^H-histamine  in  Man. 

Previous  Serial  No.:   None 

Principal  Investigator:   Michael  A.  Beaven,  Ph.  D.  and  Stephen  B.  Baylin,  M.D. 

Other  Investigators:   Harry  R.  Keiser,  M.D.  and  Albert  Sjoerdsma,  M.D. ,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   As  part  of  the  study  on  the  role  of  histamine,  the  metabolism  of 
histamine  in  man  was  investigated.   This  work  was  undertaken  to  determine  the 
relative  importance  of  the  various  enzymes  known  to  metabolize  histamine. 
With  this  information  it  is  hoped  that  more  effective  agents  can  be  chosen  to 
block  the  metabolism  of  histamine  in  man.   A  major  deficiency  in  our  knowledge 
is  whether  histaminase  (diamine  oxidase)  or  monoamine  oxidase  is  the  more 
important  enzyme  in  the  metabolism  of  histamine.   The  present  studies  utilize 
the  ability  to  distinguish  between  histaminase  and  monoamine  oxidase  activi- 
ties by   1)  the  release  of  tritiated  water  from  3-  H-histamine  by  histaminase 
and  2)  the  formation  of  tritiated  acidic  metabolites  of  g-'%-histamine  by 
monoamine  oxidase. 

Methods:   3-H-Histamine  was  prepared  by  decarboxylation  of  6-%-L-histidine 
with  bacterial  histidine  decarboxylase.   The  preparation  was  purified  in  the 
laboratory  and  solutions  for  injection  were  prepared  by  the  Pharmacy  Depart- 
ment.  The  B-'^H-histamine  was  administered  i.v.  and  urine  was  collected  over 
6-hour  periods  for  the  first  24  hours  and  for  24-hour  periods  on  the  2nd  and 
3rd  days  after  injection  of  the  labeled  amine.   Aliquots  were  removed  from 
each  collection  and  frozen.   Urinary  metabolites  of  6--^H-histamine  were 
determined  by  the  following  procedures:   1)  by  ion  exchange  chromatography  to 
separate  acidic  and  basic  metabolites;  2)  by  addition  of  unlabeled  metabolites 
or  histamine  as  carriers  and  the  crystallization  of  various  derivatives  of 
these  metabolites;   3)  by  solvent  extraction  techniques;   4)  by  thin  layer 
and  paper  chromatography;  and  5)  by  total  tritium  and  volatile  tritium 
(tritiated  water)  determinations.   Various  samples  of  authentic  histamine 
metabolites  were  obtained  from  commercial  sources  and  from  Dr.  H.  Tabor  and 
Dr.  H,  Bauer,  NIAMD. 


/<i 


Serial  No.  NHLI-87(c) 

Major  Findings;   The  data  from  six  patients  showed  the  following: 
1)  Seventy  to  eighty  percent  of  the  injected  tritiiam  label  was  excreted  into 
urine  during  the  first  6  hours;  95%  or  more  of  the  tritium  was  eliminated 
within  24  hours  after  the  injection  of  the  labeled  amine.   Over  a  3-day  period 
all  of  the  tritium  could  be  accounted  for  as  tritiated  metabolites  or  tritiated 
water.   There  was  no  evidence  of  significant  retention  of  3-  H-histamine  or 
labeled  metabolites  in  the  body.  2)   The  excretion  of  tritiated  water  accounted 
for  5  to  8%  of  the  injected  label.   This  indicated  that  the  3-  H-histamine  was 
metabolized  by  histaminase  to  only  a  minor  extent.   3)   Small  amounts  of 
unchanged  g-%-hist amine,  less  than  3%  of  the  injected  tritiated  amine,  were 
present  in  -the  first  6  hour  urine  collection.   4)  The  major  tritiated  metab- 
olites in  urine  were  methylimidazole  acetic  acid  (56  to  77%  of  the  injected 
g-^H-histamine)  and  imidazole  acetic  acid  riboside  (13  to  39%) .   These  acidic 
metabolites  accounted  for  greater  than  70%  of  the  tritium  label.   The  fact 
that  tritium  was  retained  by  these  acidic  metabolites  indicated  that  mono- 
amine oxidase  was  of  major  importance  in  the  metabolism  of  g--'H-histamine. 
5)  Administration  of  a  monoamine  oxidase  inhibitor,  pargyline  (1  mg/kg) ,  to 
one  of  the  patients  resulted  in  a  small  decrease  (20%)  in  the  amount  of 
tritiated  methylimidazole  acetic  acid  excreted.   Preliminary  results  indicated 
that  administration  of  the  histaminase  inhibitor,  aminoguanidine  (10  mg/kg), 
resulted  in  the  reduction  of  tritiated  water  formation  and  the  disappearance 
of  H- imidazole  acetic  acid  riboside  in  urine. 

Significance:   These  studies  have  revealed  that  histaminase  (diamine  oxidase) 
plays  only  a  minor  role  in  the  metabolism  of  histamine  in  vivo.  The  results 
suggest  that  the  enzyme  which  methylates  histamine  to  methylhistamine,  hista- 
mine-N-methyl-transf erase,  and  monoamine  oxidase  are  the  more  important 
enzymes  in  histamine  metabolism.   This  information  provides  a  rational  basis 
for  the  inhibition  of  histamine  metabolism  in  man. 

Proposed  Course  of  Project:   The  studies  will  be  continued  to  determine 
whether  inhibitors  of  the  enzymes  histamine-N-methyltransferase  and  monoamine 
oxidase  will  block  the  metabolism  of  histamine  and  thereby  increase  tissue 
histamine.   Since  histamine  is  a  vasodilator,  it  is  hoped  that  such  inhibitors 
will  be  of  use  in  the  treatment  of  vascular  disorders  such  as  Raynaud's 
disease.   The  use  of  these  inhibitors  will  also  be  employed  to  determine 
whether  histamine  plays  a  normal  role  in  vasodilation. 

Publications:   None 


/43 


Serial  No.   NHLI-88(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Experimental  Medicine 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Studies  of  the  Biological  Role  of  Histamine:  Application 
of  the  Enzymatic  Assay  of  Histamine  to  the  Measurement  of 
Histamine  in  Urine 

Previous  Serial  Number:   NHLI-14A 

Principal  Investigators:  Michael  A.  Beaven,  Ph.D. 

Harry  R.  Reiser,  M.D. 

Other  Investigators:   Zdenka  Horakova,  Ph.D. 
Stephen  B.  Baylin,  M.D. 
Albert  Sjoerdsma,  M.D.,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   In  order  to  further  assess  the  role  of  histamine  in  man,  the 
enzymatic  assay  of  histamine  (see  Project  Report  NllLI-144,  1969-70)  was 
niodified  for  the  direct  measurement  of  histamine  in  urine.   The  study  was 
undertaken  to  provide  a  simple  method  of  detecting  changes  in  the  excretion 
of  histamine.   Previously  published  assays  for  histamine  in  urine  are  time- 
consuming  and  require  some  prior  purification  of  the  histamine.   The  enzymatic 
assay  is  specific  and  sensitive  and  can  be  performed  on  as  many  as  60  samples 
of  untreated  urine  at  a  time.   In  the  present  study  histamine  excretion  was 
determined  in  urine  of  control  subjects  and  of  a  patient  with  mastocytosis, 
a  condition  associated  with  the  release  of  large  quantities  of  histamine.   In 
addition,  the  effect  of  oral  administration  of  L-histidine  on  histamine 
secretion  in  urine  was  studied. 

Methods:   The  assay  procedure,  which  is  based  upon  the  formation  of  "'"'^C- 
methylhistamine  from  histamine  in  the  presence  of  S-adenosylmethionine-^^C- 
methyl  and  histamine-N-methyltransf erase,  is  described  in  detail  in  Project 
Report  NHLI-144,  1969-70.   In  the  present  studies,  samples  were  removed  from 
24  hr  urine  collections  and  frozen  until  time  of  assay;  20  yl  aliquots  were 
taken  for  assay. 

Major  Findings:   1)  Adaptation  of  assay  to  measurement  of  histamine  in  urine. 
Studies  showed  that  a  20  yl  aliquot  of  urine  was  an  optimum  quantity  for  the 
assay;  larger  volumes,  50  to  100  pi,  resulted  in  a  diminution,  by  30  or  80% 
respectively,  in  the  recovery  of  histamine  as  l^C-methylhistamine.   Unlabeled 
histamine  added  to  the  20  pi  urine  sample  was  recovered  quantitatively  and 
reproducibly .   The  limit  of  sensitivity  of  the  procedure  was  0.1  ng  histamine. 

1  /if 


Serial  No.   NHLI-88(c) 

The  levels  of  histamine  in  normal  urine  were  lower  than  those  reported  pre- 
viously in  the  literature  when  the  fluorometric  assay  of  histamine  had  been 
ujjed.   Possible  reasons  for  the  lower  histamine  levels  found  in  the  present 
work  are:   (1)  the  enzymatic  assay  is  specific  for  histamine,  whereas  the 
fluorometric  assay  is  subject  to  interference  from  compounds  such  as 
spermidine;  (2)  the  isolation  of  histamine  from  urine  is  not  required  for 
the  enzymatic  assay  and  the  possibility  of  histamine  arising  from  decarboxyla- 
tion of  L-histidine  is  reduced.   The  daily  excretion  of  L-histidine  in  urine 
is  160-260  mg/day;  the  spontaneous  decarboxylation  of  0.01%  of  the  histidine 
could  yield  10  to  20  yg  histamine. 

2)  Histamine  in  normal  urine.   In  16  normal  males  and  females,  the  levels  of 
histamine  ranges  from  10  to  40  ng/ml  urine  or  3  to  40  vig/24  hr,  mean  value 

11  pg/24  hr.   An  additional  female  had  a  value  of  92  yg/24  hr. 

3)  Histamine  in  urine  of  a  patient  with  mastocytosis.   As  a  confirmation  of 
the  assay,  histamine  was  measured  in  a  patient  with  mastocytosis.   Histamine 
levels  ranged  from  160-220  yg/24  hr.   Histamine  output  in  urine  was  increased 
to  310  yg/24  hr  by  oral  administration  of  L-histidine,  4  x  10  g  daily  for  3 
days.   Urinary  histamine  excretion  returned  to  pretreatment  levels  immediately 
upon  cessation  of  L-histidine  administration.   Examination  of  histamine  levels 
in  two  suspected  cases  of  mastocytosis  failed  to  show  abnormality  in  histamine 
excretion. 

4)  Effects  of  oral  administration  of  L-histidlne  on  urinary  histamine  levels. 
A  further  confirmation  of  the  assay  was  that  the  administration  of  L-histidine 
produced  a  pronounced  rise  in  urinary  histamine  levels.   In  a  normal  volunteer, 
histamine  levels  rose  from  28  yg  to  297  yg/24  hr,  and  in  one  female  patient 
with  Rajmaud's  disease,  a  rise  from  19  yg  to  180  yg/24  hr  was  observed  after 
administration  of  40  g  L-histidine/day  for  3  days.   The  studies  with  L- 
histidine  were  carried  out  to  determine  whether  increased  histamine  production 
could  be  achieved  in  patients  with  Raynaud's  disease  for  use  as  a  possible 
form  of  therapy. 

Significance:   The  enzymatic  assay  of  histamine  in  urine  provides  a  simple 
method  of  detecting  alterations  in  synthesis  or  metabolism  of  histamine  by 
measuring  changes  in  the  excretion  of  histamine.   The  assay  makes  it  possible 
to  evaluate  agents  that  enhance  or  inhibit  histamine  synthesis  or  metabolism. 
For  example,  the  effect  of  an  inhibitor  of  histidine  decarboxylase  in  a 
patient  with  mastocytosis  can  be  determined  by  measuring  the  decrease  in 
urinary  histamine. 

Proposed  Course  of  Project:   The  assay  procedure  will  be  utilized  in  studies 
of  the  release  of  histamine  in  various  pathological  states  and  in  the  in  vivo 
evaluation  of  compounds  that  alter  the  metabolism  of  histamine. 

Honors  and  Awards:   None 


/i^ 


I 


J 


Serial  No.   NHLI-88(c) 


Publications 
1 


Horakova,  Z.,  Zierdt,  C.  H. ,  and  Beaven,  M.  A.:   Identification 

of  Lactobacillus  as  the  source  of  bacterial  histidine  decarboxylase 

in  rat  stomach.   Eur op.  J.  Pharmacol.   In  press. 


/U 


Serial  No.  NHLI-89 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Md, 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 


Project  Title: 


Isolation  and  Characterization  of  Hydroxyindole  0-methyl 
Transferase. 


Previous  Serial  Number:   None 


Principal  Investigator:   Walter  Lovenberg,  Ph.  D. 

Other  Investigators:   Richard  Jackson,  Ph.  D.  and  Joseph  Fontana,  Ph,  D. 

Project  Description: 

Objectives :   The  principle  objective  of  this  project  was  to  obtain  a  pure 
preparation  of  hydroxyindole  0-methyl  transferase  (HIOMT)  and  to  study  its 
physical  and  chemical  properties. 

Methods:  The  enzjmxe  activity  was  assayed  using  ^C-S-adenosyl  methionine  as 
the  methyl  donor  and  N-acetylserotonin  as  the  acceptor.   The  product, 
melatonin,  was  extracted  into  an  organic  solvent  and  counted.   The  protein 
was  purified  by  standard  isolation  techniques. 

Major  Findings:   Bovine  pineal  HIOMT  was  isolated  as  an  essentially  homogenous 
protein.   The  steps  developed  for  this  isolation  in  good  yield  are  summarized 
below: 


Spec 

ific  Act 

ivity 

%  Yield 

Crude  extract 

15 

100 

Ammonium  sulfate 

55 

100 

1st  DEAE  Sephadex 

122 

75 

Sephadex  G  100 

200 

43 

2nd  DEAE  Sephadex 

274 

30 

Isoelectric  Focusing 

343 

29 

The  final  product  gave  a  single  band  on  disc  gel  electrophoresis  and  appeared 
to  be  homogenous  when  studied  by  sedimentation  equilibrium  in  an  ultra- 
centrifuge.   Antibodies  to  this  apparently  pure  enzyme  were  prepared  in  sheep. 
Gel  diffusion  or  gel  electrophoresis  of  the  protein  and  reaction  with  these 
antibodies  also  indicated  that  a  single  molecular  species  had  been  isolated. 
It  is  interesting  to  note  that  at  least  3  molecular  weight  forms  of  the 
enzyme  were  observed  on  Sephadex  G-lOO  chromatography  and  that  chromatography 
of  the  major  form  on  the  2nd  DEAE  Sephadex  step  resulted  in  the  isolation  of 
2  differently  charged  enzjnnes.   Each  of  these  forms  could  be  completely 
purified  by  isoelectric  focusing.   These  two  forms  had  identical  immunologic 


fi-r 


ij'i/ 


t 

^ 


Serial  No.  NHLI-89 

properties  and  appeared  to  be  identical  in  all  other  respects.   The  molecular 
weight  of  the  enzyme  was  about  78,000  as  determined  by  sedimentation  equilib- 
rium.  Electrophoresis  in  sodium  dodecyl  sulfate  (SDS)  containing  gels 
indicated  that  the  molecular  weight  was  about  39,000.   The  native  enzyme 
i-robably  contains  2  subunits  of  about  this  molecular  weight.   Fingerprint 
analysis  of  tryptic  digests  of  the  protein  showed  that  the  two  subunits  were 
very   similar  or  identical.   The  amino  acid  content  was  determined  and  was 
unique  only  in  that  this  protein  contained  relatively  high  content  of 
leucine  (15-20).   The  enzyme  did  not  appear  to  contain  any  sugar  residues. 
Although,  other  phenolic  0-methyl  transferases  require  metal  ions  for  activity, 
examination  of  a  wide  variety  of  metal  ions  and  metal  chelators  indicate  that 
metals  do  not  participate  in  this  methyl  transfer  reaction. 

Significance:   It  was  previously  thought  that  HIOMT  was  the  rate  limiting 
enzyme  in  the  biosynthesis  of  melatonin,  and  that  diurnal  changes  in  this 
enzyme  were  responsible  for  the  corresponding  changes  in  hormone  levels. 
This  concept  has  recently  been  questioned  and  from  the  current  work  in  which 
we  find  that  4%  of  the  total  soluble  pineal  protein  is  HIOMT  and  that  the 
enzyme  has  a  low  turnover  number  it  would  appear  to  be  unlikely  that  this 
enzyme  represents  a  control  point  in  the  pathway.   The  presence  of  a  number 
of  different  molecular  species  suggest  that  possible  conversion  of  one  form 
to  another  may  be  a  means  of  controlling  enzyme  activity  ±n   vivo,  although 
there  is  yet  no  evidence  for  such  a  mechanism. 

Proposed  Course  of  Project:   With  the  availability  of  large  amounts  of  anti- 
bodies to  this  enzyme  it  should  be  possible  to  specifically  study  its  bio- 
synthesis.  This  type  of  experimentation  is  planned.   We  also  plan  to 
investigate  the  means  by  which  the  various  molecular  forms  of  the  enzyme  are 
formed  or  interconverted. 

Publications:   None 


/4e 


Serial  No.      NHLI-90 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1971  through  June  30,  1971 

Project  Title:   A  Sensitive   and  Automated  Method  for  Recording  Gastric  pH 

Changes  and  H"*"  Ion  Secretion  in  Experimental  Animals  and  the 
Use  of  this  Method  for  Studying  the  Pharmacology  of  Gastric 
Secretion. 

Previous  Serial  Number:   NHLI-150 

Principal  Investigator:   G.  H.  Besselaar,  M.D.  nJ 

„„"' 

Other  Investigators:   Ronald  Geller,  Ph.  D.  and  Walter  Lovenberg,  Ph.  D.  i«||„ 

Proiect  Description:  ,„iii, 

lull 

Objectives:  1)  To  develop  a  sensitive  method  for  measuring  pH  and  actual  IT"  "'"jl 
ion  secretion  in  the  stomach  of  experimental  animals.  2)  Use  of  this  method  ""' 
for  studying  the  effects  of  drug  administration  on  gastric  secretion.  , 

Methods:   The  model  for  continuous  recording  of  gastric  secretion  in  the  rat  '"" 

was  described  in  last  year's  project  report.   The  only  modification  was  in  |'' ^ 

the  perfusate,  which  was  changed  from  dilute  base  (.00025  N  NaOH)  to  .9% 
saline  pH  7.00.   The  endpoint  of  the  titration  unit  was  set  at  the  pH  of  the 
inflowing  saline,  so  that  in  effect  the  total  amount  of  H"*"  ions  secreted  by 
the  stomach  is  neutralized.   Gastric  secretion  in  response  to  various  pharma- 
cological agents  was  studied  in  normal  and  vagotomized  Sprague-Dawley  rats 
(250-350  grams). 

Major  Findings:   1)  Intravenous  infusions  of  small  doses  of  histamine  (8  yg/ 
min)  and  pentagastrin  (.4  pg/min)  consistently  stimulate  gastric  secretion. 
These  doses  result  in  about  half-maximum  secretion  in  most  animals.   2)   For 
vagotomized  animals  (7-10  days  post-operative)  the  response  to  the  above 
mentioned  doses  of  histamine  and  pentagastrin  is  either  absent  or  very  slight. 
With  much  higher  doses,  however,  the  peak  response  to  histamine  and  penta- 
gastrin is  the  same  as  in  control  animals.   3)   Serotonin  (3  yg/min  i.v.) 

infusions  have  an  inhibitory  effect  on  gastric  acid  output  stimulated  by  | 

histamine  (8  yg/min).   This  effect  cannot  be  demonstrated  when  gastric 
secretion  is  maximally  stimulated  by  histamine,  whereas  the  results  of  similar 
experiments  using  pentagastrin  as  the  stimulant  are  variable.   4)   L-Tryptophan 

and  5HTP  in  acute  experiments  had  no  effect  on  histamine  or  pentagastrin  C 

stimulated  gastric  secretion.   5)   Pretreatment  with  parachlorophenylalanine  t 

(PCPA  300  mg/kg  for  3  days),  a  tryptophan  hydroxylase  inhibitor,  resulting  h 

in  serotonin  depletion  in  the  proximal  duodenum,  and  a  concomitant  increase  ^ 

in  gastric  secretion  in  Shay  rats.   Perfused  rats  pretreated  with  PCPA  showed  ^ 

a  decreased  sensitivity  to  histamine  and  to  serotonin.   Further  work  along 

1  /^r 


MW 


«" 


nil 


Serial  No.  NHLI-90 

these  lines  is  in  progress.   6)  We  have  been  unable  to  demonstrate  an 
inhibitory  effect  of  secretin  on  either  histamine  or  gastric  stimulated 
secretion,  using  dose  levels  similar  to  those  used  in  man  and  dogs  where 
the  inhibitory  effect  has  been  observed.    7)   Norepinephrine,  angiotensin, 
pitressin  and  PGE,  which  have  been  shown  in  the  dog  to  decrease  gastric 
mucosal  bloodflow,  all  are  strong  inhibitors  of  gastric  secretion  in  the 
perfused  rat  stomach. 

Significance:   There  is  good  evidence  for  a  duodenal  mechanism  controlling 
gastric  acid  secretion.   Acidification  of  the  duodenum  leads  to  inhibition 
of  gastric  secretion,  and  to  release  of  serotonin  and  secretin  from  this 
area.   Both  compounds,  under  various  circumstances,  have  been  shown  to  be 
able  to  inhibit  histamine  and/or  gastric  stimulated  secretion.   Depletion  of 
endogenous  serotonin  leads  to  increased  secretion;  administration  of  exogenous 
serotonin  results  in  inhibition  of  gastric  secretion.   By  expanding  our  data 
with  PCPA  (and  reserpine)  pretreated  animals  and  studying  the  effect  of 
vagotomy  in  these  animals  we  hope  to  clarify  the  possible  physiologic  im- 
portance of  serotonin  in  the  duodenal  inhibitory  mechanism  of  gastric  acid 
secretion. 

Proposed  Course  of  Project:   Expansion  of  the  studies  of  the  effect  of  PCPA 
and  reserpine  on  gastric  acid  secretion  in  normal  and  vagotomized  animals  is 
planned.   Studies  on  the  effect  of  5HTP  pretreatment  on  gastric  secretion 
on  normal  and  vagotomized  animals  will  also  be  carried  out. 

Publications:   None 


f7c 


Serial  No.    NHLI-91 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  19  71 


Project  Title: 


Tryptophan  Hydroxylase  and  Serotonin  in  Spinal  Cord  and 
Brainstem  Before  and  After  Chronic  Transection. 


Previous  Serial  Number:   NHLI-145 


Principal  Investigator:   B.V.  Clineschmidt ,  Ph.  D. 

Other  Investigators:   Walter  Lovenberg,  Ph.  D.  and  J.E.  Pierce,  D.V.M. 

Cooperating  Units:   Section  on  Laboratory  Animal  Medicine  &  Surgery,  NHLI 

Project  Description: 

Objectives:   1)  To  ascertain  whether  the  spinal  cord  of  the  cat  contains 
serotonergic  interneurones  and  2)  to  compare  the  ratio  of  tryptophan  hydroxyl- 
ase activity  to  serotonin  (5HT)  content  in  the  area  rich  in  serotonin- 
containing  cell  bodies  (brainstem)  with  an  area  containing  predominately  axons 
(spinal  cord),  many  centimeters  removed  from  their  cell  bodies. 

Findings:   Tryptophan  hydroxylase  and  serotonin  levels  were  determined  in  normal 
and  chronically  transected  (Tg-Tg)  cats.   Lumbosacral  and  brainstem  5-HT  and 
tryptophan  hydroxylase  activity  were: 


('1/ 


Control 

3  week  transection 

5  week  transection 


5-HT  yg/gm 
cord   brainstem 


0.70 

0.94 

0.10 

1.19 

0.11 

1.04 

Tryptophan  Hydroxylase 

mpmoles  g"-*-  hr"-'- 
cord         brainstem 


8.4 
1.8 
1.4 


13.0 
15.8 
18.0 


Both  5-HT  and  tryptophan  hydroxylase  were  decreased  80  to  85%  in  the  cord 
of  animals  transected  for  3  weeks  but  significant  amounts  appeared  to  remain. 
Extending  the  duration  of  transection  from  3  weeks  to  5  weeks  caused  no 
further  decrease  in  either  parameter.   Brainstem  5-HT  was  not  affected  by 
spinal  transection.   However,  5  weeks  after  transection  brainstem  tryptophan 
hydroxylase  was  significantly  increased. 

Significance:  D   5-HT  Containing  interneurones.   According  to  workers 
employing  the  histochemical  fluorescent  technique,  all  5-HT  in  spinal  cord 
resides  in  axons  from  neurones  whose  cell  bodies  lie  in  the  Raphe  nuclei 
of  the  brainstem.   On  the  other  hand,  recent  neuropharmacological  evidence 


nf 


Serial  No.   NHLI-91 

indicates  that  spinal  cord  may  contain  serotonergic  intemeurones .   If  all 
spinal  5-HT  originates  supraspinally,  cords  removed  below  the  level  of 
transection  from  chronically  transected  animals  should  contain  insignificant 
amoup.LS  of  tryptophan  hydroxylase  (19%  of  normal)  and  5-HT  (14%  of  normal) 
•''fcer  complete  degeneration  of  descending  axons.   These  residual  levels  may 
reflect  the  existence  of  serotonergic  interneurones  in  spinal  cord. 

2.   Tryptophan  hydroxylase-5-HT  ratio  in  cell  bodies  vs.  axons.   It  was 
suggested  recently  that  the  brainstem  level  of  tryptophan  hydroxylase  exceeds 
the  amount  required  to  maintain  the  steady-state  level  of  5-HT,  perhaps 
because  the  brainstem  contains  mostly  cell  bodies  where  the  enzyme  presumably 
is  synthesized.   We  found  no  difference  in  the  ratio  of  tryptophan  hydroxylase 
to  5-HT  in  brainstem  (predominately  cell  body  area)  as  compared  with  spinal 
cord  (predominately  axon  area).   Thus,  it  might  be  proposed  that  the  level  of 
tryptophan  hydroxylase  determines  the  concentration  of  5-HT  in  a  given  area 
of  the  brain  regardless  of  whether  the  area  contains  mainly  cell  bodies  or 
axons.   However,  such  a  proposal  is  difficult  to  reconcile  with  the  increase 
in  tryptophan  hydroxylase  without  an  accompanying  rise  in  5-HT  which  we 
observed  in  brainstem  after  chronic  spinal  transection. 

Proposed  Course  of  Project:   None 

Publications: 

I.   Clineschmidt ,  B.V.,  Pierce,  J.E.  and  Lovenberg,  W. :   Tryptophan 
hydroxylase  and  serotonin  in  spinal  cord  and  brainstem  before  and 
after  chronic  transection.   J.  Neuro.  Chem.   In  Press. 


/7X 


Serial  No.  NHLI-92 

1.  Experimental  Therapeutics  Branch  B. 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  the  Biological  Role  of  Histamine:  Participation 
of  Histamine  in  Inflammation  Following  Heat  Injury 

Previous  Serial  Number:   None 

Principal  Investigator:   Zdenka  Horakova 

Other  Investigators:   Michael  A.  Beaven,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   In  a  continuing  study  of  the  role  of  histamine  in  the  body,  the 
release  of  histamine  in  inflammation  following  heat  injury  was  examined. 
These  studies  were  undertaken  with  the  aim  of  reinvestigating  the  participa- 
tion of  histamine  in  the  inflammatory  response  to  heat  injury.   The  current 
opinion  holds  that  the  role  of  histamine,  if  any,  in  the  development  of 
inflammation  is  transient  and  that  other  mediators  are  necessary  to  maintain 
inflammation.   This  opinion  is  based  upon  the  finding  that  histamine  is 
released  from  injured  tissues  at  a  rapid  rate  and  that  it  has  largely  dis- 
appeared from  tissue  at  a  time  when  inflammation  is  still  developing.   However, 
a  limitation  in  these  experiments  is  that  the  assays  were  insufficiently 
sensitive  to  measure  low  levels  of  histamine  that  may  be  present  after 
depletion  of  histamine  stores  and  which  may  represent  newly  synthesized 
histamine.   The  availability  of  the  specific  and  sensitive  enzymatic  assay 
for  histamine  makes  it  possible  to  obtain  more  precise  information  on  the 
changes  in  tissue  histamine  during  the  course  of  the  inflammatory  response  to 
in j  ury . 

Methods:   Heat  injury  was  produced  in  the  rat  paw  by  immersion  of  the  paw  in 
hot  water  (56°C)  for  30  sec.   At  various  time  intervals  after  injury,  samples 
of  the  skin  and  underlying  tissue  were  removed  from  the  paw  by  skin-punch. 
Samples  of  wound  exudate  and  aortic  blood  were  also  collected.   The  amount  of 
edema  was  assessed  by  measurement  of  paw  volume.   Histamine  was  assayed  by 
the  enzymatic  procedure  described  in  Project  Report  NHLI-144,  1969-70. 

Major  Findings:   The  following  observations  were  made  after  heat  injury: 

1)   Edema  of  the  rat  paw  was  evident  within  a  few  minutes  of  heat  injury  and 
by  one  hour  the  paw  had  swollen  to  2  to  3  times  its  normal  size.   The  edema 
persisted  for  the  remaining  24  hours  of  the  experiment. 


/r3 


\ 


pi- 


Serial  No.   NHLI-92 

2)  There  was  a  transient  increase  in  histamine  levels  in  the  paw  skin 
immediately  following  heat  injury.   This  increase  persisted  for  a  few 
minutes  and  was  followed  by  a  rapid  reduction  in  the  histamine  levels. 
Twenty  minutes  after  the  heat  injury,  histamine  levels  in  the  paw  skin 
were  depleted  by  70%  compared  to  control  and  remained  depleted  for  24  hours. 

3)  Large  amounts  of  histamine  appeared  in  the  tissue  exudate  after  heat 
injury.   In  non-heat  injured  animals,  the  normal  exudate  contained  300- 
400  ng  histamine/ml  exudate.   In  heat  injured  animals,  the  histfuiiine 
content  of  the  exudate  rose  to  2-3,000  ng/ml  by  20  min  and  remained 
elevated,  1,500  ng/uil,  at  one  hr  after  heat  injury.   The  histamine  levels 
in  the  exudate  slowly  subsided  during  the  24  hr  of  the  experiment  but  were 
still  high,  500  ng/ml,  compared  to  levels  in  non-heat  injured  animals, 

24  hr  after  injury.   Histamine  levels  in  the  circulating  blood  were 
slightly  increased  after  heat  injury. 

4)  In  animals  that  were  depleted  of  tissue  histamine  by  administration  of 
the  histamine  liberator,  compound  48/80  (1  mg/kg,  i.p.,  four  times  over 

48  hr) ,  heat  injury  resulted  in  only  a  small  increase,  30%,  in  paw  volume 
compared  to  a  2  to  3-fold  increase  in  non-48/80  treated  rats.   The  amount 
of  histamine  in  the  wound  exudate  of  the  48/80  treated  animals  was  less 
than  5%  of  that  seen  in  nontreated  rats  after  heat  injury. 

Significance;   The  application  of  heat  injury  to  the  rat  paw  provides  a 
useful  model  for  studying  the  role  of  histamine  during  inflammation.   The 
studies  show  the  increased  levels  of  histamine  persisted  in  tissue  fluid 
for  up  to  24  hr  after  heat  injury.   The  amount  of  histamine  present  could 
be  sufficient  to  account  for  the  edema  in  the  injured  paw  and  is  evidence 
that  the  action  of  histamine  in  inflammation  is  of  long  duration. 
Correspondingly,  in  animals  that  were  depleted  of  tissue  histamine,  the 
edematous  response  to  heat  injury  was  only  10%  of  that  of  normal  injured 
animals.   These  findings  are  important  in  the  consideration  of  the  mechanism 
of  edema  formation  and  will  be  the  basis  of  future  investigations  of  vascular 
responses  to  heat  injury. 

Proposed  Course  of  Project:   Studies  will  be  continued  to  determine  whether 
the  elevated  histamine  levels  in  tissue  fluids  after  heat  injury  represent 
newly  synthesized  histamine  or  histamine  released  from  tissue  stores.   The 
investigations  will  include  studies  of  mild  heat  treatment  (48°C)  in  which 
responses  such  as  vascular  dilation  and  edema  are  reversible.   The  effect 
of  agents  which  promote  histamine  synthesis  or  block  its  metabolism  and 
the  effect  of  antiinflammatory  agents  on  the  response  to  histamine  during  heat 
injury  will  be  studied.   It  is  hoped  that  such  investigations  will  provide 
information  on  whether  histamine  plays  a  role  in  vascular  physiology. 

Honors  and  Awards:   None 

Publications:   None 


(lii 


Serial  No.     NHLI-93 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report  ^i 

July  1,  1970  through  June  30,  19  71  > 

Project  Title:   Studies  on  the  Mechanisms  of  Binding  and  Release  of  Biogenic 

Amines .  f 

\ 

Previous  Serial  Number:   NHLI-148 

Principal  Investigator:   Larry  Loeffler,  Ph.  D. 

Other  Investigators:   Walter  Lovenberg,  Ph.  D.  f\f 

Cooperating  Units:   None  "11.1 

Project  Description:  i*  I'l 

Objectives:   To  further  elucidate  the  manner  in  which  biogenic  amines  are  | 

bound  in  cellular  storage  sites  and  the  mechanism  by  which  release  occurs  in  '"'" 

response  to  various  chemical  agents.   In  particular,  to  study  the  mechanism  of  ,,|i,i 

histamine  release  from  mast  cells.  },„ 

Methods:   The  peritoneal  mast  cell  system  of  the  rat  has  been  used  to  study:  '„„ 

1)   the  release  of  histamine  by  the  selective  releasing  agent  compound  48/80 
or  high  molecular  weight  dextran;   2)   the  effects  of  certain  adenine  nucleo- 
tides, phosphodiesterase  inhibitors  and  prostaglandin  E^  upon  48/80  induced 
histamine  release;   3)   the  binding  of  tritium  labelled  48/80  to  mast  cells; 
and   4)   the  nature  of  the  protein  material  in  storage  granules  of  the  mast 
cell.   Hi  vitro  cell  suspensions  and  well  established  assay  procedures  have 
been  employed  in  all  of  these  studies. 

Major  Findings:   1)   Histamine  release  studies  and  2)   The  effects  of  adenine- 
nucleotides,  phosphodiesterase  inhibitors  and  prostaglandin  E^.   Heterogeneous 
peritoneal  cell  suspensions  were  found  to  respond  uniformly  and  consistently  to 
compound  48/80  (concentration  of  about  1  Mg/ml  for  5  minutes)  as  described  in 
the  literature,  giving  a  very  rapid  release  of  70-80%  of  the  total  histamine 
content.   Preincubation  of  the  cell  suspensions  for  short  time  (i.e.  20  min) 
with  dibutyryl-3'5'-c-AMP  (DB-c-AMP)  was  found  to  cause  inhibition  of  histamine 
release  by  compound  48/80.   Strong  inhibition  was  observed  at  10~^M  concentra- 
tion, statistically  significant  inhibition  at  10~%.   The  following  adenine 
nucleotides  were  found  to  be  ineffective  as  inhibitors  at  similar  concentra- 
tions: 5'-AMP;  5'-ADP;  5'-ATP;  and  3'5'-c-AMP.   Adenosine,  phosphate  and 
pyrophosphate  were  also  ineffective. 


nr 


Serial  No.  NHLI-93 

In  an  effort  to  obtain  effects  with  DB-c-AMP  at  concentrations  approaching 
physiological,  inhibitors  of  c-AJCP  phosphodiesterase  were  studied  and  were 
also  found  to  inhibit  48/80  induced  release,  even  in  the  absence  of  added 
DB-c-AMP.   Theophylline  was  completely  inhibitory  at  10-2m  concentration  and 
oirtiaily  effective  at  10~3m.   The  phosphodiesterase  inhibitors  reserpine, 
DEAE-reserpine  and  perphenazine  were  very  significantly  inhibitory  at  con- 
centrations of  3  X  10~^M,  5  X  10"%  and  6  x  10"%,  respectively.   These  con- 
centration levels  are  similar  to  those  required  for  phosphodiesterase 
inhibition.   The  latter  three  inhibitors,  but  not  theophylline,  themselves 
promoted  a  less  dramatic  release  of  histamine  compared  with  48/80  during 
prolonged  incubation  periods.   The  inhibitory  effects  of  theophylline  or 
DB-c-AflP  could  be  removed  by  washing  of  centrifuged  cells  and  the  response 
of  resuspended  cells  to  the  releasing  agent  48/80  nearly  completely  restored. 
Prostaglandin  Ei,  which  would  be  expected  to  stimulate  adenyl  cyclase  and 
elevate  c-AMP  levels,  was  also  found  to  be  an  effective  histamine  release 
inhibitor,  at  concentrations  of  3  x  10~^M  and  above. 

The  above  interest  in  the  ^lU  vitro  effects  of  DB-c-AMP  and  theophylline  was 
stiiTiulated  by  the  work  of  Drs.  H.  H.  Baxter,  M.A.  Beaven  and  Z.  Horakova,  who 
found  that  these  agents  protect  rats  against  the  anaphylactoid  response  to 
dextran  and  effectively  block  the  normal  elevation  of  blood  histamine  found 
after  administration  of  dextran. 

3)  Binding  of  tritium  labelled  48/80  to  mast  cells.   Compound  48/80  was 
randomly  labelled  with  tritium  via  the  Wilzbach  procedure  and  the  product  was 
found  to  retain  its  histamine  releasing  activity.   By  means  of  column 
chromatography  on  CM  Sephadex  and  Sephadex,  four  major  peaks  of  radioactivity 
were  obtained.   All  of  the  biological  activity  resided  in  the  major  or 
highest  molecular  weight  fraction.   At  a  concentration  of  1  pg/ml,  only  about 
3-4%  of  the  labelled  material  became  "bound"  to  the  cells  after  incubation. 
No  significant  differences  in  binding  were  observed  upon  preincubation  of  the 
cells  with  the  release  inhibitors  DB-c-AMP  and  theophylline.   Therefore,  these 
experiments  offer  no  evidence  that  these  compounds  inhibit  histamine  release 
by  inhibiting  the  binding  of  48/80. 

4)  Investigations  of  the  nature  of  the  protein  component  of  the  mast  cell 
storage  granule.   Homogenous  mast  cells,  obtained  by  density  gradient 
centrifugation  of  peritoneal  cell  mixtures,  were  ruptured  by  sonication  and 
the  granules  isolated  by  differential  centrifugation.   Disc  gel  electro- 
phoresis studies  on  the  granular  material  (heparin-protein-his tamine  complex) 
indicated  that  the  protein  material  was  a  complex  multicomponent  one,  and  no 
further  work  was  initiated. 

Significance:   Previously  reported  studies  indicate  that  release  of  histamine 
from  mast  cells  by  48/80  involves:   1)  attachment  of  48/80  to  the  cell  membrane, 
2)  specific  and  extremely  rapid  degranulation  by  an  energy  requiring 
"exocytosis"  of  granules  which  contain  a  histamine-heparin-protein  complex, 
and  3)  liberation  of  the  heparin  bound  histamine  from  the  granules  by  an  ion 
exchange  process  with  cations  of  the  extracellular  fluid.   The  effects  of 
DB-c-AMP,  various  phosphodiesterase  inhibitors  and  prostaglandin  E  on  this 


/7^ 


Serial  No.   NHLI-93 

release  process  can  be  interpreted  as  circumstantial  evidence  supporting  a 
role  for  the  adenyl  cyclase  system.   Higher  levels  of  cyclic  AMP  would  appear 
to  protect  the  cell  against  the  effects  of  48/80,  perhaps  by  altering  the 
energy  requiring  release  process.   Elevated  levels  of  c-AMP  can  be  promoted 
by  DB-c-AMP  which  presumably  enters  the  cell  rapidly,  phosphodiesterase 
inhibitors,  which  prevent  the  destruction  of  c-AMP  and  PGEj^,  which  stimulates 
adenyl  cyclase.   Definitive  biochemical  studies  on  the  adenyl  cyclase  system 
of  homogeneous  mast  cells  and  measurement  of  c-AMP  levels  in  response  to 
release  iniiibitors  are  required  to  establish  definitely  the  role  of  the  adenyl 
cyclase  system  if  any. 

Proposed  Course  of  Project:   No  further  work  is  contemplated.   Extension  of 
these  observations  to  antigen-antibody  promoted  histamine  release,  and 
definitive  biochemical  studies  of  the  adenyl  cyclase  system  in  the  mast  cell, 
would  be  of  considerable  interest  in  the  understanding  of  allergic  responses. 

Publications ;  ,11, if 

1.   Loeffler,  L. J.  ,  Lovenberg,  W.  ,  and  Sjoerdsma,  A.:   Effects  of  dlbutyryl-  '"»'* 

3'5'-cyclic-adenosine  monophosphate,  phosphodiesterase  inhibitors  and  i!!]],,, 

prostaglandin  E^   on  histamine  release  from  rat  peritoneal  mast  cells  l''l''" 

in  vitro.   Biochemical  Pharmacol.   In  Press.  '!!' 


/77 


Serial  No.   NHLI-94(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Studies  on  the  Biosynthesis  and  Metabolism  o£  Physiologically 
Active  Amines. 

Previous  Serial  Number:  NHLI  149 

Principal  Investigator:  Walter  M.  Lovenberg,  Ph.D. 

Other  Investigators:  B.  Van  Clineschmidt,  Ph.D.,  Paul  J.  Schechter,  M.D. , 
Ph.D.,  David  Horwitz,  M.D. ,  and  Richard  Wyatt,  M.D. 

Cooperating  Units:  Adult  Psychiatry  Branch,  National  Institute  of  Mental 
Health. 

Project  Description: 

Objectives:  A  number  of  phenylethyl-  and  indolealkylamines  have  physiological 
roles  or  are  pharmacologically  active.  The  objectives  of  this  study  are  to 
learn  more  about  the  physiologic  roles  of  amines  and  to  understand  what  con- 
trols their  synthesis  and  degradation.  The  studies  are  directed  specifically 
towards  serotonin,  dopamine,  and  norepinephrine. 

It  has  been  reported  recently  that  moa"phine  addiction  in  mice  results  in 
an  increased  synthesis  of  serotonin  in  the  brain.  In  this  study  this  finding 
is  reexamined  and  the  rate  limiting  enzyme  (tryptophan  hydroxylase)  in  serotonin 
synthesis  is  measured  in  morphine  addicted  mice.  The  effect  of  chronic 
administration  of  a  serotonin  antagonist  (methysergide)  or  a  serotonin  de- 
pletor  (reserpine)  on  tryptophan  hydrox>4ase  in  the  brain  is  measured  in  an 
effort  to  further  understand  the  mechanisms  controlling  serotonin  synthesis. 
In  addition,  the  levels  of  tryptophan  hydroxylase  have  been  measured  in  REM 
deprived  rats.  Finally,  the  level  of  dopamine- 6 -hydroxylase  has  been  examined 
in  normal  and  hypertensive  human  serum. 

Methods :  Mice  were  addicted  to  morphine  by  subcutaneous  implantation  of  a 
morphine  pellet  (75  mg)  for  5  days.  Rats  were  treated  with  reserpine  2.5 
mg/kg  daily  for  3  days  or  with  methysergide  5  mg/kg  twice  daily  for  5  days. 
Serotonin  synthetic  rates  in  mice  were  measured  by  the  rate  of  accumulation  of 
serotonin  in  the  brain  following  administration  of  a  monoamine  oxidase  inhibi- 
tor. Tryptophan  hydroxylase  was  measured  in  the  supernatant  fraction  of  brain 
homogenates  using  a  tritium  release  assay  developed  in  this  laboratory.  Depri- 
vation of  REM  in  rats  was  achieved  by  confining  the  animals  to  6  cm  flower  pots 
inverted  in  containers  of  tepid  water. 


/T^ 


Serial  No.   NHLI-94(c) 


Major  Findings:  The  tryptophan  hydroxylase  activity  in  the  brain  o£  mice 
addicted  to  morphine  (95  +_  9  yymoles/mg)  showed  no  significant  difference 
from  mice  implanted  with  control  pellets  (102  +_  7   yymoles/mg/hr) .  The 
adequacy  of  the  addiction  treatment  was  confirmed  by  measuring  Naloxone 
withdrawal  (jumping  test) .  Since  there  was  no  difference  in  the  rate  con- 
trolling enzyme,  the  original  observation  on  increased  brain  serotonin 
synthesis  in  addicted  mice  was  reinvestigated.  In  contrast  to  the  reports  in 
the  literature,  we  find  no  difference  in  the  rate  of  serotonin  accumulation 
following  a, monoamine  oxidase  inhibitor  in  morphine  addicted  animals. 

Rats  treated  chronically  with  reserpine  have  brainstem  tryptophan 
hydroxylase  levels  which  are  identical  with  normal  rats.  The  brainstem 
tryptophan  hydroxylase  levels  in  rats  treated  with  methysergide ,  however, 
appeared  to  be  slightly  lower  than  controls  (0.25  vs  0.35  mymoles/mg/hr) 
in  preliminary  experiments.  It  was  expected  that  tryptophan  hydroxylase 
activity  might  be  increased  following  REM  deprivation  since  it  has  been  re- 
ported that  serotonin  turnover  increases.  In  this  study,  however,  the  REM 
deprived  animals  did  not  show  a  significant  difference  from  the  appropriate 
control  animals  (0.27  vs  0.31  mymoles/mg/hr).  The  stressed  control  animals 
(large  pots  surrounded  by  water)  showed  significantly  higher  tryptophan 
hydroxylase  than  nonstressed  controls  (0.23  vs  0.31  mymole/mg/hr) . 

Since  the  enzyme  dopamine- B -hydroxylase  is  released  into  plasma  during 
sympathetic  activity  and  one  of  the  factors  in  some  types  of  human  hyperten- 
sion is  increased  "sympathetic"  activity,  a  study  measuring  dopamine-6- 
hydroxylase  in  control  and  hypertensive  patients  was  started.  Normal  levels 
of  enzyme  activity  were  found  to  range  from  130  to  500  mymoles  of  tyramine 
hydroxylated  per  20  minutes  per  ml  of  plasma.  Two  patients  with  pheochromo- 
cytoma  and  2  hypertensives  had  levels  in  this  range.  A  third  patient  with 
hypertension  had  a  very  low  level  of  this  enzyme  (2-4  mymoles  tyramine/ml/ 
20  min) . 

Significance:  The  increasing  use  of  psychoactive  drugs  medically  and  abuse 
of  these  compounds  by  the  general  population  makes  it  imperative  that  we 
understand  the  mechanism  by  which  these  drugs  act.  Many  of  these  compounds 
appear  to  relate  in  one  way  or  another  to  serotonin  metabolism  in  the  central 
nervous  system.  Although  the  results  are  largely  negative  they  are  of  value 
to  our  understanding  of  a  relatively  unknown  aspect  of  the  mechanism  of  action 
of  psychoactive  drugs.  The  studies  in  the  REM  deprived  animals  do  little  to 
further  our  understanding  of  the  role  of  serotonin  in  REM  sleep.  They  do  pro- 
vide evidence  that  tryptophan  hydroxylase  levels  are  not  necessarily  the  con- 
trolling factor  in  serotonin  turnover. 

Proposed  Course  of  Project:  Studies  will  be  continued  on  the  possible  decrease 
in  tryptophan  hydroxylase  after  methysergide  treatment,  and  the  effect  of  other 
serotonin  antagonists  will  be  evaluated.  A  much  wider  study  on  the  dopamine- 
s-hydroxylase in  plasma  is  planned.  The  patient  with  the  extremely  low  levels 
of  this  enzyme  will  be  further  studied  to  determine  if  the  essential  absence 
of  dopamine- 6-hydroxylase  is  in  any  way  related  to  his  hypertension. 

2  /y? 


4 


Serial  No.   NHLI-94(c) 


Honors  and  Awards:  None 
Pi'ilications: 

1.  Lovenberg,  W.  and  Engelman,  K. :  Serotonin.  In:  Handbook  of 
Clinical  Laboratory  Data  (Ed.  3) .  Cleveland,  Ohio,  Chemical 
Rubber  Co.   In  press . 

2.  Lovenberg,  W. :  Some  vaso-  and  psychoactive  substances  in  food  - 
amines,  stimulants,  depressants  and  hallucinogens.  In:  Toxicants 
Occurring  Naturally  in  Foods.  Washington,  D.C.  National  Research 
Council  Publication.  In  press . 


/go 


mmmmmmmmmmmmmmmmmmmmmimammm  n 


Serial  No.    NHLI-95 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Shock- induced  Aggression  in  Hypertensive  Rats. 

Previous  Serial  Number:   None 

Principal  Investigator:   Wybren  DeJong,  M.D. ,  Ph.  D. 

Other  Investigators:   B.  Eichelman,  M.D.  ,  Ph.  D.  (LCP-NIMH)  and  R.  B.  "|l„ 

Williams,  M.D.  (LCP-NIMH).  'f.,/ 

Ill* 
Cooperating  Unit:   Laboratory  of  Clinical  Psychobiology,  National  Institute  C\l 

of  Mental  Health.  i',/ 

11  li'f 

""ii 
Project  Description:  ,„" 

Objectives:   Behaviour  is  an  important  aspect  of  hypertension  and  little  i" 
information  is  available  on  the  behaviour  of  hypertensive  rats.   We  felt 

that  a  study  of  aggression  in  the  genetically  pure  strain  of  spontaneously  I 

hypertensive  rats  (SHR)  might  give  valuable  data  when  compared  to  adequate  n" 

normotensive  and  hypertensive  controls.  !l'' 

Methods:   Fighting  was  induced  in  paired  female  rats  by  applying  50  electric 
shocks  (2mA)  to  their  feet  in  each  session  on  four  consecutive  days.   In 
addition  mouse  killing  was  studied  in  all  rats  used  and  we  also  determined 
the  jump-flinch  thresholds  by  a  standardized  method.   The  rats  used  were 
10-12  weeks  old.   Renal  hypertension  was  induced  by  application  of  a  solid 
silver  clip  (0.25  mm  int.  diameter)  on  the  left  renal  artery  in  8  weeks  old 
rats.   These  rats  were  studied  5-7  weeks  after  the  operation. 

Major  Findings:   In  normotensive  Wistar  rats  (NIH)  a  high  fighting  rate  was 
observed  on  all  four  days,  while  another  normotensive  strain  (Sprague-Dawley 
rats,  Zivic  Miller  Laboratories)  showed  low  fighting  rates  on  all  four  days. 
Spontaneously  hypertensive  rats  had  a  high  fighting  rate  on  the  first  day 
studied  which  decreased  to  low  levels  on  the  subsequent  days.   Comparison  to 
renal  hypertensive  rats  (Wistar  strain,  NIH)  indicated  that  this  decrease  was 
not  a  direct  effect  of  the  high  blood  pressure  since  fighting  rates  of  the 
renal  hypertensive  rats  remained  high  as  observed  in  normotensive  Wistar 
controls.   No  significant  differences  in  mouse  killing  were  observed  between 
the  different  groups  of  rats  studied.   A  slightly  lower  jump-flinch  threshold 
was  observed  in  the  S.H.  rats. 


/«/ 


^ 


Serial  No.  NHLI-95 

Significance:  These  findings  show  that  high  blood  pressure  by  itself  does  not 
affect  shock-induced  fighting  of  paired  rats.  The  decrease  of  fighting  rates 
of  SHR  may  be  specific  for  genetic  hypertensive  rats. 

Proposed  Course  of  Project:   Shock- induced  aggression  in  another  type  of 
genetic  hypertensive  rats  (salt  dependent  hypertension)  will  be  studied  to 
obtain  information  about  the  specificity  of  the  observed  decrease  in  fighting 
rate  in  SHR. 

Publications:   None 


/^l 


mm 


Serial  No.       NHLI-96 


1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Renin -angiotensin  System  of  the  Spontaneously  Hypertensive 
Rat. 

Previous  Serial  Number:  None 

Principal  Investigator:  Wybren  De  Jong,  M.D. ,  Ph.D.  (Guest  Worker) 

Other  Investigators:  Walter  M.  Lovenberg,  Ph.D.  and  Albert  Sjoerdsma,  M.D. , 
Ph.D. 

Cooperating  Units:  None 

Project  Description: 

Ob j ectives :  The  renin-angiotensin  system  is  o£  importance  in  hypertension  and 
its  role  is  insufficiently  documented  in  the  genetically  pure  strain  of 
spontaneously  hypertensive  rats  (SHR) .  To  obtain  a  greater  understanding  of 
"die  contribution  of  this  system  to  the  high  blood  pressure  in  these  animals 
we  investigated  this  system  in  male  SHR  at  different  ages. 

Major  Findings:  Plasma  renin  activity  of  SHR  (3.9  ng/ml)  was  found  to  be  high- 
er  than  in  controls  (1.5  ng/ml)  at  12  weeks  of  age  and  this  difference  re- 
mained up  to  35  weeks  of  age.  At  8  weeks  in  the  initial  experiment  no  signifi- 
cant difference  occurred.  However,  in  additional  experiments  an  increase  of 
plasma  renin  activity  of  SHR  was  found  as  early  as  6  weeks  of  age.  An  increase 
in  plasma  renin  substrate  was  observed  in  SHR  of  16  weeks  and  older.  At  the 
age  of  20  weeks  renin  substrate  levels  were  665  +  18  ng/ml  in  Wistars  and 
905  +_  43  ng/ml  in  the  SHR.  A  marked  decrease  inlcidney  renin  content  was  found 
in  SH  rats  at  35  weeks  of  age.  When  renal  renin  release  was  stimulated  by 
pentobarbital,  plasma  renin  activity  of  renal  venous  blood  of  SHR  did  not 
differ  from  that  of  normotensive  Wistar  rats.  Also  no  difference  in  plasma 
renin  activity  was  observed  \^^en  measured  at  the  time  of  peak  activity  during 
normal  diurnal  rhythm.  Preliminary  studies  indicated  that  the  elevated  plasma 
renin  in  SHR  may  arise  from  an  extrarenal  source,  since  it  is  still  present  in 
plasma  of  these  rats  18  hours  after  nephrectomy. 

Significance :  Several  investigators  suggested  that  the  renin  angiotensin 
system  is  depressed  by  the  high  blood  pressure  level  in  genetic  hypertensive 
rats.  The  decrease  of  kidney  renin  content  in  older  animals  might  be  a 
reflection  of  such  a  mechanism.  The  present  findings,  however,  do  not  confirm 
such  a  mechanism  in  SH  rats.  The  significance  of  the  observed  increased 
plasma  renin  activity  in  SPIR  for  the  high  blood  pressure  remains  to  be 
ascertained.  Occurrence  of  a  extrarenal  renin- like  enzyme  that  contributes  to 

1  ^83 


f  \/ 


rn  7 

Pi 

V 


Serial  No.   NHLI-96 


plcisma  renin  activitv'  is  unusual  but  has  been  shown  in  nephrectomized 
patients  and  in  different  animal  species  after  a  bilateral  nephrectomy. 

Proposed  Course  of  Project:  It  is  proposed  to  clarify  the  mechanism  which 
induces  increased  plasma  renin  activity  in  SH  rats ,  to  determine  the  source 
of  the  presumably  extra-renal  renin  like  enzyme  and  to  evaluate  the  con- 
tribution of  the  increased  plasma  renin  activity  to  the  hypertension. 

Honors  and  Awards:  None 

Publications:  None 


/B^ 


Serial  No.      NHLI-97 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Tryptophan  Hydroxylase  and  Phenylalanine  Hydroxylase: 
Comparative  Properties,  Purification  and  Interactions. 

Previous  Serial  Number:  None 

Principal  Investigator:  Richard  E.  Bensinger,  M.D. 

Other  Investigators:  Walter  M.  Lovenberg,  Ph.D. 

Cooperating  Units:  None 

Project  Description: 

Ob j  ectives :  Tryptophan  hydroxylase  and  phenylalanine  hydroxylase  catalyze  an 
important  step  in  the  biogenesis  of  clinically  significant  intermediary  sub- 
stances in  amine  metabolism.  The  fundamental  properties  appear  similar  for 
the  two  enzymes  but  little  work  has  been  done  comparing  the  two.  This  project 
will  investigate  these  interrelationships  and  attempt  to  purify  the  enzymes  to 
accomplish  this  purpose.  In  addition,  clinically  useful  information  related 
to  diagnosis  of  disorders  involving  these  two  enzymes  is  being  sought. 

Major  Findings:  A  new  assay  for  tryptophan  hydroxylase  has  been  developed. 
This  assay  is  based  on  the  release  of  tritium  atoms  from  5 -^H- tryptophan  fol- 
lowing hydroxylation.  Although  the  "NIH  shift"  occurs,  incubation  of  the 
product,  4-2H-5-hydroxytryptophan,  under  acid  conditions  results  in  complete 
release  of  the  tritium.  The  assay  can  measure  the  formation  of  as  little  as 
0,2  mymoles  of  5 -hydroxy tryptophan  and  can  be  used  with  small  tissue  samples 
from  the  central  nervous  system,  liver,  or  kidney.  This  assay  and  a  similar 
one  for  phenylalanine  hydroxylase  has  facilitated  the  remainder  of  the  studies. 

Tryptophan  hydroxylase  from  bovine  pineal  has  been  purified  some  30 -fold 
using  standard  protein  fractionation  procedures.  Phenylalanine  hydroxylase 
has  also  been  partially  purified  from  rat  liver  by  a  published  procedure. 
Attempts  to  more  easily  prepare  this  enzyme  using  affinity  chromatography  re- 
sulted in  the  isolation  of  a  fraction  from  rat  liver  which  had  very  high 
specific  activity  using  phenylalanine  as  a  substrate,  but  little  activity  to- 
ward tryptophan.  This  fraction,  however,  strongly  stimulated  tryptophan 
hydroxylase  which  had  been  partially  purified  from  bovine  pineals.  The  trypto- 
phan hydroxylase  stimulating  factor  (THS)  was  present  in  crude  tissues  and 
appeared  to  be  purified  simultaneously  with  phenylalanine  hydroxylase  activity. 
It  was  both  heat  labile  and  non-dialyzable.  It  is  possible  that  THS  is 

1  /0sr 


f\/ 


Serial  No.  NHLI-97 


s)Tionomous  with  liver  phenylalanine  hydroxylase.  THS  both  stimulates 
tryptophan  hydroxylase  and  shifts  the  pH  optijnum  from  7.3  to  8.2.  Although 
the  partially  purified  fractions  from  beef  pineal  hydroxylate  both  phenyl- 
alanine and  tryptophan,  only  tr)'ptophan  hydroxylating  activity  is  stimulated. 
Studies  on  cofactor  specificity  indicate  that  only  fenrous  iron  satisfies 
the  metal  requirement  and  that  ascorbic  acid  will  not  replace  the  reduced 
pteridine  requirement. 

Another  part  of  this  project  is  designed  to  develop  specific  radio- 
pharmaceuticals which  will  localize  tumors  having  an  excess  of  these  enzymes, 
or  enzymes  of  related  systems.  The  labeled  nuclide  is  attached  to  substrates 
or  inhibitors  having  a  high  affinity  (low  I^  or  Kj)   for  the  enzyme,  thus 
binding  there  and  identifying  the  neoplastic  tissue.  Preliminary  experiments 
are  being  conducted  by  measuring  tlie  tissue  distribution  of  131-iodotyrosine 
in  rats.  Since  this  is  an  inhibitor  with  high  affinity  for  tyrosine 
hydroxylase,  it  might  be  expected  to  be  differentially  retained  by  the  adrenal 
glands  which  have  a  large  amount  of  this  enzyme.  No  such  distribution  is  yet 
evident . 

Significance:  The  development  of  a  simple  and  sensitive  radioassay  for 
tryptophan  hydroxylase  will  greatly  enlarge  the  scope  of  the  work  that  can  be 
done  with  this  important  enzyme.  The  finding  of  a  second  protein  factor  that 
stimulates  tryptophan  hydroxylase  may  in  part  explain  the  great  difficulties 
previously  encountered  in  attempts  to  purify  this  enzyme.  Since  the 
stimulating  factor  appears  to  be  identical  with  phenylalanine  hydroxylase,  it 
suggests  that  mammalian  hydroxylating  enzymes  may  consist  of  several  subunits 
and  that  the  substrate  specificity  in  individual  organs  may  reflect  a  particu- 
lar combination  of  these  units.  These  findings  also  have  implications  for  the 
disease  phenylketonuria  in  which  individuals  lack  the  enzyme  phenylalanine 
hydroxylase  but  appear  to  be  capable  of  forming  hydroxyindoles .  Further  under- 
standing of  the  hydroxylase  reactions  are  inportant  in  the  design  of  com- 
pounds to  help  regulate  serotonin  synthesis  in  m.ental  disorders  and  in  hydroxy- 
indole  producing  tumors  (carcinoid) . 

The  development  of  techniques  using  radionuclides  to  detect  areas  of 
relatively  high  concentration  of  certain  enzymes  would  be  of  obvious  benefit 
both  in  the  diagnosis  and  surgical  removal  of  tumors. 

Proposed  Course  of  Project:  The  complete  isolation  of  the  tryptophan 
hydroxylase  stimulating  factor  will  be  attempted.  Using  this  factor  attempts 
to  isolate  pineal  tryptophan  hydroxylase  will  be  made.  Continued  attention 
will  be  devoted  to  possible  inhibitors  and  their  possible  clinical  significance. 

Honors  and  Awards:  None 

Publications: 

1.  Lovenberg,  W.  and  Jackson,  R.L.:  Tryptophan- 5 -hydroxylase.  In: 
Methods  in  Investigative  and  Diagnostic  Endocrinology.  In  press. 


Serial  No.   NHLI-97 


2.  Lovenberg,  W. ,  Bensinger,  R.E.,  Jackson,  R.L.,  and  Daly,  J,W. :  Rapid 
analysis  o£  tryptophan  hydroxylase  in  rat  tissue  using  5 -3h- tryptophan. 
Anal.  Biochem.  In  press. 


i 


^37 


I 


Serial  No.    NHLI-98 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Studies  on  the  Isolation  and  Characterization  of  Clostridial 
Electron  Transfer  Proteins  and  Other  Iron-Sulfur  Proteins. 

Previous  Serial  Number:  NHLI-154 

Principal  Investigator:  Walter  M.  Lovenberg,  Ph.D. 

Other  Investigators:  William  A.  Eaton,  M.D. ,  Ph.D. 

Cooperating  Units:  Laboratory  of  Physical  Biology,  NIAMD;  Central  Research 
Department  of  DuPont,  Wilmington,  Delaware;  Department 
of  Chemistry,  Pennsylvania  State  University,  University 
Park,  Pennsylvania. 

Project  Description: 

Objectives:  Iron-sulfur  proteins  are  a  large  class  of  proteins  that  catalyze 
many  vital  redox  functions  in  living  organisms.  The  major  objectives  of  this 
project  are  to  elucidate  the  structure  of  the  active  center  of  this  type  of 
protein,  and  to  learn  how  this  relates  to  its  role  in  accepting  or  donating 
electrons.  Both  rubredoxin,  a  single-iron  protein,  and  the  ferredoxin  type 
multi-iron  proteins  have  been  used  in  this  work.  Clostridial  rubredoxin  which 
was  first  isolated  in  this  laboratory  several  years  ago  is  the  most  simple  of 
the  iron-sulfur  proteins,  consisting  of  a  single  polypeptide  chain  which  con- 
tains four  cysteine  residues,  positioned  in  such  a  way  that  the  sulfhydryl 
groups  tightly  chelate  a  single  iron  atom.  The  ferredoxin  type  protein  also 
consist  of  a  single  polypeptide,  but  these  contain  several  sulfhydryl  groups 
which  fonn  a  much  more  complex  structure  with  2  or  more  iron  and  inorganic 
sulfide  atoms.  These  proteins  normally  occur  in  the  oxidized  form  and  trie 
addition  of  one  or  two  electrons  to  the  iron-sulfur  complex  results  in  changes 
in  the  physical -chemical  properties  that  provide  clues  for  determining  the 
nature  of  the  active  center.  Several  chemical  and  physical  techniques  have 
been  used  to  further  understand  the  nature  of  the  iron  binding  site. 

Methods:  A.  Rubredoxin.  Rubredoxin  from  Clostridium  pasteurianum  consists 
of  54  amino  acids.  Using  the  classical  amino  acid  sequencing  techniques  and 
data  provided  by  x-ray  crystallographic  analysis  the  following  tentative 
sequence  has  been  determined: 


fee 


Serial  No.   NHLI-98 


F-Met  Lys  Lys  Tyr  Thr  Cys  Thr  Val  Cys  Gly  Tyr  Ilu  Tyr  Asp  Pro  Glu  Asx  Gly  Asx 
Pro  Val  Asx  Gly  Asx  Asx  Asp  Gly  Thr  Asp  Phe  Lys  Asp  Ilu  Pro  Asp  Asp  Try  Val 
Cys  Pro  Leu  Cys  Gly  Val  Gly  Lys  Asp  Glu  Phe  Glu  Glu  Val  Gin  Glu 

This  sequence  shows  a  number  of  homologies  with  the.  2  other  rubredoxins  from 
anaerobes  that  have  been  sequenced,  and  with  each  half  of  the  much  larger 
Pseudomonad  oleovoran  rubredoxin.  Most  important,  however,  is  the  observa- 
tion  that  the   cysteine  residues  occur  in  homologous  positions  since  it  is  the 
sulfhydryl  groups  that  hold  the  iron  atom.  Also  of  interest  is  the  finding 
that  the  initiator  amino  acid  of  bacterial  polypeptide  synthesis,  N  formyl- 
methionine,  is  retained  at  the  N  terminus.  This  is  the  first  native  protein 
that  has  been  found  to  retain  this  residue. 

The  geometry  of  the  ligand  field  of  the  high  spin  iron  atom  in  rubredoxin 
was  reported  previously  to  be  approximately  tetrahedral.  This  was  determined 
by  x-ray  crystallographic  analysis,  and  by  the  measurement  of  a  very  optical- 
ly active  d  ^  d  transition  in  the  near  infrared  using  the  technique  for 
measuring  circular  dichroism  in  the  infrared  developed  in  this  project.  This 
work  has  been  continued  and  extended.  The  original  observations  indicated  an 
optically  active  absorption  band  at  about  1.6  p  in  reduced  rubredoxin.  Be- 
cause of  a  "\/indow"  in  the  opacity  of  D2O,  we  now  have  been  able  to  extend  the 
observed  spectra  to  include  0.8  to  1.8  y  and  2.1  to  2.5  \i.     The  data  indicate 
that  another  optically  active  band  in  reduced  rubredoxin  is  centered  at  a 
somewhat  lower  energy  than  2.5  vi.  This  band  is  also  interpreted  as  arising 
from  a  d  ^  d  transition  in  the  reduced  rubredoxin.  The  splitting  of  the  d  ->■  d 
absorption  bands  probably  results  from  a  distortion  in  perfect  tetrahedral 
symmetry  around  the  iron  atom.  This  distortion  has  been  confirmed  by  the 
large  degree  of  didiroism  in  the  visible  absorption  spectra  of  single  crystals 
observed  parallel  and  perpendicular  to  the  crystal  axis.  The  x-ray  analysis 
also  suggests  some  distortion.  Another  approach  to  the  ligand  geometry  of 
rubredoxin  has  been  examination  of  the  Laser-Raman  spectra  in  collaboration 
with  Dr.  Thomas  V.  Long.  Excitation  of  the  molecule  with  the  488  nm  line  of 
an  Ar"*"  laser  results  in  Raman  spectra  which  are  consistent  with  a  tetra- 
hedrally  bound  iron  atom. 

Examination  of  the  magnetic  susceptibility,   the  nuclear  magnetic 
resonance  spectra  and  the  Mossbauer  spectra  of  rubredoxin  all  were  consistent 
with  the  following  conclusions:  1)  the  iron  of  rubredoxin  is  high  spin  ferric 
and  high  spin  ferrous  in  the  oxidized  and  reduced  forms  respectively,  and  2) 
the  iron  is  held  in  a  ligand  field  consisting  of  the  four  cysteinyl  sulf- 
hydryls . 

B.  Ferredoxin  Type  Proteins.  It  was  reported  previously  that  in  C. 
pasteurianum  ferredoxin  all  the  g  protons  of  the  cysteinyl  residues  were  very 
strikingly  contact  shifted  in  the  NMR  spectrum.  This  was  interpreted  as 
resulting  from  the  bonding  of  the  sulfhydryl  groups  to  the  iron  atoms  since 

2  /8f 


~*7=^'^iC'!i'.itri 


Serial  No.   NHLI-98 


these  contact  shifted  resonances  exhibited  anti-Curie  law  behavior.  Studies 
on  this  phenomenon  have  been  further  refined  and  extended  over  broader 
temperature  ranges,  but  with  no  new  fundamental  discoveries. 

More  significant,  however,  has  been  the  application  of  the  teclmiques  for 
measuring  infrared  absorption  and  circular  dichroism  developed  in  the 
rubredoxin  study  to  plant  ferredoxin  and  adrenodoxia.  Tliese  two  electron 
carriers  which  serve  key  roles  in  plant  and  mammalian  metabolism  each  have  2 
iron  and  2  inorganic  sulfide  atoms  and  are  similar  in  many  physical  properties, 
altliough  the  structure  of  their  active  centers  is  not  known.  The  following 
proteins  were  examined:  spinach  ferredoxin,  parsley  ferredoxin  and  beef 
adrenodoxin.  Each  of  these  proteins  had  an  optically  active  band  at  approxi- 
mately 1.6  u  when  in  the  reduced  state.  Although  the  extinction  coefficient 
and  anisotropy  factor  v/ere  not  identical  to  rubredoxin,  these  band^  were 
qualitatively  similar  and  suggested  the  possibility  that  a  high  spin  ferrous 
atom  was  tetrahedrally  coordinated.  Like  rubredoxin  these  proteins  also  had 
absorption  bands  at  energies  lower  than  2.2  u.  '/\(hile  the  evidence  is  somewhat 
less  convincing  than  in  the  case  of  rubredoxin,  it  is  consistent  with  a  recent- 
ly proposed  model  for  the  active  center  of  the  2  iron-2  sulfur  protein  depicted 
in  2  dimensions: 


Significance:  The  development  of  indirect  techniques  using  the  simple  iron- 
sulfur  protein  rubredoxin  for  probing  the  nature  of  the  iron  binding  in  other 
iron-sulfur  proteins  has  been  successful.  Use  of  such  techniques  has  facil- 
itated a  greater  understanding  of  the  active  center  in  the  very  important  iron- 
sulfur  protein  plant  ferredoxin  and  adrenodoxin. 

Proposed  Course  of  Project:  Work  will  continue  on  the  use  of  infrared  absorp- 
tion  and  circular  dichroism  to  elucidate  the  structure  of  the  more  complex 
iron-sulfur  proteins.  Studies  on  the  role  of  various  amino  acid  side  chains 
in  maintaining  the  conformation  necessar)'  for  iron  binding  will  be  initiated. 
The  single  crystal  spectra  of  rubredoxin  will  be  examined  carefully  in  an 
attempt  to  use  this  means  to  define  the  nature  of  the  distortion  in  the  tetra- 
hedral  ligand  field. 

Honors  and  Awards:  None 

Publications: 

1.  Phillips,  W.D. ,  Poe,  M. ,  Weiher,  J.F.,  McDonald,  C.C.,  and  Lovenberg,  W.: 
Proton  magnetic  resonance,  magnetic  susceptibility  and  Mossbauer  studies 
of  Clostridium  pasteurianum  rubredoxin.  Nature  227:  574-577,  1970, 

3  /9a 


Serial  No.   NHLI-98 


2.  McCarthy,  K.  F.  and  Lovenberg,  W. :  N-£ormy Methionine:  The  N  terminus 
o£  Clostridium  pasteurianum  nibredoxin.  Biochem.  Biophys.  Res.  Commun. 
40:  1053-1057,  1970.  ' 

3.  Eaton,  W.  A.  and  Lovenberg,  W. :  Near-infrared  circular  dichroism  o£  an 
iron- sulfur  protein,  d  ->-  d  transitions  in  rubredoxin.  J.  Am.  Chem. 
Soc.  92:  7195-7198,  1970.  ' 

4.  Long,  T.  v.,  II,  Loehr,  T.  M. ,  Allkins,  J.R. ,  and  Lovenberg,  W. :  The 
determination  of  iron  coordination  in  nonheme  iron  proteins  using 
Laser-Raman  spectroscopy.  II.  Clostridium  pasteurianum  rubredoxin  in 
aqueous  solution.  J.  Am.  Chem.  Soc.  In  press. 


^^/ 


i«eWS«3E»-K-^.'^  %,;.«.  .^•<^£.' 


Serial  No.    NHLI-99 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  19  70  through  June  30,  1971 

Project  Title:   Catecholamine  Metabolism  and  Blood  Pressure  of  Spontaneously 
Hypertensive  Rats. 

Previous  Serial  Number:   NHLI-156 

Principal  Investigator:   Hirohiko  Yamabe,  M.D. 

Other  Investigators:   Wybren  DeJong,  M.D. ,  Ph.  D, ,  B.  Van  Clineschmidt,  Ph.D., 
Yukio  Yamori,  M.D.,  Walter  Lovenberg,  Ph.  D.  and 
Albert  Sjoerdsma,  M.D. ,  Ph.  D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Previous  studies  have  shown  that  a  genetically  pure  strain  of 
spontaneously  hypertensive  rats  (SHR)  had  lower  levels  of  brainstem  norepi- 
nephrine (NE)  than  similar  Wistar  control  animals.   The  objective  of  this 
study  was  tc  attempt  to  correlate  the  reduced  brainstem  NE  with  the  develop- 
ment of  hypertension. 

Major  Findings:   Norepinephrine  Synthesis.   The  mechanism  which  results  in 
decreased  brainstem  NE  in  the  SHR  is  unknown.   An  examination  of  the  enzymes 
responsible  for  the  biosynthesis  of  NE  in  SHR  and  control  rats  indicated  that 
tyrosine  hydroxylase  was  similar  in  each,  but  the  level  of  aromatic  L^  amino 
acid  decarboxylase  was  reduced  to  about  50%  in  SHR.   The  decrease  in  decarboxyl- 
ase was  apparent  even  in  newly  born  animals.   Crosses  between  SHR  and  control 
animals  gave  a  F-^   generation  that  had  decarboxylase  levels  that  were  inter- 
mediate between  the  hypertensive  and  control  animals.   Examination  of  the 
kinetic  properties  of  the  enzyme  from  each  source  revealed  that  the  Km  values 
for  ]^-5-hydroxytryptophan  and  L^-dopa  were  essentially  the  same  from  either 
strain.   The  values  for  L-5-HTP  were  about  8  x  lO-^M  and  for  L-dopa  2.5  x  10"%. 
Thus,  it  appears  that  the  gross  amounts  of  aromatic  I^-amino  acid  decarboxylase 
in  these  two  strains  of  Wistar  rats  are  different  and  that  these  levels  are 
genetically  controlled. 

Effects  of  6-Hydroxydopamine  (6-HDA) .   It  is  known  that  6-HDA  destroys 
sympathetic  nerve  endings  when  administered  peripherally.   If  the  hypertension 
in  the  SHR  was  in  any  way  related  to  over-activity  of  the  sympathetic  nervous 
system,  then  destruction  of  the  peripheral  sympathetic  nerves  should  result 
in  a  reduction  in  blood  pressure.   Administration  of  6-HDA  resulted  in 


/f2 


TO 


Serial  No.  NHLI-99 

destruction  of  up  to  80%  of  the  sympathetic  nerves  in  heart,  spleen,  and 
kidney  as  determined  by  catecholamine  content  of  these  organs.   The  treatment 
resulted  in  no  more  than  a  very  transient  effect  on  blood  pressure.   Con- 
versely it  was  felt  that  if  levels  of  catecholamines  in  the  brainstem  were 
inversely  related  to  blood  pressure  that  central  administration  of  6-HDA  in 
normal  animals  should  result  in  the  development  of  hypertension.   Several 
experiments  were  done  in  which  various  doses  (50  to  800  yg)  of  6-HDA  were 
injected  intraventricular ly.    In  one  experiment  (100  ]sg   6-HDA  centrally) 
the  animals  developed  significant  increases  in  blood  pressure,  however, 
several  subsequent  experiments  failed  to  confirm  this  finding.   In  all 
experiments,  however,  substantial  (>  50%)  decreases  in  brainstem  NE  were 
found.   From  these  experiments  one  could  not  substantiate  a  clear  inverse 
relationship  between  brainstem  NE  nor  could  the  peripheral  sympathetic  system 
be  implicated  in  the  development  of  hypertension. 

Catecholamine  Repletion.   Attempts  were  made  to  elevate  the  levels  of 
catecholamines  in  the  central  nervous  system  of  the  SHR.   Administration  of 
the  catecholamine  precursor  L^-dopa  (200  mg/kg)  to  SHR  with  or  without  the 
peripheral  decarboxylase  inhibitor  MK-486  (L^-hydrazino-a-methyldopa) ,  100  mg/kg, 
resulted  in  an  acute  and  significant  decrease  in  blood  pressure.   Continued 
administration  of  MK-486  and  L^-dopa  for  several  days  in  the  SHR  continued  to 
depress  blood  pressure.   Chronic  treatment  of  animals,  however,  did  not  appear 
to  be  feasible  since  the  animals  receiving  this  drug  treatment  ceased  to  grow 
normally.   Using  several  different  dosage  schedules  of  dopa  with  or  without 
a  decarboxylase  inhibitor  and/or  a  monoamine  oxidase  inhibitor,  a  significant 
inverse  correlation  between  blood  pressure  and  brainstem  NE  was  observed. 
Since  treatment  of  animals  with  L^-dopa  causes  a  decreased  serotonin  content 
of  the  brainstem,  the  effect  of  the  specific  serotonin  depletor  p-chloro- 
phenylalanine  (PCPA)  was  studied.   In  normotensive  Wistars  and  SHR  a  slight 
but  significant  increase  in  blood  pressure  occurred  following  a  serotonin 
depleting  dose  of  300  mg/kg  of  PCPA.   Administration  of  the  normal  serotonin 
precursor,  5-HTP,  to  normal  or  SHR  did  not  affect  blood  pressure.   It  was 
therefore  concluded  that  the  ability  of  L-dopa  to  reduce  blood  pressure  was 
perhaps  related  to  catecholamine  repletion,  but  probably  not  to  serotonin 
depletion. 

Significance:   Hypertension  in  man  is  a  complex  disorder  probably  resulting 
from  a  variety  of  factors.   The  genetically  hypertensive  rats  appear  to  be 
an  excellent  animal  model  of  essential  hypertension  in  man.   The  current  work 
raises  the  possibility  that  one  of  the  factors  in  the  hypertension  of  these 
animals  is  a  decrease  in  the  number  of  or  activity  of  noradrenergic  neurons 
in  the  central  nervous  system.   It  is  interesting  to  note  that  one  of  the 
side  effects  in  patients  receiving  L^-dopa  for  Parkinsonism  is  hypotension. 
At  a  metabolic  level  this  work  also  raises  the  possibility  that  of  the 
enzymes  involved  in  the  biosynthesis  of  NE  from  tyrosine,  tyrosine  hydroxylase 
may  not  be  the  rate  limiting  enzyme  in  all  conditions.   In  other  words  the  NE 
biosynthetic  enzymes  may  be  reasonably  well  balanced  in  vivo  and  a  50%  decrease 
in  aromatic  L^  amino  acid  decarboxylase  may  be  sufficient  to  affect  the  level 
of  NE  in  tissues. 


I 


^3 


Serial  No.  NriLI-99 

Proposed  Course  of  Project:   Studies  are  now  in  progress  using  H-dopa  and 
jI-^C- tyrosine  as  precursors  of  ME  to  determine  if  the  SHR  have  a  lower  NE 
synthetic  rate  and  if  in  this  instance  the  decarboxylase  could  be  rate 
limiting  in  the  central  nervous  system.   Systems  have  been  reestablished  for 
the  complete  separation  and  measurement  of  tyrosine,  dopa,  dopamine  and  nor- 
epinephrine in  tissues  and  experiments  are  being  done  to  evaluate  central  and 
peripheral  catecholamine  metabolism  in  SHR. 

Publications; 

1.   Yamori,  Y.,  Lovenberg,  W. ,  and  Sjoerdsma,  A.:   Norepinephrine  metabolism 
in  brainstem  of  spontaneously  hypertensive  rats.   Science  170:  544-5''^6, 
1970. 


^^y 


i 


Serial  No.      NHLI-100 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


Project  Title:   Studies  on  a  Pineal  Gland  Protein  Kinase 

Previous  Serial  Number:   None 

Principal  Investigator:   Joseph  Fontana,  Ph.  D. 

Other  Investigators:   Walter  Lovenberg,  Ph.  D. 

Project  Description: 

Objectives:   It  has  been  observed  that  norepinephrine  stimulates  the  pro- 
duction of  melatonin  in  the  pineal  gland.   This  stimulation  appears  to  operate 
through  a  cyclic  AMP  mechanism.   The  objective  of  the  present  investigation  is 
to  probe  for  the  existence  of  a  cyclic-AMP  sensitive  control  mechanism  in 
pineal  extracts,  characterize  it  and  determine  its  physiological  significance. 

Methods:   Cyclic-AMP-dependent  protein  kinases  which  catalyze  the  phosphoryla- 
tion of  casein,  protamine  and/or  histone  by  ATP  have  been  found  in  muscle, 
liver,  brain,  rabbit,  reticulocytes,  adipose  tissue  and  bacteria.   Utilizing 
the  hypothesis  that  such  a  cyclic-AMP  dependent  kinase  also  exists  in  the 
pineal  gland,  a  protein  kinase  assay  was  adapted  to  the  pineal  system.   The 
assay  simply  involves  the  phosphorylation  by  ATP-yP-^^  of  either  endogenous  or 
exogenous  (histone,  casein,  etc.)  substrate  in  the  absence  or  presence  of 
cyclic  AMP. 

The  sources  of  pineals  employed  in  the  study  are  Pel-Freeze  and  Swift  and 
Co.   Rat  pineals  are  freshly  obtained  from  Sprague-Dawley  rats. 

Major  Findings:   Cyclic-AMP  dependent  kinase  activity  was  found  in  bovine 
pineal  homogenates.   The  activity  was  partially  purified  by  (NH4)2S04 
precipitation  (0-33%)  and  passage  through  DEAE-cellulose  using  step  elution 
with  0.1  and  0.3M  potassium  phosphate  pH=7.0  buffers.   Two  peaks  of  protein 
kinase  activity  were  eluted  from  the  DEAE-cellulose  column-one  eluting  a 
O.IM  potassium  phosphate  and  the  second  at  0.3M  potassium  phosphate.   The 
entire  activity  from  the  latter  peak  was  stimulated  from  10-30  fold  by  cyclic- 
AMP.   Maximum  stimulation  occurred  at  SxlO'^M  cyclic-AMP.   Although  a  variety 
of  proteins,  including  histone,  phosvitin,  casein  and  bovine  serum  albumin 
were  able  to  act  as  phosphate  acceptors  for  the  enzyme,  histone  was  far  more 
effective  in  low  amounts  on  a  weight  basis  than  were  any  of  the  other  proteins 
studied.   Lysine  rich  histone  and  histone  seem  to  give  similar  results  and 
both  are  better  substrates  than  arginine  rich  histone. 


L 


/?r 


;:i 


Serial  No.  NHLI-100 

It  has  also  been  possible  to  detect  protein-kinase  activity  in  individual 
rat  pineals  (=  1  mg  wet  weight).   Preliminary  experiments  show  that  this 
activity  is  slightly  stimulated  by  cyclic-AMP.   Conditions  must  be  further 
developed  to  confirm  this  cyclic-AJIP  stimulation. 

Significance  and  Proposed  Course  of  Project:   The  diurnal  variation  of  the 
enzyme  levels  in  the  melatonin  pathway  in  pineals  offers  a  unique  means  of 
examining  one  possible  physiological  significance  of  the  cyclic-AMP  dependent 
protein  kinase  activity.   Further  studies  will  examine  the  response  of  the 
cyclic-AMP  dependent  protein  kinase  to  variations  in  environmental  lighting 
and  stimuli  from  the  central  nervous  system  and  correlate  these  responses  to 
variations  in  the  levels  of  the  melatonin  biosynthetic  enzymes  and  phosphoryla- 
tion of  endogenous  histones.   These  studies  may  elucidate  the  role  of  histone 
phosphorylation.   We  will  also  attempt  to  purify  and  characterize  the  protein 
kinase (s)  from  the  pineal. 

Pubiicacions :   None 


/f^ 


Serial  No.       NHTJ-101     

1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 

PHS-NBQliI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  Naturally  Occurring  Vasoactive 
Substances 

Previous  Serial  Number:   NHLI-141 

Principal  Investigator:   Ronald  G.  Geller,  Ph.D. 

Other  Investigators:   Wybren  DeJong,  M.D.,  Ph.D. 

Renee  Ray-Chung  Chen,  Ph.D. 
Joseph  Pierce,  D.V.M. 
Michael  Heaven,  Ph.D. 
Takenori  Tanimura,  Ph.D. 
John  J.  Pisano,  Ph.D. 

Cooperating  Units:   Laboratory  of  Animal  Medicine  and  Surgery, 

NHLI 


Project  Description: 

Objectives;   To  characterize  the  vasoactive  substances  1)  found 
in  dog  plasma  following  bilateral  renal  artery  ligation,  2)  in 
the  venom  of  the  bald-faced  hornet,  and  3)  which  are  synthetic 
analogs  of  ranatensin,  an  undecapeptide  isolated  from  frog  skin. 

Methods:   Various  isolated  smooth  muscle  preparations  and  blood 
pressure  recordings  from  experimental  animals  were  employed. 

Major  Findings:   A  substance  which  raised  blood  pressure  in  the 
pentolinium  treated  rat  was  found  in  the  plasma  of  dogs  after 
bilateral  renal  artery  ligation.   It  was  isolated  from  the  40-70% 
ammonium  sulfate  fraction.   It  has  been  suggested  that  this 
pressor  substance  may  be  a  protein,  angiotensin  I,  angiotensin  II, 
or  a  peptide  bound  to  a  protein.   The  nature  of  the  blood  pressure 
response  in  the  rat  and  the  lack  of  a  response  on  isolated  rat 
uterus  or  rat  colon  suggests  that  the  substance  is  like  angiotensin 
I,  but  is  not  angiotensin  II.   The  pressor  activity  is  abolished 
by  boiling  the  plasma  indicating  the  possibility  of  an  angiotensin 
I-protein  complex. 

1  /f7 


NHLI-101 
The  venom  of  the  bald-faced  hornet  was  found  to  contain  a 
potent  hypotensive  agent  on  rat  blood  pressure.   Experiments 
using  the  isolated  rat  uterus  and  guinea  pig  ileum  indicated 
that  the  venom  contained  in  addition  serotonin  (1-5  ug/sac)  and 
and  histamine  (2.5  iig/sac)  .   It  did  not  contain  acetylcholine  or 
bradykinin.   Histamine  and  serotonin  were  also  measured  using 
fluorescence  assays.   The  hypotensive  response  in  the  rat  was 
not  completely  blocked  by  antihistamine  and  anti-serotoninergic 
agents.   This  suggests  that  another  substance  possibly  a  peptide, 
is  present,  but  has  no  action  on  the  isolated  preparations. 

Ranatensin  is  an  undecapeptide  isolated  from  frog  skin  with 
the  amino-acid  sequence  PYR-VAL-PRO-GLN-TRP-ALA-VAL-GLY-HIS-PHE- 
MET-NH2.   It  has  a  characteristic  spectrum  of  pharmacological 
activity  which  includes  a  potent  depressor  action  on  the  blood 
pressure  of  monkeys  and  a  potent  stimulant  action  on  the  isolated 
rat  uterus.   The  following  analogs  of  ranatensin  have  been  synthe- 
sized and  their  biologic  activities  studied.   (See  table). 

Analogs  which  lowered  blood  pressure  were  also  active  on 
the  uterus,  but  one,  C-8,  was  much  more  active  in  lowering  blood 
pressure  than  in  contracting  the  uterus. 

Significance  to  Biom.edical  Research  and  Institute  Program:   An 
analog  of  ranatensin  which  lowers  blood  pressure,  but  does  not 
contract  other  smooth  muscles  could  be  a  suitable  one  for  testing 
in  hypertension.   One  which  acts  on  the  uterus,  but  does  not  lower 
blood  pressure  would  be  of  interest  as  an  abortive  agent.   The 
isolation  and  characterization  of  the  pressor  peptide  (or  protein) 
associated  with  the  initiation  and  maintenance  of  hypertension  of 
renal  origin  could  help  clarify  the  role  of  the  renin-angiotensin 
system  in  renal  artery  stenosis. 

Proposed  Course  of  Project;   Other  bioassay  systems  will  be  used 
to  study  the  structure-activity  relationships  of  ranatensin  and 
its  synthetic  analogs.   The  vasoactive  substances  from  dog  plasma 
and  hornet  venom  will  be  further  characterized.   Attempts  to  find 
other  naturally  occurring  vasoactive  substances  in  frog  skin  and 
insect  venom  will  continue. 

Honors  and  Awards:   None 


-9 


NHLI-101 


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NHLI-101 


Publications: 


Geller,  R.G.,  Govier,  W.C.,  Pisano,  J.J.,  Tanimura,  T., 
and  Clineschmidt,  B.V.:   The  action  of  ranatensin,  a  new 
polypeptide  from  amphibian  skin,  on  the  blood  pressure  of 
experimental  animals.   Brit.  J.  Pharmacol.   40:   605-616, 
1970. 

Clineschmidt,  B.V.,  Geller,  R.G.,  Govier,  W.C.,  Pisano,  J.J 
and  Tanimura,  T.:   Effects  of  ranatensin,  a  polypeptide 
from  frog  skin  on  isolated  smooth  muscle.   Brit.  J.  Pharm. 
In  press,  1971. 


SCO 


Serial  No.       NHLI-102 

1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


3 


PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


Project  Title: 


A  Sensitive  Assay  for  Esterase  Activity  Employing 
Radioactive  Substrates:   Application  to  Plasma 
Kallikrein 


Previous  Serial  Number:   NHLI-132 

Principal  Investigator:   Vida  H.  Heaven,  Ph.D. 

Other  Investigators:   John  J.  Pisano,  Ph.D. 

Jack  V.  Pierce,  Ph.D. 
Ronald  G.  Geller,  Ph.D. 
Harry  Margolius,  M.D.,  Ph.D. 


Cooperating  Units:   None 

Project  Description: 

Objectives ;   A  sensitive  ass 
employed  p-tosyl-L-arginine- 
and  measured  the  ^H-methanol 
veloped  in  this  laboratory  ( 
This  assay  was  successfully 
urinary  kallikrein  esterase 
1970-71) .   The  objective  of 
of  the  assay  conditions  for 
esterase  activity. 


ay  for  esterase  activity,  which 
^H-methylester  (^H-TAMe)  as  substrate 

released  upon  hydrolysis,  was  de- 
see  Project  Report  NHLI-132,  1969-70). 
applied  to  the  measurement  of  human 
activity  (see  Project  Report -Margolius, 
the  present  work  is  the  modification 
the  measurement  of  plasma  kallikrein 


Method:   The  method  of  assay  for 
in  the  previous  report;  however, 
were  adapted.   The  standard  incub 
soybean  trypsin  inhibitor  or  buff 
purified  human  plasma  kallikrein 
was  separated  from  the  ^H-methano 
from  the  incubation  mixture  into 
The  toluene  solution  was  assayed 
was  considered  to  be  that  esteras 
by  soybean  trypsin  inhibitor. 


i 


esterase  activity  was  described 
the  following  modifications 
ation  contained  ^n-TAMe,  buffer, 
er,  and  plasma  or  partially 
(HPK) .   The  unchanged  ^n-substrate 
1  by  extraction  of  the  ^H-methanol 
a  toluene-Liquifluor  solution, 
for  tritium.   Kallikrein  esterase 
e  activity  which  was  inhibited 


AO/ 


NHLI-102 

Major  Findings;   The  optimum  assay  conditions  for  plasma  kallikrein 
esterase  were  as  follows:   The  HPK  production  of  ^H-methanol  was 
linear  with  HPK  concentration  to  0.0004  EU/0.05  ml  incubation 
(1  EU  =  amount  of  enzyme  which  hydrolyzed  1  umole  ester/min)  and 
with  time  to  30  minutes.   The  HPK  esterase  activity  had  a  pH 
optimum  of  7.5  and  temperature  optimum  of  25 °C.   The  activation 
of  plasma  kallikrein  esterase  was  compared  in  human  and  dog  plasma 
using  (1)  acetone  (20%,  v/v,  for  4  hr  at  25°C)  or  (2)  kaolin 
(5  mg/ml,  for  one  min  at  25°C) .   Human  plasma  kallikrein  esterase 
was  activated  1.5  times  more  with  acetone  than  with  kaolin r  dog 
plasma  kallikrein  esterase  was  activated  3  times  more  with  kaolin 
than  with  acetone.   Maximum  activation  of  dog  plasma  kallikrein 
esterase  activity  was  inhibited  40%  in  5  minutes  and  75%  in 
30  minutes  after  the  addition  of  kaolin  to  the  plasma.   The  maxi- 
mum activation  of  dog  plasma  kallikrein  with  kaolin  was  also  seen 
in  one  minute  when  measured  in  the  rat  uterus  bioassay.   Preliminary 
experiments  with  kaolin  activated  dog  plasma  indicated  that  the 
plasma  kallikrein  esterase  activity,  from  both  arterial  and  renal 
venous  samples,  was  not  influenced  by  short-term  infusions  of 
angiotensin  (0.5  |j,g/kg/min,  i.v.)   or  norepinephrine  (10  ug/kg/min, 
i.v.).   The  same  results  were  observed  in  plasma  kallikrein 
measured  in  the  bioassay. 

Significance  to  Biomedical  Research  and  Institute  Program;   The 
development  of  a  reliable,  rapid  and  sensitive  esterase  assay 
which  can  be  applied  to  the  measurement  of  plasma  kallikrein 
esterase  activity  is  crucial  to  establishing  the  significance  of 
this  enzyme  in  physiological  and  pathological  conditions. 

Proposed  Course:   Further  evaluation  of  the  assay  is  planned  to 
establish  that  the  esterase  activity  that  is  measured  in  the 
presence  of  plasma  is  all  attributable  to  plasma  kallikrein 
esterase.   For  example,  specific  activators  and  inhibitors  of 
plasma  kallikrein  esterase  will  be  tested  to  establish  that  the 
esterase  activity  inhibited  by  soybean  trypsin  inhibitor  is  kalli- 
krein esterase  and  no  other.   Should  a  lack  of  specificity  be 
discovered,  other  radioactive  ester  or  peptide  substrates  will  be 
evaluated.   The  selection  of  substrate  will  be  based  upon  the 
ability  of  antibody  to  plasma  kallikrein  to  inhibit  the  hydro- 
lysis of  the  substrate;  the  hydrolysis  of  TAMe  by  urine  kallikrein 
esterase  is  not  inhibited  by  a ntibody  to  urine  kallikrein.   After 
the  plasma  assay  is  established  it  will  be  used  to  study  a  variety 
of  clinical  conditions  including  hypertension  (See  Project  Report 
Margolius)  asthma,  edema,  shock,  fever,  inflammation,  pain,  and 
arthritides  of  various  etiologies. 

o  AOA 


Publications; 


Beaven,  V.H.,  Pierce,  J.V.,  and  Pisano,  J.J.:   A  sensitive 
isotopic  procedure  for  the  assay  of  esterase  activity: 
Measurement  of  human  urinary  kallikrein.   Clin.  Chim.  Acta, 
32:   67-73,  1971. 


^1 


W 


NHLI-102 


Honors  and  Awards:   None 


Serial  No.    NHLI-103 

1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1970  thiDugh  June  30,  1971 

Project  Title:   Studies  on  the  Enzymes  Involved  in  the  Activation 
of  Human  Plasma  Kallikrein 

Previous  Serial  Number:   NHLI-133 

Principle  Investigator:   Marion  E.  Webster,  Ph.D. 

Other  Investigators:   Vida  H.  Heaven,  Ph.D. 

Yuiniko  Nagai,  Ph.D. 
Jack  V.  Pierce,  Ph.D. 
John  J.  Pisano,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  kallikrein  which  is  present  in  human  plasma  is 
normally  found  as  an  inactive  precursor  called  prekallikrein. 
The  mechanism  by  which  this  enzyme  is  activated  in  human  blood 
is  not  fully  understood.   Nothing  is  known  of  the  chemical  bonds 
cleaved  during  the  conversion  of  prekallikrein  to  kallikrein 
although  it  is  assumed  to  be  mediated  by  an  enzyme.   This  inac- 
tive enzyme  precursor  is  believed  to  be  activated  by  other  en- 
zymes, and  it  is  currently  proposed  (Webster,  Fed.  Proc.  21-*    ^4, 
1968)  that  a  series  of  pre-enzyme  to  enzyme  conversions  results 
in  the  activation  of  plasma  kallikrein  which  acts  on  the  final 
substrate,  kininogen,  to  release  bradykinin.   Two  of  the  enzymes 
believed  to  be  involved  in  the  activation  of  prekallikrein  are 
Hageman  factor  and  pF/dil.   This  study  is  an  investigation  of  the 
enzymes  involved  in  the  activation  of  prekallikrein.   Attempts 
are  being  made  to  prepare  both  the  active  form  and  inactive  pre- 
cursor of  the  different  enzymes  and  to  arrange  them  in  an  orderly 
sequence . 

Methods:   Plasma  kallikrein  activity  was  determined  by  measuring 
the  generation  of  kinins  using  the  direct  bioassay  on  the  isolated 
guinea  pig  ileum  with  heated  (60°  for  1/2  hour)  plasma  as  substrate, 


NHLI-103 

When  intact  plasma  is  used  as  substrate  in  this  bioassay,  the 
activity  due  to  both  plasma  kallikrein  and  the  activator  of  pre- 
kallikrein  are  determined.   TAMe  esterase  activity  was  determined 
using  ^H-TAMe  as  previously  described  (Beaven,  NHLI  Project  Report 
No.  132,  1970).   Hageman  factor  and  PTA  activity  are  performed  in 
the  usual  manner  using  human  and  bovine  plasma  deficient  in  these 
factors.   Both  inactive  and  active  factors  are  estimated  in  the 
presence  of  kaolin  whereas  in  the  absence  of  kaolin  only  the  active 
factor  is  determined. 


3. 


Major  Findings;   Previous  results  had  shown  that  chromatography 
of  acetone  activated  human  plasma  on  DEAE-cellulose  resulted  in 
the  separation  of  a  niomber  of  peaks  (II,  III,  IV,  and  V)  which 
would  generate  kinin  activity  from  both  heated  and  non-heated 
plasma  suggesting  that  they  were  activators  of  prekallikrein. 
Attempts  to  arrange  these  enzymes  in  an  orderly  sequence  by  kinetic 
studies  or  by  differentiating  them,  by  inhibitors  was  unsuccessful, 
although  they  could  readily  be  differentiated  from  plasma  kalli- 
krein, (peak  I) .   Active  Hageman  factor  and/or  active  PTA  were 
also  present  in  the  fractions  from  this  column.   The  relative  ac- 
tivity of  the  clotting  factors  was  similar  in  the  varioE  fractions, 
but  varied  from  that  shown  by  the  activators  of  prekallikrein. 
Peak  V,  which  in  some  columns  represented  the  major  portion  of 
the  activator  activity,  was  further  purified  by  chromatography  on 
hydroxyappatite.   A  single  sharp  peak  of  activity  was  obtained 
and  the  enzyme  had  been  purified  2-6, 000- fold  when  compared  to 
the  starting  plasma  with  an  over-all  yield  of  36%.   These  fractions 
still  retained  TAMe  esterase  activity,  although  only  a  small 
portion  of  that  found  in  Peak  V.   Repeated  freezing  and  thawing 
of  these  fractions  resulted  in  loss  of  activity,  possibly  due  to 
the  low  concentration  of  protein  in  the  fractions.   Peaks  II  and 
III  were  rechromatographed  on  DEAE-cellulose  to  investigate  their 
possible  conversion  to  peak  V  as  found  by  other  investigators 
(Kaplan  and  Austen,  J.  Immunology  105,  802,  1970) .   However,  in 
our  hands,  only  a  small  portion  of  the  activity  (30%)  rechromato- 
graphed in  a  position  which  might  be  considered  equivalent  to 
peak  V.   The  remaining  activity  remained  associated  with  the  major 
protein  peak  and  could  now  be  clearly  differentiated  from  the 
major  TAMe  esterase  peak  found  in  these  fractions. 


Since  the  recovery  of  plasma  kallikrein  and  of  the  clotting 
factors  from  the  DEAE-cellulose  columns  was  poor  (30%  or  less) , 
alternate  methods  for  the  purification  of  these  active  enzymes 
were  investigated.   Plasma  kallikrein  can  be  recovered  600-fold 
purified  with  a  50%  yield  by  adsorption  and  elution  from  a  soy 


r 


-a^r 


NHLI-103 
bean  trypsin  inhibitor-sepharose  (STI-sepharose)  column 
NHLI  Project  Report-Pierce  ) .   The  activators  of  prekallikrein, 
on  the  other  hand,  are  found  in  the  filtrates  from  these  colun\ns 
and  can  be  purified  300-fold  by  adsorption  and  elution  on  a  lima 
bean  trypsin  inhibitor-sepharose  (LTI-sepharose)  column.   The 
observation  that  all  of  the  activator  activity  can  be  removed  by 
a  single  inhibitor  column,  together  with  the  earlier  inability  to 
differentiate  peaks  II,  III,  IV  and  V  by  kinetic  measurements  or 
by  inhibitors,  suggests  that  multiple-forms  of  the  same  enzyme 
may  be  formed  during  acetone  activation  of  human  plasma.   This 
view  was  strengthened  when  it  was  found  that  treatment  of  the 
STI-sepharose  filtrates  with  urea,  guanidine  or  potassium,  thio- 
cyanate  resulted  in  a  two-fold  increase  in  biological  activity. 

The  above  procedures,  however,  cause  a  loss  in  the  clotting 
factors.   For  example,  in  one  such  preparation  only  38  and  62% 
of  the  active  Hageman  and  PTA  activities  were  recovered  in  the 
STI-sepharose  filtrates  and  only  2,6  and  10%  respectively  in  the 
final  LTI-sepharose  filtrates.   The  method  of  Speer  et_  a_l.  (Thromb. 
Diath.  Haera.  1_4,  i-11,  1965)  for  the  preparation  of  active  Hageman 
factor  was  first  investigated.   By  this  procedure  the  Hageman 
factor  is  adsorbed  and  eluted  from  deactivated  supercel  and  further 
purified  by  isoelectric  precipitation,  ammonium  sulfate  precipi- 
tation and  chromatography  on  CM-Sephadex.   Following  this  pro- 
cedure active  Hageman  factor  was  obtained  from  the  supercel  eluates , 
However,  adjustment  of  the  pH  to  5.2  for  the  isoelectric  preci- 
pitation caused  losses  in  activity  and  no  active  precipitate 
formed.   Ammonium  sulfate  precipitation  of  the  supercel  eluates 
resulted  in  the  recovery  of  most  of  the  active  Hageman  factor 
activity  while  achieving  a  300-fold  purification.   Attempts  to 
further  purify  by  CM-Sephadex  chromatography  as  described  by 
these  authors  were  unsuccessful  and  all  activity  was  lost.   Also, 
it  was  found  that  the  major  portion  of  active  Hageman  factor 
(PTA  activity  was  not  measured)  could  be  removed  from  acetone 
activated  human  plasma  by  adsorption  on  deactivated  supercel, 
leaving  most  of  the  prekallikrein  activator  and  plasma  kallikrein 
in  the  filtrate.   Since  acetone  activated  plasma  appears  to  contain 
all  of  the  Hageman  factor  activity  of  the  starting  plasma,  this 
separation  of  active  Hageman  factor  and  prekallikrein  activator 
makes  it  unlikely  that  the  activator  is  derived  from  active  Hage- 
man factor  as  proposed  by  others  (Kaplan  and  Austen,  J.  Immunol. 
105,  803,  1970;  Wuepper  et  aj^. ,  J.  Immunol.  105,  1307,  1970). 

In  addition  to  isolating  the  active  components  of  this  enzyme 
system  as  described  above,  preliminary  attempts  have  been  made  to 

3  ^4 


;  ,00 


NHLI-103 

isolate  the  inactive  components.   Plasma  deficient  in  Hageman 
factor  contains  prekallikr ein  (Webster  and  Ratnoff ,  Nature  192, 
180,  1961) ,  but  does  not  form  active  enzyme  on  contact  with  nega- 
tively charged  surfaces  or  on  treatment  with  acetone.   Chromatogra- 
phy of  this  plasma  on  DEAE-cellulose  gave  a  partially  purified 
prekallikrein  preparation,  the  activity  being  associated  only 
with  those  proteins  which  did  not  bind  to  DSAE-cellulose.   The 
activity  of  these  fractions  was  determined  by  measuring  the  in- 
crease in  esterase  activity  generated  by  the  prekallikrein  acti- 
vator. .Preliminary  experiments  indicated  that  the  pH  of  the 
activation  step  could  be  varied  from  7.0  to  8.5  with  no  change 
in  activity.   The  amount  of  esterase  activity  generated  at  room 
temperature  by  this  pre-incubation  increased  from  three  to  ten 
minutes  and  remained  constant  or  decreased  slightly  if  the  incu- 
bation was  prolonged  for  peria3 s  of  time  up  to  30  minutes.   With 
the  concentration  of  enzyme  chosen  in  these  studies  a  linear 
relationship  between  concentration  of  substrate  and  generation  of 
esterase  activity  was  obtained  using  a  five-minute  incubation 
period  of  pH  8.5.   The  fractions  from  this  column  also  generated 
esterase  activity  when  trypsin,  rather  than  the  activator  of  pre- 
kallikrein, was  used  as  the  enzyme.   Trypsin,  however,  was  not  as 
specific  as  the  activator  of  prekallikrein  since  it  also  generated 
large  amounts  of  esterase  activity  from  another  unidentified 
pre-enzyme  which  adsorbed  to  the  DEAE-cellulose. 

Attempts  were  also  made  to  find  an  inactive  precursor  of  the 
activator  of  prekallikrein.   In  these  experiments,  the  fractions 
from  the  DEAE-cellulose  coliomns  of  Hageman  deficient  plasma  and 
of  acetone  activated  Hageman  deficient  plasma  were  incubated  with 
partially  purified  active  Hageman  factor  and  with  a  source  of  pre- 
kallikrein.  No  increased  generation  of  esterase  activity  was 
found.   However,  duplicate  determinations  were  poor  as  were  attempts 
to  repeat  the  results  on  subsequent  days.   It  was  found  that  part 
of  the  difficulty  was  due  to  the  extreme  lability  of  the  prekalli- 
krein fractions  which  can  only  be  frozen  and  thawed  once  and  the 
remaining  difficulties  due  to  the  large  amount  of  protein  adsorbed 
to  the  IRC-50  resin.   These  observations  led  to  the  development 
of  the  extraction  method  for  the  determination  of  human  plasma 
kallikrein  (Beaven,  NHLI  project  Report  No.     ) . 

The  preparation  of  inactive  Hageman  factor  was  also  attempted. 
Initially,  plasma  containing  40  ug/ml  hexadimethrine  bromide  was 
adsorbed  with  deactivated  supercel  and  eluted  in  a  manner  similar 
to  that  described  for  the  preparation  of  active  Hageman  factor. 
The  eluates  from  these  adsorptions  contained  much  higher  Hageman 


0. 


o 


I 


^*r 


NHLI-103 

factor  activity  (4-5  fold)  than  those  eluates  prepared  from  plasma 
in  the  absence  of  hexadimethrine  bromide.   They  also  contained 
a  4-fold  increase  in  content  of  activator  of  prekallikrein, 
while  the  kallikrein  content  of  the  two  eluates  varied  by  less 
than  two- fold.   Since  the  supernatant  from  these  adsorptions 
were  essentially  devoid  of  Hageman  factor  activity,  this  suggested 
that  the  presence  of  hexadimethrine  bromide  was  either  directly 
preventing  the  inactivation  of  Hageman  factor  and/or  prekallikrein 
activator  or  was  delaying  the  activation  of  the  Hageman  factor  on 
the  supercel  and  thus  preventing  its  inactivation  by  the  plasma 
proteins  in  the  filtrate.   Hexadimethrine  bromide  at  this  concen- 
tration did  not  prevent  the  activation  of  Hageman  factor  since 
only  50%  or  less  of  the  Hageman  factor  was  estimated  to  be  in  its 
inactive  form.   Also  when  these  eluates  were  fractionated  with 
anmonium  sulfate  in  the  presence  of  hexadimethrine  bromide  they 
appeared  to  become  fully  activated,  giving  a  two- fold  increase 
in  both  active  Hageman  factor  and  prekallikrein  activator.   The 
parallelism  is  these  fractions  between  the  active  Hageman  factor 
activity  and  the  content  of  prekallikrein  activator  was  striking 
suggesting  that  both  were  derived  from  the  same  molecule  (inac- 
tive Hageman  factor) .   In  order  to  further  substantiate  this 
hypothesis,  efforts  are  currently  being  made  to  prepare  inactive 
Hageman  factor  essentially  devoid  of  active  Hageman  factor.   Pre- 
liminary results  have  provided  such  a  preparation  but  require 
confirmation. 

In  summary,  progress  is  being  made  in  isolating  and  charac- 
terizing both  the  active  and  inactive  forms  of  the  various  enzymes 
involved  in  the  activation  of  plasma  prekallikrein.   No  conclusive 
evidence  is  yet  available  that  more  than  one  activator  is  involved. 
In  fact  the  current  evidence  would  suggest  that  multiple  forms  of 
the  same  activator  are  generated  during  acetone  activation  of 
plasma.   Also,  the  present  evidence  does  not  favor  the  view  that 
the  prekallikrein  activator  is  formed  from  active  Hageman  factor 
as  proposed  by  others,  but  rather  suggests  that  inactive  Hageman 
factor  forms  at  least  two  active  products,  the  activator  of  pre- 
kallikrein (pF/dil?)  and  active  Hageman  factor. 

Significance  to  Biomedical  Research  and  Institute  Program:   The 
generation  of  kinins  by  plasma  kallikrein  has  been  implicated  in 
a  number  of  pathological  conditions  such  as  hereditary  angio- 
neurotic edema,  gouty  and  rhetimatic  arthritis,  asthma,  pulmonary 
edema,  reactions  to  blood  transfusions,  infection  by  pathogenic 
organism,  pancreatitis,  etc.   A  complete  understanding  of  this 
system  together  with  isolation  and  characterization  of  the  various 


SaS 


NHLI-103 

enzymes,  could  lead  to  the  development  of  inhibitors  which  would 
be  therapeutically  useful. 

Proposed  Course  of  Project;   Continued  investigation  of  the  enzymes 
involved  in  the  activation  of  prekallikrein.   Methods  will  be 
developed  for  the  further  purification  of  both  the  active  form  and 
inactive  precursor  of  the  different  enzymes  and  an  attempt  will 
be  made  to  arrange  them  in  an  orderly  sequence. 


« 


Honors  and  Awards:   None 


Publications: 


Webster,  MoEo,  Pierce,  J.V.,  and  Sarapaio,  M.U.:   Studies 
on  antibody  to  bradykinin.   Adv.  Exp.  Med.  Biol»  8:   57-64, 
1970o 


Webster,  M.E.:   Recommendation  for  nomenclature  and  units. 
In  Erdos.  E.G.  (Ed),  "Bradykinin,  kallidin  and  kallikrein" . 
Handbook  of  Experimental  Pharmacology,  25:   659-665,  1970. 

Webster,  M.E.:   Kallikreins  in  glandular  tissues.   In  Erdos, 
E.G.  (Ed),  "Bradykinin,  kallidin  and  kallikrein".  Handbook 
of  Experimental  Pharmacology,  25:   131-155,  1970. 

Webster,  M.E.,  and  Prado,  E.S.:   Glandular  kallikreins 
from  horse  and  human  urine  and  from  hog  pancreas.   In 
Lorand,  L.  and  Perlman,  E.G.  (Eds),  Methods  in  Enzymology 
19:   681-699,  1970. 

Goodfriend,  T.L.,  Webster,  M.E.,  and  McGuire,  J.S.:   Complex 
effects  of  antibodies  to  polypeptide  hormones.   J.  Clin. 
Endocrinol.  Metab.   30:   565-572,  1970. 

Skinner,  S.No,  Jr.,  and  Webster,  M.E.:   Acetylcholine  and 
functional  vasodilatation  in  the  submaxillary  gland  of  the 
cat,   J.  Europ.  Pharmacolo  12:   271-275,  1970. 


Suaf 


P. 


Serial  No.  NHI.I-104 

1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 

FHS-NtlLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Analysis  of  Amino  Acid  Phenylthiohydantoin  by 
Gas  Chromatography 

Previous  Serial  Number:   NHI-105 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  analysis  of  ammo  acid  phenylthiohydantoin  deri- 
vatives (PTH)  is  a  major  problem  in  the  determination  of  the 
amino  acid  sequences  of  polypeptides  and  proteins.   Current  methods 
of  analysis  involve  thin  layer  and  paper  chromatographic  tech- 
niques which  are  inadequate.   The  present  work  was  undertaken  to 
improve  the  analysis  of  amino  acid  PTHs  and  in  particular  to 
explore  the  potential  of  gas  chromatography,  a  technique  noted 
for  its  high  speed  of  analysis,  resolving  power,  sensitivity,  and 
ease  of  quantitation. 

Major  Findings:   Advances  in  the  gas  chromatography  procedure  for 
the  identification  of  amino  acid  thriohydantoins  include:   1)  a 
nev;  blend  of  silicone  stationary  phases  (7.33%  SP-400,  5.33% 
OV-210,  and  0.66%  OV-225)  which  provides  superior  resolving  power 
than  higherto  obtained  with  single  phases  or  earlier  blends; 
2)  the  use  of  helium  as  carrier  gas  which  is  superior  to  the  more 
commonly  used  nitrogen  andargon,  in  that  it  gives  the  best  reso- 
lution and  the  greatest  range  of  flow  rates  with  no  sacrifice  in 
efficiency;  and  3)  high  temperature  conditioning  which  gives  more 
efficient  columns  with  significantly  less  bleed  (baseline  rise) 
during  temperature  programming.   The  methylthiohydantoins  were 
also  examined.   All  derivatives,  except  the  seryl  (unstable)  and 
arginyl  (non-volatile)  were  separable  on  a  column  using  the  sta- 
tionary phase,  OV-225. 


Sac 


«  I 


(."' 


NHLI-104 
Automated  sample  injection  has  been  evaluated  and  its  feasi- 
bility demonstrated  with  standard  PTH  derivatives  obtained  for 
the  automated  degradation  currently  under  study.   The  latter 
samples  contain  impurities  which  may  hasten  the  destruction  of 
the  derivatives  in  the  sample  holder. 

A  new  solvent,  dioxane,  containing  the  antioxidant  diethyl- 
dithiocarbamate  is  superior  to  all  other  solvents  tested  for 
preparing  standard  solutions  of  the  amino  acid  PTHs .   Unlike  any 
previous  solvent  tested  (or  mixture  of  solvents)  it  dissolves  all 
the  derivatives.   An  added  advantage  (due  to  the  antioxidant)  is 
the  greater  stability  of  the  derivatives  in  this  solvent, 

A  comparison  has  been  made  between  the  gas  chromatographic 
and  a  new  mass  spectrometric  method  developed  by  Dr.  Henry  Fales, 
Laboratory  of  Chemistry,  NHLI.   The  latter  technique  employs 
chemical  ionization  mass  spectrometry.   Sperm  whale  myoglobin 
was  degraded  by  Dr.  Bryan  Brewer,  Molecular  Diseases  Branch, 
NHLI,  using  the  protein  sequenator.   It  was  shown  that  the  gas 
chromatographic  method  is  much  simpler  to  employ,  but  the  MS 
method  has  the  potential  of  greater  sensitivity  and  speed  of 
analysis. 

Significance  to  Biomedical  Research  and  Institute  Program;   The 
technique  greatly  facilitates  the  analysis  of  amino  acid  PTH 
derivatives  obtained  in  the  sequential  degradation  of  polypeptides 
and  proteins.   Included  in  these  classes  of  substances  are  hormones, 
antibodies,  enzymes,  structural  proteins,  peptides,  and  other 
substances  of  biomedical  importance.   The  structural  determination 
of  these  important  substances  has  been  greatly  hampered  by  ana- 
lytical difficulties.   With  automation  of  the  Edman  degradation 
by  Edman  and  the  now  commercially  available  protein  sequenators, 
the  gas  chromatographic  procedure  for  the  analysis  of  the  amino 
acid  PTH  derivatives  (Edman  derivatives)  is  even  more  attractive 
because  of  the  speed,  sensitivity,  resolving  power  and  ease  of  1  j 

quantitation  of  the  technique.  I 

Proposed  Course  of  Project;   Extension  of  the  automation  of  the  ,  ] 

gas  chromatographic  procedure  developed  with  standards  to  PTH  > 

derivatives  obtained  from  the  protein  sequenator.   Development  of  i 

a  protocal  which  would  allow  identification  of  a  PTH  amino  acid 

in  less  than  50  minutes.   Development  of  a  gas  chromatographic  ^ 

procedure  for  the  analysis  of  pyroglutamic  acid,  N-acetyl  and  t 

N- formal  amino  acids.   Assists  other  laboratories  at  NIH  in  V 

setting  up  the  procedure.   Collaborate  with  other  laboratories  in  £^' 

polypeptide  sequence  analysis.  ^ 

f 
2  .       A^/ 


NHLI-104 
Honors  and  Awards:   None 

Publications: 

Pisano,  J.J.,  Bronzert,  T.J.,  and  Brewer,  H.B.,  Jr.: 
Advances  in  the  gas  chromatographic  analysis  of  amino  acid 
phenyl-  and  methylthiohydantoins.   Anal.  Biochemo 
In  press. 

Fales,  H.M.,  Nagai,  Y.,  Milne,  G.W.A.,  Brewer,  H.B.,  Jr., 
Bronzert,  T.J.,  and  Pisano,  J, J.:   The  use  of  chemical 
ionization  mass  spectrometry  in  the  analysis  of  the  amino 
acid  phenylthichydantoin  derivatives  formed  during  the 
Edman  degradation  of  proteins.   Anal.  Biochem.   In  press. 


AU 


I 


Serial   No.         NHLI-105 

1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 


Project  Title; 


Application  of  Countercurrent  Chromatography  to 
the  Isolation  and  Characterization  of  Substances 
of  Biochemical  Interests 


Previous  Serial  Number; 


NHLI-136 


Principle  Investigator:   Hisanobu  Yoshida,  Ph.D. 

Other  Investigators:   John  J.  Pisano,  Ph.D. 

Yoichiro  Ito,  M.D. 
Robert  L.  Bowman,  M.D. 

Cooperating  Units:   Laboratory  of  Technical  Development 

Project  Description: 

Objectives:   To  perfect  and  apply  the  technique  of  countercurrent 
chromatography  to  the  solution  of  problems  in  the  isolation  and 
characterization  of  compounds  of  biomedical  interests. 

Methods:   Design  and  construction  of  suitable  equipment.   Develop- 
ment of  theory  and  practice  of  countercurrent  chromatography. 

Major  Findings:   Droplet  countercurrent  chromatography  (DCCC) ,  a 
new  chromatographic  technique  developed  in  our  laboratory,  was 
applied  to  the  separation  of  milligram  quantities  of  2,  4  dini- 
trophenyl  derivatives  of  amino  acid  (NHLI-136)  with  an  efficiency 
comparable  to  gas  chromatography.   The  promise  of  this  method  is 
being  fulfilled  with  its  application  to  the  analysis  of  new 
classes  of  compounds  and  with  the  construction  of  an  even  simpler 
unit  consisting  only  of  teflon  tubing.   This  unit  has  been  used 
in  the  separation  of  the  following  model  peptides:   bradykinin, 
kallidin,  and  angiotensin;  ranatensin  from  crude  methanolic 
extracts  of  frog  skin  and  insulin  from  its  A  and  B  chains.   Sol- 
vent systems  used  are  similar  to  those  employed  in  countercurrent 
distribution  and  include  sec-butanol-trifluoracetic  acid-  water, 
n-butanol-acetic  acid-water,  sec-butanol-1%  dichloreacetic  acid 


^^3 


iftlisaWWWK.-tsxei'jA- 


NHLI-105 
and  n-butanol-0.1%  acetic  acid-pyridine .   Reproducibility  of  the 
method  is  excellent.   It  has  also  been  shown  that  the  elution 
volume  may  be  calculated  knowing  the  partition  coefficient  and 
the  volume  of  stationary  phase.   Recovery  of  the  sample  is  quan- 
titative with  as  little  as  1  ug  of  material. 

Another  form  of  countercurrent  chromatography,  gyration 
locular  countercurrent  chromatography  (developed  by  Drs.  Ito  and 
Bowman),  is  being  refined  and  evaluated.   Several  kinds  of  locular 
columns  (all  teflon,  teflon-glass,  glass-lined  thru  hole  in  the 
teflon  spacers,  etc.)  have  been  constructed  and  applied  to  the 
separation  of  bradykinin,  kallidin,  and  angiotensin  and  Val-t-RNA 
or  Phe-t-RNA  from  a  mixture  of  amino  acyl-t-RNAs .   In  general 
the  method  has  higher  resolving  power  and  speed  of  analysis  than 
droplet  countercurrent  chromatography.   However,  it  is  more  diffi- 
cult to  execute  and  has  a  lower  capacity. 

Significance  to  Biomedical  Research  and  Institute  Problems: 
Standard  ion  exchange,  and  liquid-liquid  chromatographic  procedures 
are  often  not  satisfactory  for  the  separation  of  neutral,  organic- 
soluble  compounds  of  biological  interest.   The  classic  counter- 
current  distribution  technique  is  often  used,  but  it  is  ciombersome. 
Liquid-liquid  chromatographic  methods  employing  solid  supports 
often  exhibit  tailing  due  to  adsorption  of  the  solutes  to  the 
support,  and  the  capacity  of  this  technique  is  low. 

Countercurrent  chromatography  on  the  other  hand  is  an  all- 
liquid  separation  technique  ideally  suited  for  any  substance 
which  partitions  between  two  immiscible  solvents  and  is  free  of 
adsorptive  effects.   The  latter  is  most  important  in  the  separation 
of  peptides  and  many  other  biological  substances  which  adsorb 
(often  irreversibly)  to  the  solid  supports  used  in  conventional 
chromatography . 

Proposed  Course  of  Project:   Having  established  the  usefulness  of 
countercurrent  chromatography  in  the  separation  of  peptides  and 
ribonucleic  acids,  the  technique  will  be  extended  to  other  classes 
of  substance  such  as  lipids,  pesticides,  carbohydrates,  etc. 
Practical  applications  are  anticipated  in  the  isolation  and  iden- 
tification of  naturally  occurring  peptides  and  peptides  obtained 
in  the  degradation  of  proteins  undergoing  amino  acid  sequence 
analysis. 

Honors  and  Awards:   None 


Ac^ 


NHLI-105 


Publications: 


Tanimura,  T.,  Pisano,  J.J.,  Ito,  Y.,  and  Bowman,  R.L, 
Droplet  countercurrent  chromatography.  Science,  169: 
54-56,  1970. 


Vt 


JUS" 


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wmnTBTnmFWfAatfaniim-^ni/^T'  :f^^»\ 


Serial  No.  NHLI-106 

1.  Experimental  Therapeutics  Branch  I 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  the  Structure  of  Villikinin 

Previous  Serial  Number:   NHLI-143 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Other  Investigators:   Esztar  Kokas,  Ph.D. 

Cooperating  Units:   Physiology  Department 

University  of  North  Carolina 
Medical  School 

Project  Description: 

Objectives:   To  isolate  and  determine  the  structure  of  villikinin, 
a  substance  obtained  from  intestinal  mucosal  extract  which  has 
a  specific  stimulant  action  on  intestinal  villous  motility. 

Methods:   Crude  intestinal  mucosal  extract  is  desalted  on  Dowex-50 
further  purified  by  gel  filtration  on  Sephadex  G-25  or  Bio-Gel 
P-4  and  finally  chromatographed  on  Beckman  UR-30  resin  using  a 
pyridine  acetate  buffer  or  on  SE  Sephadex  using  an  ammonium 
acetate  gradient.   Material  is  assayed  in  vivo  by  measuring  its 
activity  on  pumping  action  of  exposed  intestinal  villi  of  dogs. 

Major  Findings:   Although  the  previously  reported  procedxire  for 
obtaining  an  active  villikinin  fraction  from  crude  mucosal  extract 
resulted  in  over  a  100-fold  purification,  the  peaks  obtained  from 
both  gel  filtration  on  Sephadex  G-25  and  cation  exchange  chroma- 
tography in  a  pyridine  acetate  buffer  have  been  unsatisfactorily 
broad.   Bio-gel  P-4  is  currently  under  evaluation  as  a  substitute 
for  Sephadex  G-25.   A  well-defined  peak  of  activity  with  an 
apparent  molecular  weight  between  2000  and  4000  was  obtained  on  a 
Bio-gel  P-4  column  using  bovine  mucosal  extract.   Canine  mucosal 
extract  on  Sephadex  G-25  gave  a  broad  peak  with  an  apparent 
molecular  weight  of  about  1000.   At  this  time  it  is  impossible  to 
judge  whether  the  apparent  disparity  is  due  to  a  real  difference 


^^ 


NHLI-106 

between  canine  and  bovine  villikinin,  or  is  a  result  of  differing 
experimental  procedures .   Canine  extract  must  be  tested  on 
Bio-gel  P-4. 

When  either  active  product  obtained  by  gel  filtration  is 
chromatographed  in  a  pyridine  acetate  buffer  on  cation  exchange 
resin,  it  is  strongly  retarded  and  elutes  slowly  in  a  very  broad 
peak.   Cation  exchange  chromatography  using  SE-Sephadex  is  now 
being  studied  as  a  purification  step  to  follow  gel  filtration. 
Bovine  mucosal  extract  which  had  been  processed  through  the 
Dowex-50  desalting  step  and  Bio-gel  P-4  chromatography  was  tested 
with  several  enzymes.   Like  canine  material  it  was  inactivated 
by  Pronase,  papain  and  chymotrypsin  and  not  inactivated  by  trypsin. 
These  results  are  significant  because  they  indicate  that  we  are 
purifying  a  specific  biologically  active  substance  in  as  much  as 
the  partially  purified  material  behaves  identically  to  the  crude 
extract  treated  with  the  above  enzymes.   What  is  more,  finding 
the  same  material  in  canine  and  bovine  intestinal  extracts  suggests 
its  widespread  occurrence  and  physiological  importance. 

Significance  to  Biomedical  Research  and  Institute  Program;   Villi- 
kinin may  be  a  specific  hormone  which  controls  intestinal  villous 
motility.   The  theory  can  only  be  proven  when  the  substance  is 
isolated  and  its  structure  deteinnined. 

Proposed  Course  of  Project;   With  the  discovery  of  villikinin  in 
bovine  intestinal  extracts  abundant  supplies  of  material  is 
assured.   Various  purification  schemes  will  be  tested.   When  pure 
material  is  obtained  its  amino  acid  sequence  will  be  determined 
and  confirmed  by  synthesis.   Pharmacological  studies  will  be  also 
undertaken  to  determine  the  physiological  importance  of  villikinin 
in  man. 

Honors  and  Awards:   None 

Publications:   None 


r 


«a/7 


Serial  No.   NHLI-107 


1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 
3„   Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Unusual  Linkages  in  Peptides 

Previous  Serial  Number:   NHLI-140 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Other  Investigators:   John  S,  Finlayson,  Ph.D. 

Yumiko  Nagai,  Ph.D. 

Cooperating  Units:   Division  of  Biological  Standards 

Laboratory  of  Blood  and  Blood  Products 

Project  Description: 

Objectives:   The  objectives  in  this  study  were  to  determine  the 
biochemical  lesion  in  Factor  XIII  deficiency,  to  measure  the 
degree  of  crosslinking  in  normal  human  plasma  and  in  plasma  from 
patients  with  Factor  XIII  deficiency,  and  to  determine  the  extent 
of  the  a  and  y   chain  involvement  in  crosslinking,  for  example, 
the  number  of  crosslinks  contributed  by  each  chain. 

Methods:   A  technique  has  been  developed  for  clotting  fibrin 
directly  in  human  plasma  and  removing  the  ciDt  by  winding  it  on  a 
stirring  rod  as  it  forms  so  that  the  clots  can  be  washed,  solu- 
bilized  and  carried  through  previously  described  procedures  for 
measuring  €- (-^/-glutamyl)  lysine  crosslinks  o   Both  an  enzymic  di- 
gestion method  for  measuring  crosslinks  directly  and  a  chemical 
procedure  for  measuring  crosslinked  lysine  by  cyanoethylation  and 
subsequent  acid  hydrolysis  are  used.   Qualitative  determination  of 
the  extent  of  crosslinking  was  determined  by  disc  gel  electro- 
phoresis. 

Major  Findings:   Fibrin  clots  which  were  formed  with  normal  plasma 
contained  approximately  6  moles  e- ('y-glutamyl)  lysine/mole  fibrin, 
whereas  those  formed  in  plasma  from  individuals  with  Factor  XIII 
deficiency  contained  little  or  none  of  this  crosslink  (0„02-0,64 
moles/mole  fibrin) .   Partial  supplementation  of  the  deficient  plasma 
with  Factor  XIII  commensurately  increased  the  number  of  crosslinks. 

1  ^e 


NHLI-107 
To  determine  the  extent  that  each  of  the  chains  of  fibrin 
participated  in  normal  crosslinking,  polymerized  fibrin  was  re- 
duced in  the  presence  of  denaturing  agents.   7-Chains  were  re- 
covered as  dimers  which  contain  a  maximxam  of  two  crosslinks/dimer . 
a-Chains  were  recovered  as  polymers.   Examination  of  high  molecular 
weight  and  intermediate  molecular  weight  a-polymers  revealed  4.8 
and  3.2  crosslinks/2  a  chains,  respectively. 


*^. 


Significance;   The  fact  that  little  or  no  crosslink  formation 
occurred,  in  the  plasma  of  Factor  Xlll-def  icien  t  patients  furnishes 
an  explanation  and  biochemical  basis  for  the  laboratory  findings 
usually  associated  with  this  deficiency  (viz.,  friable  clots, 
solubility  of  clots  in  5  M  urea,  1%  monochloroacetic  acid) . 
Although  it  is  not  certain  that  the  inability  to  form  e-  (^-glutamyl) 
lysine  is  the  sole  molecular  defect  in  Factor  XIII  deficiency,  the 
importance  of  the  crosslink  in  hemostasis  appears  established. 

The  finding  of  6  crosslinks  per  mole  for  normal  fibrin  has 
shed  new  light  on  the  nature  of  fibrin  crosslinking.   The  a-chains 
are  more  highly  crosslinked  than  7-chains  and  contribute  an 
average  of  4  crosslinks  per  mole. 

Proposed  Course  of  Project;   This  project  will  be  continued  to 
determine  the  exact  extent  of  involvement  of  the  a-chains  in 
fibrin  crosslinking,  to  determine  the  amount  of  normal  plasma  or 
purified  factor  needed  to  supplement  Factor  Xlll-deficient  plasma 
to  form  the  normal  6  crosslinks/mole,  to  examine  fibrin  cross- 
linking  in  clots  formed  in  vivo,  and  to  examine  other  proteins 
for  the  e- (7-glutamyl) lysine  crosslink. 

Honors  and  Awards;   None 

Publications; 


Pisano,  J.J.,  Finlayson,  J.S.,  Peyton,  M.P.,  and  Nagai,  Y.; 
e- ('Y-Glutamyl)  lysine  in  fibrin:   lack  of  crosslink  formation 
in  factor  XIII  deficiency.   Proc.  Nat.  Acad.  Sci.   68; 
4,  (1971)  in  press. 


J/f 


Serial  No.   mht  .7-108 


1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1S70  through  June  30,  1971 

Project  Title:   Preparation  of  Affinity  Adsorbents 

Previous  Serial  Number:   NHLI-137 

Principal  Investigator:   Jack  V.  Pierce,  Ph.D. 

Other  Investigators:   Larry  J.  Loeffler,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives;   To  prepare  chemically  reactive  derivatives  of 
agarose,  glass  beads,  or  other  materials  useful  in  affinity 
chromatography,  which  can  be  coupled  under  mild  conditions  with 
a  wide  variety  of  proteins,  peptides,  and  inhibitors  of  interest 
to  provide  useful  affinity  adsorbents  for  the  purification  of 
materials  of  biological  interest.   Initially,  to  prepare  an  in- 
soluble trypsin  adsorbent  of  sufficient  binding  capacity  for 
practical  use  in  the  isolation  and  purification  of  trypsin  inhi- 
bitors from  potatoes,  peanuts,  and  other  sources.   To  bind  these 
inhibitors  themselves  to  insoluble  supports  for  possible  use  in 
purifying  enzymes  of  the  plasma  kallikrein  system. 

Method:   Covalent  binding  of  trypsin  to  Sepharose  or  porous 
glass  beads,  using  strategies  which  will  result  in  the  trypsin 
molecule  being  at  a  sufficient  distance  from  the  support  backbone 
to  allow  full  activity  in  binding  inhibitors. 

Major  Findings:   Trypsin  insolubilized  by  direct  coupling  to 
cyanogen  bromide-activated  Sepharose  4B  was  found  to  have  less 
than  50%  of  the  theoretical  activity  in  binding  such  inhibitors 
as  soybean  trypsin  inhibitor  and  lima  bean  trypsin  inhibitor, 
probably  due  to  the  proximity  of  the  trypsin  molecule  to  the 
support  backbone . 


Aa« 


NHLI-108 
A  high  capacity  trypsin  column  has  now  been  prepared,  employing 
Sepharose  4B  as  the  support,  but  interposing  a  chain  of  about  16 
atoms  between  the  trypsin  and  the  support.   The  methyl  ester  of 
11-aminoundecanoic  acid  was  prepared  by  esterif ication  using 
thionyl  chloride  in  methanol.   The  product  was  coupled  to  cyanogen 
bromide-activated  Sepharose  4B,  affording  an  insoluble  ester 
derivative  containing  0.30  m  equivalents  of  ester  groups  per  100  g 
of  Sepharose  (original  wet  weight) .   Treatment  of  this  material 
with  hydrazine  hydrate  in  methanol  gave  a.hydrazide  derivative. 
Treatment  of  the  hydrazide  in  dilute  HCl  with  NaN02  gave  the  azide, 
which  was  reacted  with  trypsin  in  buffer  at  pH  8-9.   Under  the 
most  favorable  conditions,  an  insoluble  trypsin  was  obtained  which 
contained  about  7.1  mg  of  trypsin  bound  per  g  of  Sepharose  and 
which  bound  3.2  mg  of  soybean  trypsin  inhibitor  per  g  of  Sepharose 
(about  70%  of  theory) . 


,,1 

,  CO 


I  2 

^ 

3 


A  similar  series  of  reactions  was  carried  out  using  glycine 
methyl  ester  and  afforded  an  insoluble  trypsin  with  a  considerably 
lower  capacity  for  soybean  trypsin  inhibitor. 

A  trypsin-porous  glass  derivative,  prepared  by  a  literature 
procedure,  was  found  to  be  totally  inactive  in  binding  soybean 
trypsin  inhibitor,  although  a  large  amount  of  trypsin  had  been 
bound . 

Significance  and  Proposed  Course  of  Project:   The  Sepharose- 
trypsin  column  discussed  above  promises  to  be  of  considerable 
utility  for  the  large  scale  isolation  of  various  trypsin  inhibitors, 
some  of  which  may  also  inhibit  enzymes  of  the  kallikrein  system. 
Such  inhibitors  would  be  coupled  to  an  insoluble  support,  perhaps 
using  the  Sepharose-azide  intermediate  mentioned  above,  to  yield 
adsorbents  for  isolating  the  inhibitable  enzymes.   The  Sepharose- 
azide  intermediate  may  also  prove  useful  for  preparing  a  lysyl- 
bradykinin  derivative  for  use  in  the  purification  of  antibrady- 
kinin,  which  then  in  turn  may  be  coupled  to  serve  as  the  basis 
for  a  kinin  radioimmuno-assay  and  to  be  used  in  the  direct  iso- 
lation of  plasma  kininogens  terminating  in  the  kinin  moiety. 
Further  developmental  work  on  the  chemistry  of  potentially 
useful  coupling  reactions  on  Sepharose,  porous  glass,  and  other 
suitable  materials  is  planned. 

Honors  and  Awards:   None 


Piiblications:      None 


5<a/ 


fimiBfatinKiv*T«  • 


Serial  No.      NHTJ-109 


1.  Experimental  Therapeutics  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 

PHS-NHLI 

Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Biochemistry  of  the  Kallikrein-kininogen-kinin 
System 

Previous  Serial  Number:   NHLI~134 

Principal  Investigator:   Jack  V.  Pierce,  Ph.D. 

Other  Investigators:   Kjell  Nustad,  M.D,  (Visiting  Scientist) 

Marion  E.  Webster,  Ph.D. 
Vida  H.  Beaven,  Ph.D. 
Ronald  Geller,  Ph.D. 
Renne  Chen,  Ph.D. 

Cooperating  Units:   Ungulate  Unit,  Animal  Center  Section, 

Laboratory  Aids  Branch,  Division  of  Research 
Services  (L .  Stuart) 

Project  Description: 

Obj  ectives:   Purification  of  human  urinary  and  plasma  kallikreins, 
Pf/dil,  and  Hageman  Factor  for  purposes  of  characterization  and 
of  production  of  specific  antiserums.   Preparation  from  specific 
antibodies  of  affinity  adsorbents  for  isolating  the  enzymes  and 
possibly  the  proenzymes  directly  and  specifically  from  human  plasma 
and  other  fluids,  and  for  devising  specific  biochemical  and  radio- 
immunochemical  assays.   Application  of  these  assays  to  studies 
of  human  circulatory  disease  states,  aich  as  hypertention. 

Purification  of  high  molecular  weight  (HMW)  and  low  molecular 
weight  (LMW)  kininogens  from  human  and  other  mammalian  plasma  by 
means  of  affinity  chromatography.   Characterization  of  the  isolated 
proteins.   Isolation  of  piece  B  from  kininogen  II  for  physico- 
chemical  and  biological  studies.   Preparation  of  an  affinity 
adsorbent  from  piece  B  to  isolate  specific  antibodies  either  from 
antiserums  to  kininogen  II  or  from  antiserums  prepared  to  B. 

Methods:   Affinity  chromatography  on  inhibitor-Sepharose  columns 
of  activated  human  plasma  to  obtain  human  plasma  kallikrein  and 

1  Ji^iA 


''■^m 


NHLI-109 
Pf/dil;  and  on  kininogen  antibody-Sepharose  coliomns  of  heated 
human  plasma  to  obtain  human  plasma  kininogens.   Devising  elution 
schemes  for  recovering  the  adsorbed  proteins  in  biologically 
active  form. 


\^. 


P- 


Major  Findings;   1)  Urinary  Kallikreins.   Human.   Highly  purified 
kallikrein  from  Type  B  urine  was  obtained  by  pressure  dialysis, 
hydroxyapatite  chromatography,  and  isoelectric  focusing.   The 
last  step  gave  five  TAMe  esterase  and  biologically  active  peaks 
which  were  indistinguishable  by  disc  gel  electrophoresis:   each 
gave  three  bands  with  the  same  mobilities.   The  bands  from  un- 
stained disc  gels  of  the  main  electrofocusing  peak  (HUK-B3)  were 
cut  out,  macerated,  suspended  in  Freund ' s  complete  adjuvant,  and 
injected  intramuscularly  into  a  sheep.   Precipitating  antibody 
was  obtained  after  two  booster  injections.   The  antiserum  inhi- 
bited the  kinin-raleasing  ability  of  HUK,  whereas  normal  sheep 
serum  was  not  inhibitory.   Since  normal  sheep  serum  inhibits  the 
biological  activity  of  HPK  (hiiman  plasma  kallikrein)  ,  antibody 
to  HUK  must  be  purified  before  it  can  be  tested  on  HPK.   A  speci- 
fic precipitate  of  HUK  and  antibody  to  HUK  has  been  prepared  as 
the  first  step  in  the  isolation  of  pure,  specific  antibody. 


Rat .   Fractionation  of  a  large  lot  of  rat  urine  by  methods 
described  in  our  previous  report  gave  two  main  TAMe  esterase 
peaks,  A  and  B;  only  B  had  kallikrein  activity.   Electrofocusing 
of  peak  B  after  hydroxyapatite  chromatography  gave  four  peaks  of 
TAMe  esterase  and  biological  activity.   Peaks  B-j^  and  B3  showed 
single  disc  gel  bands,  whereas  B2  and  B^  showed  two  bands:   the 
lighter  bands  of  B2  and  B4  had  the  same  mobilities  as  the  B^  and 
B3  bands,  respectively.   All  the  bands  were  localized  in  a  narrow 
mobility  range.   Molecular  weight  determinations  of  these  RUK 
isoenzymes  were  obtained  by  SDS  polyacrylamide  disc  gel  electro- 
phoresis, using  ovalbumin  and  chymotrypsinogen  A  as  markers. 
Bio~Gel  P-200  gel  filtration  of  B3  showed  coincident  TAMe  esterase 
activity  and  A28O  peaks  and  no  indication  of  impurities. 

Antibody  was  elicited  in  a  sheep  by  the  disc  gel  method  on 
RUK-B3.   A  specific  precipitate  was  prepared  by  mixing  antiserum 
with  pressure-dialyzed  rat  urine  at  equivalence.   Separation  of 
antibody  and  antigen  was  achieved  by  Sephadex  G-lOO  gel  filtration 
on  8  M  urea.   The  enzyme  was  recovered  free  of  antibody  in  30% 
yield  with  a  specific  activity  of  20  (p.mole  TAMe/min/A280  ^^^^)  • 
The  A28O  peak  appearing  shortly  after  the  column  void  volume 
contained  only  antibody.   Another  14%  of  the  starting  enzyme 
activity  with  a  specific  activity  of  10  was  found  in  the  region 


1-5  "-c: 


<a»j 


NHLI-109 
between  the  two  A28O  peaks.   Disc  gel  electrophoresis  showed  two 
sharp  bands  in  the  7-globulin  region  for  the  antibody  peak,  three 
bands  for  the  enzyme  peak  with  the  same  mobilities  found  for  a 
mixture  of  B,  through  B4,  and  the  same  three  bands  plus  a  broad 
band  with  a  mobility  intermediate  between  the  enzyme  and  the 
7-globulin  bands.   The  intermediate  band  had  TAMe  esterase  acti- 
vity, so  was  probably  an  antigen-antibody  complex.   The  purified 
antibody  was  covalently  bound  to  Sepharose  by  means  of  cyanogen 
bromide:   the  derivative  was  highly  active  in  binding  not  only 
rat  urinary  kallikreins,  but  also  the  kallikreins  in  extracts  of 
rat  submandibular  gland  and  pancreas. 


Rat  Urinary 
Kal  likrein 


Specific 
Activity 


Recovery  of 
Kallikrein 
Activity  % 


Molecular  Weight 
SDS  Gel   Gel  Filtration 


Bl 
B2 

Ba 


14.5 
20.4 
22.8 
18.8 


2.2 

9.5 

20.2 

9.4 


36,200 
34,000 
33,400 
32,600 


38,500 


From   Imrriune   Precipitate 
Enzyme 

Frac.    1  20.0  31 

Enz^TTie 

Frac.    2  10.4  14 


2)  Human  Plasma  Kallikrein.   Small  amounts  of  this  enzyme 
have  been  isolated  by  affinity  chromatography  of  acetone-activated 
hujnan  plasma  on  STI  (soybean  trypsin  inhibitor) -Sepharose  columns. 
After  washing  the  column  with  suitable  solvents,  about  25%  of  the 
starting  H?K  TAMe  esterase  and  biological  activities  were  recovered 
in  the  5  M  guanidine  hydrochloride  eluate.   The  degree  of  purity 

of  such  preparations  has  not  yet  been  tested. 

3)  Human  Pf/dil.   This  enzyme  is  thought  to  effect  the  acti- 
vation of  plasma  kallikrein  and  is  itself  present  in  fresh  human 
plasma  in  an  inactive  form.   It  appears  to  be  derived  from  Hageman 
factor.   The  Pf/dil  activity  in  acetone-activated  plasma  is  not 
bound  to  STI-Sepharose,  being  quantitatively  recovered  in  the 
filtrate,  but  can  be  weakly  bound  to  an  LTI  (lima  bean  trypsin 
inhibitor) -Sepharose  column  m  the  presence  of  0.15  M  NaCl.   A 
purification  of  between  200-  and  400-fold  in  nearly  quantitative 


JLa*/ 


NHLI-109 
yield  has  been  achieved  on  a  small  scale.   The  Pf/dil  activity  of 
the  STI-Sepharose  column  filtrate  can  be  doubled  by  treatment  with 
the  chaotropic  solvents  8  M  urea,  5  M  guanidine  hydrochloride, 
and  4  M  sodium  thiocyanate.   After  dialysis  against  saline,  then 
0.01  M  Tris  CI,  pH  7.5,  the  Pf/dil  activity  behaves  the  same  on 
LTI-Sepharose  columns  as  the  untreated  material. 

4)  Mammalian  Plasma  Kininoqens.   Human.   H\aman  plasma 
collected  under  silicone  conditions  can  be  heated  at  60°  for  0,5 
hour  without  apparent  effect  on  either  the  HMW  or  LMW  kininogens, 
whereas  the  enzymes  of  the  kinin-generating  system  are  destroyed. 
Tte  kininogens  present  in  such  heated  plasma  have  been  adsorbed 
to  antikininogen-Sepharose,  either  on  coliomns  or  in  batch  operation. 
Elution  with  urea,  sodium  thiocyanate,  or  guanidine  hydrochloride 
gave  fractions  containing  both  HMW  and  LMW  kininogens  in  about 
10%  yield.   Disc  gel  electrophoresis  showed  the  presence  of  conta- 
minants with  the  mobility  of  y-globulins;  whether  these  are  specific 
antibody  from  the  Sepharose  col\amn  or  plasma  proteins  co-adsorbed 
with  the  kininogens  has  not  yet  been  determined. 

Bovine.   Sheep  entiserum  has  been  prepared  from  highly  puri- 
fied bovine  LMW  kininogen  (supplied  by  E.  Habermann)  by  the  disc 
gel  method.   The  specific  precipitate  formed  at  equivalence  by 
mixing  antiserum  and  bovine  serum  has  been  dissolved  in  8  M  urea 
and  is  being  chromatographed  on  hydroxyapatite  to  separate  the 
antibody  from  the  antigen.   As  found  earlier  for  the  human  kinin- 
ogen-antibody  precipitate,  the  antibody  is  not  adsorbed  and  appears 
in  the  column  filtrate  free  of  antigen;  antigen  could  be  subse- 
quently eluted  free  of  antibody.   Ouchterlony  double  diffusion 
plates  can  be  used  to  follow  the  fractionation  directly.   An  8  M 
urea  solution  of  antigen-antibody  precipitate  gives  a  precipitin 
ring,  whereas  an  8  M  urea  solution  of  only  antigen  (or  antibody) 
gives  a  sharp  precipitin  line  with  antibody  (or  antigen) ;  if  both 
are  present,  but  one  is  in  excess  of  equivalence,  a  precipitin 
line  and  a  partial  ring  are  found. 

Significance  to  Biomedical  Research  and  Institute  Program:   The 
purification  of  the  components  of  the  plasma  kallikrein  system  is 
crucial  to  investigations  of  its  physiological  function (s).   This 
system  is  activated  simultaneously  with  the  blood  coagulation 
system:   Factor  XII  (Hageman  Factor)  of  the  latter  appears  to  be 
an  integral  part  of  the  former.   There  are  a  number  of  observa- 
tions implicating  the  kallikrein  system  in  inflammation,  pain, 
immune  reactions,  the  carcinoid  syndrome,  arthritides  of  various 
etiologies,  and  hereditary  angioneurotic  edema. 


to 


aaC 


NHLI-109 
Proposed  Course  of  Project;   Specific  antibodies  to  human  urinary 
kallikrein  will  be  isolated  from  the  immune  precipitate,  bound  to 
Sepharose  for  isolation  of  the  enzyme  directly  from  pressure- 
dialyzed  urine  and  possibly  of  other  glandular  kallikreins,  and 
tested  for  inhibition  and  binding  of  plasma  kallikrein.   Further 
purification  of  human  plasma  kallikrein,  Pf/dil,  and  Hageman 
Factor  will  be  performed  for  purposes  of  characterization  and  of 
production  of  specific  antibodies  for  making  affinity  adsorbents. 

Honors  and  Awards:   None 

Publications: 

Webster,  M.E.,  Pierce,  J.V.,  and  Sampaio,  M.U.:   Studies 
on  antibody  to  bradykinin.   Bradykinin  and  Related  Kinins: 
Cardiovascular,  Biochemical,  and  Neural  Actions.   Plenum 
Press,  pp.  57-64  (1970). 

Beaven,  V.H-,  Pierce,  J.V.,  and  Pisano,  J.J.:   A  sensitive 
isotopic  procedure  for  the  assay  of  esterase  activity: 
measurement  of  human  urinary  kallikrein.   Clin.  Chim. 
Acta  32:   67-73,  1971. 


AS4 


Serial  No.  NHLI-llO(c) 

1.  Experimental  Therapeutics  Branch 

2 .  Section  on  Neuroendocrinology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Effects  of  Steroid  Hormones,  Protein  Hormones 
and  Electrolytes  on  Neural  Function  . 

Previous  Serial  Number:   NHLI-166(c) 

Principal  Investigator:   Robert  I.  Henkin,  M .D . ,  Ph.D. 


Other  Investigators; 


M.  Buchsbaum,  M.D. 
J.  Fontana,  Ph.D. 
C.  Gillen,  M.D. 
N.  Jacobs,  M.D. 
M.  D.  Walker,  M.D. 


Cooperating  Units: 


Department  of  Medicine,  University  of 
Cincinnati  School  of  Medicine,  Cincinnati, 
Ohio;  National  Cancer  Institute,  Baltimore, 
Maryland;  Polytechnic  Institute  of  Brooklyn, 
Brooklyn,  N.  Y.;  National  Institute  of  Mental 
Health,  Bethesda,  Md . 


Project  Description: 

Objectives:   To  investigate  systematically  the  interrelationships 
between  steroid  hormones,  protein  hormones  and  electrolytes  on 
neural  function  with  respect  to  sensory  detection  and  recognition 
and  to  investigate  the  mechanisms  by  which  these  interrelationships 
occur . 

Major  Findings:   1 .   Effects  of  adrenocorticosteroids  on  NE  and 
Ch  uptake  of  brain  synaptosomes  .  It  has  long  been  known  that  the 
excess  of  and  lack  of  carbohydrate-active  steroids  (CAS)  greatly 
affect  central  nervous  system  activity.   The  mechanism  by  which 
CAS  influences  CNS  activity  remains  relatively  obscure.   It  appears 
however  that  one  possible  mechanism  may  involve  an  interaction 
between  the  steroid  molecules  and  the  actual  nerve  endings.   The 
object  of  this  investigation  is  to  study  the  possibility  of  this 


3L^7 


Serial  No.  NHLI-llO(c) 

interaction . 

In  light  of  the  difficulty  involved  in  examining  a  steroid- 
nerve  ending  interaction  in_  vivo,  synaptosomes  (pinched  off  nerve 
endings  consisting  of  intact  nerve  membranes  enclosing  synaptic 
vesicles)  were  utilized  since  these  particles  provide  a  convenient 
in  vitro  system  for  the  study  of  the  effects  of  drugs  on  neuro- 
transmitter function.   Therefore,  the  effect  of  CAS  on  the  synap- 
tosomal uptake  and  release  of  various  neurotransmitters  was  in- 
vestigated-  Cortisol  hemisuccinate  was  chosen  as  the  adrenal 
steroid  although  it  does  not  possess  high  CAS  activity.   However, 
it  is  water  soluble  and  allows  a  completely  aquaeous  system  to 
be  employed . 

Active  uptake  of  NE  and  Ch  by  synaptosomes  was  demonstrated . 
Cortisol  hemisuccinate  inhibited  NE  and  Ch  uptake,  and  results 
are  summarized  below: 

Inhibition  by  Cortisol  Hemisuccinate  of  NE  Uptake 

Concentration  of  Steroid        %   Inhibition  of  NE  Uptake 

0.8  X  10""^  M  0 


2  .0  X  10-4  M  2.6 
4.0  X  IQ-'^  M  3.4 
8.0  X  10"'*  M  4.8 
1.6  X  10-3  M  13  ,2 

3  .2  X  10"-^  M  18.2 
6.4  X  10-3  lyi  ^2  .J 
1.2  X  10"2  M  62.0 


*a^ 


! 


Serial  No.  NHLI-llO(c) 

Inhibition  by  Cortisol  Hemisuccinate  of  Ch  Uptake 
Concentration  of  Steroid         %   Inhibition  of  Ch  Uptake 
4  X  10~4  0 


X  10 


1.6  X  10 


3.2  X  10 


-4 


-3 


-3 


6.4  X  10"^ 


2.8 
3.8 

26 

61 


Inhibition  by  Cortisol  hemisuccinate  of  NE  and  Ch  uptake  re- 
quired high  concentrations  of  steroid. 

It  has  been  postulated  that  part  of  mechanism  concerned  with 
neurotransmitter  uptake  involves  ATPase  activity.   It  was  de- 
termined that  our  synaptosomal  preparations  contained  appreciable 
Na-K  ATPase  activity  (See  Table)  . 


Fraction 
Nuclei  +  cell  debris 
17,000g  Super nate 
Synaptosome 


Total  Activity  of  Na-K  ATPase 
22.5  Units 
2  .1  Units 
7.0  Units 


Therefore,  the  effect  of  Cortisol  21  hemisuccinate  on  synaptosomal 

Na-K  ATPase  activity  was  investigated.   The  results  are  summarized 
below: 

Concentration  of  Steroid    %   Inhibition  of  Ha-K  ATPase  Activity 
5  X  10-4  M  18 


1  X  10 


in-3 


M 


2  X  10~3  M 

4  X  10"-^  M 
8  X  10-3  M 


23 

35 

44 
58 


^f 


Serial  No.  NHLI-llO(c) 

Thus,  Cortisol  hemisuccinate  inhibited  Na-K  ATPase  activity  and 
this  inhibition  took  place  at  steroid  levels  which  also  inhibited 
NE  and  Ch  uptake. 

2 .   EEG  changes  in  man  in  the  absence  of  adrenocorticosteroid 
secretion  and  during  administration  of  ACTH.   These  studies  comprise 
two  major  efforts;  one,  the  effect  of  these  changes  on  visual 
evoked  response  activity  (AER)  in  man,  the  second,  the  effect  of 
these  changes  on  sleep  phenomena  in  man.   In  the  first  part  of 
this  study  patients  with  several  forms  of  adrenal  cortical  insuf- 
ficiency had  measurements  made  of  their  visual  evoked  responses 
to  graded  light  intensities  before  and  during  withdrawal  from  the 
effects  of  their  replacement  therapy.   In  addition,  these  measure- 
ments were  made  during  administration  of  ACTH.   Results  of  these 
studies  indicate  that  during  ACTH  administration  there  is  signi- 
ficant enhancement  of  the  AER  which  is  dependent  of  the  ACTH 
effect  on  the  adrenal  gland  and  appears  to  be  a  direct  effect  of 
ACTH  on  brain  activity.   In  the  second  part  of  this  study,  the 
sleep  activity  of  patients  with  various  forms  of  adrenal  cortical 
insufficiency  was  measured  under  the  same  conditions  as  noted  in 
part  one.   Results  of  these  studies  indicate  that  REM  sleep  de- 
creases significantly  during  the  time  which  replacement  therapy 
is  withdrawn,  is  further  decreased  during  ACTH  administration 
and  return  to  normal  during  readministration  of  steroid  hormone 
therapy.   These  studies  differ  from  those  carried  out  in  normal 
volunteers  which  indicate  that  administration  of  carbohydrate- 
active  steroids  is  correlated  with  increases  rather  than  decreases 
in  REM  activity. 

3o   Distribution  and  metabolism  of  H   steroids  in  nervous 
tissues  of  the  cat.   In  continuation  of  previous  studies  the 
distribution  of  H-^  testosterone  and  progesterone  in  various  tissues 
of  the  eviscreted  cat  has  been  studied,,   Following  a  controlled 
rate  infusion  of  H-^  testosterone  concentrations  of  hormone  in  the 
brain  followed  a  pattern  similar  to  that  previously  shown  for  H^ 
Cortisol  but  quite  different  from  that  of  H-^  progesterone.   The 
metabolites  of  H  testosterone  in  the  tissues  studied  are  being 
measured  by  gas-liquid  chromatography  by  Dr«  Leon  Sholiton, 
University  of  Cincinnati. 

4„   EEG  changes  in  patients  with  serum  calcium  abnormalities 
and  with  pseudohypoparathyroidism.   The  effects  of  serum  calcium 
concentration  on  the  visual  average  evoked  responses  (AER)  was 
studied  in  24  patients  with  various  disorders  of  calcium  metabolism 

4  33^ 


\u, 


Serial  No„  NHLI-llO(c) 


HfD 


and  in  5  normal  volunt 
4  days.  Low  serum  cal 
were  associated  with  g 
than  were  high  serum  c 
Administration  of  para 
calcium  concentration, 
latency  or  amplitude, 
correlated  with  serum 
patients'  diagnostic  c 


eers  given  450  U  of  parathyroid  extract  for 
cium  concentrations  (below  8  mg/100  ml) 
reater  amplitude  and  shorter  latency  AERs 
alcium  concentrations  (above  12  mg/100  ml) . 
thyroid  extract  in  doses  that  raised  serum 
albeit  minimally,  had  no  effect  on  the  AER 
AER  latency  and  amplitude  changes  were 
calcium  concentration  rather  than  with  the 
ategory . 


t 


Perceptual  function  in  8  patients  with  pseudohypoparathyrodism 
(PHP)  was  studied  using  two  neurophysiological  measurement  tech- 
niqueSj,  the  average  evoked  response  (AER)  and  motor  nerve  conduction 
velocity.   A  battery  of  psychophysical  tasks  including  reaction 
time,  size  estimation,  hidden  pictures  and  the  rod  and  frame 
procedure  were  also  used.   Patients  with  PHP  had  significantly 
longer  latency  visual  AER  and  slower  reaction  times  than  did  a 
group  of  normal  volunteers  and  the  patients  performed  erratically 
and  poorly  on  the  psychophysical  tasks.   Differentiating  these 
patients  from  patients  with  diffuse  mental  deficiency  were  two 
relatively  specific  perceptual  response  patterns:   (1)  AER  amplitude 
decreased  with  increasing  stimulus  intensity  and  (2)  Reaction  time 
showed  abnormally  strong  effects  of  the  duration  of  the  preparatory 
interval.   These  results  could  not  be  attributed  to  alterations  in 
serum  calcium  concentration.   Each  of  these  patients  did  exhibit 
decreases  in  cyclic  AMP  in  the  urine.   Since  cyclic  AMP  appears  to 
play  some  role  at  synaptic  junctions  it  may  be  useful  to  speculate 
about  the  role  which  cyclic  AMP  may  play  in  sensory  function  in 
the  future.,   Future  studies  are  designed  to  investigate  this 
possible  relationship* 

5.   The  role  of  adrenocorticosteroids  in  neural  functions 
During  this  past  year  work  in  laboratory  has  focused  on  one  sen- 
sory modality,  taste,  and  the  effects  of  removal  and  replacement 
of  carbohydrate-active  steroids  on  taste  responses  in  man.   Two 
general  kinds  of  measurements  were  made,  threshold  measurements 
and  intensity  measurements.   We  tried  to  relate  these  two  types 
of  measurements  to  each  other  and  to  show  the  effects  of  adrenal 
corticosteroids  on  each.   Based  upon  observations  in  man  we  derived 
a  taste  equation  which  appears  to  fit  the  taste  responses  of 
normal  man  and  of  patients  with  various  forms  of  adrenal  cortical 
insufficiency  and  Gushing 's  syndrome  in  their  treated  as  well  as 
untreated  state. 


^3/ 


Serial   No.     NHLI-1 10(c) 
This    equation    is: 

I/I 


K  (T)^ 


max    K  (T)  ^  +  1 

where  I  is  the  intensity  of  tastant  at  concentration  (T)  ,  Ijnax' 
the  maximum  intensity  at  highest  (T)  and  K  is  a  constant^   Based 
upon  our  previous  data  and  hypotheses  there  is  a  specific  inter- 
action between  tastant  and  receptor  molecule  at  the  taste  bud 
membrane  such  that  a  tastant-receptor  complex  is  formed  (RT) .   If 
we  assume  that,  one  tastant  reacts  with  one  receptor  molecule  in  a 
simple  equilibrium  it  is  possible  to  derive  the  taste  equation  1 
shown  above  as  follows: 


K  =  Ki/Ki    eqn  2 


Kl 
R   +   T    ^  RT 

and 

iSi 

(RT) 

(R)    +     (RT) 

K  (T) 


K  (T)  +  1 


eqn  3 


The  forced  scaling  intensity  data  in  the  normal  volunteers, 
and  to  a  lesser  extent  in  the  patients,  was  described  by  eqn  1 
which  is 

j/j  =         K  (T)^ 

-"/■'max    K  (T)2  +  1 

^1 
The  binding  stoichiometry        R  +  2T  =J  RT2  K  =  K^/K-^  eqn  4 

-JMU =     -JUni _„  5 

(R)  +  (RT)^      K  (t2)'^  +  1  ^^" 


would  lead  to  eqn  1  if  one  assumes  that  the  intensity  is  propor- 
tional to  the  concentration  of  RT  complexes.   In  this  model  the 
single  parameter  K  obtained  by  fitting  eqn  1  to  the  experimental 
data  is  the  binding  constant  for  the  formation  of  the  RT2  complex. 
Equation  1  can  be  derived  as  follows:   At  equilibrium,  eqn  4 
becomes  (RT2)  =  K  (R)  (T)"^.   Now  (R)  +  (RT2)  =  Rq  where  Rq  is 
the  total  number  of  receptor  molecules  present.   Therefore, 


(RT2)  =  k[Ro-(RT)2](T) 

_KRa_ 
K  (T) 

6  3l3^ 


<-.'  =  FtrBV 


Serial  No.  NHLI-llO(c) 

and  if  we  assume  that  the  intensity  is  proportional  to  the  number 
of  (RT2)  complexes,  i.e.,  I  =  C (RT2) ,  then 

CRn  K  (T)^         , 

I  =  TT^ — r~7        where 

K  (T)^  +  1 

C  is  a  proportionality  constant.   As  (T)  becomes  very  large,  I 
approaches  its  maximum  value  Ij^ax  ~  '--^O'   Therefore, 


I/I 


max   K  (T)  2  +  1 


which  is  eqn  1.   Eqn  4  is  of  course  only  one  of  many  possible 
models  from  which  eqn  1  can  be  derived.   However,  from  the  simplest 
model  (eqn  2)  eqn  1  cannot  be  derived. 


This  model  would  explain  why  the  intensity  function  reaches 
a  maximum  at  high  tastant  concentrations;  the  tastant  molecules 
would  complex  with  all  the  available  receptor  molecules  and  intro- 
duction of  more  tastant  could  not  form  more  complexes.   As  the 
tastant  concentration  decreases  the  number  of  receptor  molecules 
complexed  would  also  decrease  and  at  a  sufficiently  low  concen- 
tration the  signal  produced  would  no  longer  be  recognized,  albeit 
detected,  and  if  tastant  concentration  is  decreased  further,  it 
would  not  be  detected  from  background. 

The  signal  produced  by  this  complex  formation  presumably 
occurs  at  the  taste  receptor  and  is  anatomically,  spatially  and 
temporally  separated  from  the  neural  events  (Fig.  6).   Thus,  the 
classification  of  pre-neural  and  neural  events  has  physical  bases., 

CAS  deficiency  and  excess  states  may  affect  the  preneural 
events  of  taste  by  affecting  the  binding  constants  by  which 
tastant-receptor  molecule  complexes  occur  or  by  affecting  the 
stoichiometry  of  binding.   Excessive  and  inadequate  amounts  of 
CAS  decrease  the  binding  constants  for  all  taste  qualities  be- 
cause the  intensity  curves  are  all  shifted  to  higher  concentrations „ 
From  the  second  point  of  view  the  possible  deviation  of  the  data 
from  eqn  1  toward  a  lower  order  (in  (T) )  equation  such  as  eqn  3 
would  imply  that  the  stoichiometry  of  binding  is  altered.   Apparently 
there  is  a  concentration  of  CAS  at  which  the  intensity  is  maximal 
for  a  given  tastant  concentration  and  either  an  excess  or  a  de- 
ficiency of  CAS  lowers  the  intensity. 


I 


SLZi 


Serial  No=   NHLI-llO(c) 

The  fit  of  equation  1  to  the  data  suggests  that  two  tastants 
are  required  for  the  recognition  process  to  occur  normally.   The 
equation  does  not  specify  the  spatial  relationships  among  the 
tastants  and  receptors.   However,  these  relationships  are  sugges- 
tive of  the  existence  of  cooperative  binding  in  which  K  would 
be  the  product  of  the  square  (Ki^)  of  the  intrinsic  binding 
constants  (Ki)  of  the  tastants  and  a  coupling  term  Kc;  i.e., 
K  =  Ki^Kc.   According  to  this  interpretation  excess  or  deficiency 
CAS  states  would  shift  the  intensity  curves  by  reducing  the 
coupling  constant  which  would,  in  the  limit  of  Kc  =  1,  also  shift 
the  binding  stoichiometry  to  a  lower  order.   High  tastant  con- 
centrations can  overcome  this  decoupling  by  sheer  mass  action. 

Significance:   1 .   Role  of  adrenocorticosteroids  in  neural  con- 
duction .   We  have  shown  that  NE  and  Ch  uptake  by  brain  synapto- 
somes  are  markedly  inhibited  by  the  presence  of  carbohydrate- 
active  steroids .   This  effect  appears  to  be  mediated  by  inhibition 
of  Na-K  activated  ATPase  activity  by  carbohydrate-active  steroids  . 
Since  no  effect  of  pharmacological  doses  of  these  steroids  could 
be  observed  on  the  Na  or  K  currents  of  the  nerve  it  is  clear  that 
the  effect  of  these  corticosteroids  on  neural  function  is  not  on 
the  excitable  portion  of  the  nerve  membrance,  per  se,  but  rather 
on  the  metabolism  of  the  nerve;  i.e.,  in  maintaining  the  mem- 
brane potential  and  also  on  synaptic  conduction .   Whatever  effect 
these  steroids  may  have  on  axonal  conduction  is  probably  through 
their  effects  on  myelin . 

2.   Role  of  adrenocorticosteroids  in  neural  function.   We 
have  described  an  equation  by  which  taste  phenomena  occur  in 
normal  man  and  in  patients  with  various  abnormalities  of  adrenal 
cortical  function.   This  equation  is: 

I/Imax  =   ^  (^)' 


K  (T)2  +  1 

where  I  is  the  intensity  of  tastant  at  concentration  (T)  ,  Ij^ax' 
the  maximum  intensity  at  highest  (T)  and  K  is  a  constant .   This 
equation  can  be  derived  from  simple  relationships  of  the  theory 
which  we  have  developed  over  the  past  3  years .   The  model  which 
is  described  by  this  equation  would  explain  why  intensity  functions 
in  taste  reach  a  maximum  at  high  tastant  concentrations;  taste 
molecules  would  complex  with  all  available  receptor  molecules  and 
introduction  of  more  tastant  could  not  form  more  complexes.   De- 


Jl3^ 


I' 


Serial  No.  NHLT-llO(c) 

ficiency  and  excesses  of  carbohydrate-active  steroids  in  man 
may  affect  the  pre-neural  events  of  taste  by  directly  affecting 
the  binding  constants  by  which  tastant -receptor  molecule  complexes 
occur  or  by  affecting  the  stoichiometry  of  binding.   Our  data 
demonstrate  that  excessive  and  inadequate  amounts  of  these 
steroids  decrease  the  binding  constants  for  all  taste  qualities 
because  the  intensity  curves  are  all  shifted  to  higher  concen- 
trations .   Apparently  there  is  a  concentration  of  steroid  at 
which  intensity  is  maximal  for  a  given  tastant  concentration  and 
either  an  excess  or  a  deficiency  of  steroid  lowers  the  intensity. 

3.   Role  of  calcium  in  EEG .   Through  the  application  of  the 
quantitative  technique  of  visual  averaged  evoked  responses  to 
clinical  investigative  problems  it  has  been  possible  to  study 
the  EEG  changes  in  man  during  several  physiological  states  in- 
volving calcium  metabolism.   Our  studies  have  shown  that  low 
serum  calcium  concentrations  (below  8  mg/100  ml)  are  associated 
with  greater  amplitude  and  shorter  latency  AER  than  high  serum 
calcium  concentrations  (above  12  mg/100  ml) . 

Proposed  Course  of  Project;   Specific  work  will  be  carried  out 
which  will  identify  the  role  which  carbohydrate-active  steroids 
play  at  the  synapse  through  their  effects  on  several  neurotrans- 
mitter agents.   In  man,  the  theories  and  hypotheses  made  during 
this  past  year  with  respect  to  the  effects  of  these  steroids  on 
taste  will  be  tested  and  evaluated . 


Honors  and  Awards; 


None 


Publications; 


Henkin,  R.I.:  The  neuroendocrine  control  of  perception.  In 
Hamburg,  D.  (Ed.):  Perception  and  its  Disorders.  Baltimore, 
Md.,  Williams  &  Watkins,  1970,  pp.  54-107. 

Zisman,  E.,  Henkin,  R.I.,  Ross,  G .T .  and  Bartter,  F.C.  :   A 
biochemical  similarity  between  chromatin  negative  gonadal 
dysgenesis  and  pseydohypoparathyroidism.  Acta  Endocrin .  Panam. 
1:  49-71,  1970. 


[ 


Henkin,  R.I.:  The  effects  of  corticosteroids  and  ACTH  on  sen- 
sory systems.   Prog  .  in  Brain  Research   32:  270-294,  1970. 


c^sr" 


mpBBIWiiiawynBi^ 


Serial  No.  NHLI-llO(c) 

Walker,  M.D.,  Henkin,  R.I.,  Harlan,  B  .A .  and  Casper,  A.G.T.: 
The  distribution  of  tritiated  Cortisol  in  blood,  brain,  CSF 
and  other  tissues  of  the  cat.   Endocrinology  88:  224-232,  1971. 

Buchsbaum,  M.  and  Heiokin,  R.I.:  Serum  calcium  concentration 
and  the  average  evoked  response.   Electroencephalography 
and  Clin.  Neurophysiology.   30:  10-16,  1971. 

Buchsbaum,  M.,  Silverman,  J.  and  Henkin,  R.I.:  Contrast  effects 
on  the  auditory  evoked  response  and  its  relation  to  psycho- 
physical judgment.   Perception  and  Psychophysics   In  press, 
1971. 

Henkin,  R.I.,  Fontana,  J .A .  and  Walker,  M.D.:   On  the  mechanism 
of  the  presence  and  distribution  of  adrenal  corticosteroids 
in  the  central  and  peripheral  nervous  system.   Proceedings 
of  the  III  International  Congress  on  Hormonal  Steroids. 
Excerpta  Medica.   Elsevier,  Amsterdam,  Holland,  In  press,  1971. 


10 


^3^ 


Serial  No.   NHLI-lll(c) 

1.  Experimental  Therapeutics  Branch 

2.  Section  on  Neuroendocrinology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Trace  Metal  Metabolism 

Previous  Serial  Number:   NHLI-164  (c) 

Principal  Investigator:   Robert  I.  Henkin,  M.D«,  Ph=D. 

Other  Investigators:   Joseph  Fontana,  Ph.D. 

Eugene  Giroux,  Ph.D. 
N.  Prakash,  Ph<.D. 
R.  Michell,  Ph.D.,  D.V.M. 
J.  Pierce,  D.V.M. 


Cooperating  Units: 


Department  of  Chemistry,  Charlottesville, . Va . 
Department  of  Medicine,  Presbyterian  Hospital, 
N.Y.,  N.Y. 


Objectives:   This  project  was  designed  to  study  the  physiology, 
metabolism,  biochemistry  and  pathology  of  copper,  zinc  and  other 
trace  metals  in  physiological  fluids  and  tissues  of  normal  sub- 
jects in  patients  with  various  diseases  and  in  animals.   We 
have  also  studied  the  interaction  between  metals  and  their  binding 
proteins. 

Major  Findings;   Physiology 

1.   Copper  depletion  and  corticosteroid  production  in  the 
adrenal  gland  of  cats  and  rats.   The  a5-3  ketosteroid  isomerase 
activity  in  guinea  pig  adrenals  is  dependent  on  the  presence  of 
copper  ions.   This  enzyme  activity  along  with  3  P-hydroxysteroid 
dehydrogenase  activity  is  responsible  for  the  conversion  of  preg- 
nenolone to  progesterone  in  the  early  biosynthesis  of  nearly  all 
biologically  active  steroid  hormones.   In  conjunction  with  our 
laboratory's  interest  in  the  interrelationship  of  trace  metals  and 
normal  body  function,  the  effect  of  copper  deficiency  in  cats  and 
rats  on  adrenal  steroid  production  was  investigated. 


I 


^37 


NHLI-l]l(c) 
Adrenals  obtained  from  normal  and  copper  deficient  cats  and 
rats  were  sliced  and  incubated  with  tritiated  cholesterol.   In 
one  experiment  involving  rat  adrenals,  pregnenolone^  progesterone, 
corticosterone  and  Cortisol  were  isolated,  using  paper  and  thin 
layer  chromatography.   In  a  second  axperim.ent  involving  cat  adrenals, 
Cortisol,  cortisone  and  corticosterone  were  isolated,  again  using 
paper  and  thin  layer  chromatography o   Fxnal  identification  of  all 
compounds  was  made  by  successive  crystalizations  to  a  constant 
specific  activity  using  authentic  carrier. 

The  effect  of  copper  deficiency  on  adrenal  weight  is 
described  in  Table  I . 

TABLE  I 

Experiment  Wet  Weight  of  Adrenals 

|,  Copper  Deficient  Normal 

Cats  (3)  177.4  ±  9.5  (s.e.m.)*    102.8  ±  4.3  (s.e.m.)* 

Rats  (6)  93.0  ±  4.3  (s.e.m.)**    64.0  ±  7.7  (s.e.m.)** 

*  Each  adrenal  compared. 
,  ]  i'       **  Each  pair  of  adrenals  from  a  rat  compared. 

\C " 

These  results  indicate  that  copper  deficiency  results  in  a 
marked  adrenal  hyperplasia. 

The  results  of  the  incubation  experiments  indicate  that 
copper  depleted  animals  produce  markedly  reduced  levels  of  Cortisol, 
cortisone  and  corticosterone  when  incubated  with  H^  cholesterol 
and  compared  to  adrenals  of  normal  animals  (Table  II) ,  but  both 
groups  produce  similar  levels  of  pregnenolone  (Table  III) . 


^36 


TSTTTXTT^BranZ! 


TABLE 

II 

NHLI-lll(c) 

Type 

Corticosterone* 

Cortisol* 

Cortisone* 

Norma 1-1 

30,926 

6,318 

6,690 

Copper  Depleted-1 

10,083 

3,813 

3,778 

Norma 1-2** 

91,709 

14,370 

— 

Copper  Depleted-2** 

32,166 

5,362 

— 

Normal-3 

12,010 

3,267 

— 

Copper  Depleted-3 

7,568 

2,452 

— 

Norma 1-4** 

19,098 

13,149 

— 

Copper  Depleted-4** 

7,601 

2,744 

— 

Normal-5 

13,246 

14,593 

— 

Copper  Depleted-5 

8,888 

2,584 

~ 

Norma 1-6** 

27,252 

12,972 

— 

Copper  Depleted-5 

6,900 

3,319 

— 

^^. 


*dpm/gm  tissue,  net  weight 
**Cu(N03=)  1  X  10~5  M  added  to  reaction  vessel 


TABLE  III 

In  Vitro  Steroid  Production  by  Adrenals  from  Normal 
and  Copper  Deficient  Rats 


L^ 


Cortisol*  Corticosterone*Preqnenolone*Proqesterone'^ 
Copper 
Deficient(5)  31,234±7,295  42,133±4,476  75, 112±26, 600    50,197 

Normals{5)     48,609±6,277  76, 614±21, 138  84, 182±29, 437  185,014 

*gpm/gm  tissue,  net  weight 


^3f 


..«'  •:' 


NHLI-l  11(c) 
These  results  indicate  that  the  block  in  steroidogeneses 
caused  by  copper  depletion  occurs  at  the  conversion  of  preg- 
nenolone to  progesterone,  at  the  A^-3  hydroxysteroid-dehydrogenase- 
iscinerase  step. 

2 .  Metal-protein  complexes  in  physiological  fluids.   This 
project  was  designed  to  quantitate  the  various  metal-ligand 
complexes  which  occur  in  physiological  fluids.   Earlier  work  in- 
volved fractionating  serum  into  stable  metalloproteins  and  rela- 
tively easily  dissociable  metal  complexes  could  not  be  determined  <> 
We  were  interested  in  the  manner  by  which  various  trace  metals 
formed  complexes  with  protein  or  peptide  moeities  and  we  have 
studied  these  complexes  in  terms  of  their  ability  to  pass  a  variety 
of  semi-permeable  membranes. 

Urine  was  examined  initially.   It  was  stored  at  4°C,  then 
warmed  to  37 °C,  brought  to  pH  5.0  with  HAc,  centrifuged  and  fil- 
tered.,  The  filtrate  was  passed  through  membranes  of  nominal  pore 
sizes  46,  38,  30  and  24  A °  diameter.   The  apparatus  and  membranes 
for  carrying  out  these  procedures  came  from  the  Amicon  Corp., 
Boston,  Mass.   All  metal  measurements  were  made  by  atomic  absorption 
spectrophtometry  or  flame  spectrophotometry.   Only  the  smallest 
membrane  significantly  retained  calcium,  magnesium  and  zinc 
complexes.   These  results  indicated  that  metaLs  are  bound  to  small 
ligands  in  urine.   In  an  effort  to  evaluate  the  character  and  size 
of  these  ligands  model  studies  of  the  interraction  of  metal  com- 
plexes with  known  ligands  are  in  progress,  as  noted  below. 

In  combination  with  gel  filtration  studies  it  is  possible  to 
identify  amino  acid,  carboxylate  and  inorganic  acid  anion  ligands; 
however,  because  of  their  lack  of  homogeneity  it  may  not  be  possible 
to  quantitatively  describe  the  metal  distribution  among  these 
ligands.   These  studies  can  be  carried  out  easily  in  blood  and  we 
are  in  the  process  of  doing  so.   We  are  also  evaluating  the  manner 
by  which  metals  pass  these  membranes  in  conjunction  with  the  assays 
of  various  protein,  polypeptides  and  peptides  in  the  ultraf iltrate 
and  retentate  which  should  lead  to  kinetic  information  related  to 
metal  distribution.   This  information  has  particular  relevance  to 
the  manner  by  which  protein-metal  complexes  cross  the  glomerulus 
of  the  kidney. 

3.  Metal-proteL n  complexes  in  tissue.   Kidney  and  liver 
contain  sulfhydryl  rich  polypeptides  which  are  capable  of  binding 
zinc,  copper,  mercury,  cadmium  and  other  metals.   The  character 
of  the  polypeptide  and  the  metals  which  it  contains  appear  to 


3t¥^ 


vl 


NHLI-1 11(c) 

differ  in  different  tissues  and  in  different  animal  species.   Vallee 
has  stated  that  metallothionein  is  a  cadmium  containing  polypeptide 
and  he  has  suggested  that  this  protein  plays  a  role  in  metal  de- 
toxification in  the  kidney.   Horse  and  human  kidney  are  rich  in 
cadmium  and  are  excellent  sources  of  metallothionein. 


Our  results  indicate  that  dog  kidney  contains  about  15  ppm  Zn, 
3  ppm  Cu  and  less  than  0.2  ppm  cadmium  per  gm  wet  weight  of  tissue. 
Valle's  data  from  human  kidney  cortex  indicates  there  is  350  ppm 
Zn  and  140  ppm  Cd  per  gm  dry  weight  of  tissue.   Purified  human 
kidney  metallothionein  contains  3.7-3.9  g-atoms  Cd,  3.2-4.3  g-atoms 
Zn,  0  g-atoms  Cu  and  0.3  g-atoms  Hg .   In  a  preliminary  experiment 
dog  kidney  cortex  was  homogenized  in  dilute  phosphate  buffer  and 
the  homogenate  prepared  in  a  manner  similar  to  that  used  by  Vallee 
for  preparation  of  metallothionein.   Various  fractions  were  filtered 
through  a  Sephadex  G-75  column.   Two  low  molecular  weight  metallo- 
proteins  were  observed.   One,  MW  34,000,  with  equal  amounts  of 
copper  and  zinc,  is  probably  the  cytocuprein  described  previously 
by  Carrico  and  Deutsch.   The  second,  MW  10,000  corresponds  to 
metallothionein  in  terms  of  size,  but  contains  Cu  without  any  Zn 
or  Cd.   This  metalloprotein  will  be  isolated  and  characterized  in 
future  studies,  its  sulfhydryl  content  determined  and  its  avidity 
for  various  metal  ions  identified. 

In  a  similar  manner  protein  complexes  with  zinc  and  copper 
in  tongue  of  rat  are  being  identified  and  characterized. 


4o   Circadian  variation  of  copper  and  zinc  in  man.   Copper 
and  zinc  metabolism  was  studied  in  ten  normal  volunteers  on  a 
constant  regimen  consisting  of  an  alternate  4  hourly  intake  of  a 
liquid  diet  or  distilled  water  and  a  regulated  amount  of  physical 
activity.   Urine  was  collected  in  4  hour  periods  and  blood  samples 
drawn  in  the  middle  of  these  periods,   A  circadian  pattern  of 
variation  for  serum  copper  and  zinc  concentration  was  demonstrated; 
serum  copper  was  above  the  mean  at  10:00  am  and  2:00  pm,  at  the 
mean  at  6:00  pm  and  10:00  pm,  and  below  the  mean  at  2:00  am  and 
6:00  am.   This  pattern  persisted  in  2  subjects  who  received  2.5  mg 
prednisolone  every  6  hours  for  3  days.   A  circadian  pattern  for 
the  urinary  excretion  of  copper  but  not  for  zinc  was  demonstrated. 
Serum  ceruloplasmin  tended  to  follow  serum  copper  concentrations 
and  suggested  that  the  regulation  of  metal  binding  proteins  may 
be  important  in  the  circadian  pattern  of  these  metals  observed  in 
serum.   To  our  knowledge  this  is  the  first  demonstration  of  a 
circadian  pattern  of  variation  in  ceruloplasmin. 


av^ 


r 


NHLI-lll(c) 

Data  were  collected  over  a  period  of  5  days  in  each  subject 
studied.   We  are  now  in  the  process  of  analyzing  these  data  over 
this  time  period  to  determine  whether  or  not  a  longer  cycling 
process  occurs  and  whether  phase  relationships  between  changes  in 
blood  and  urine  can  be  determined. 

5.   Pituitary-qonadal  regulation  of  copper  and  zinc  metabolism 
in  the  female  rat.   This  study  was  undertaken  to  specify  the  changes 
in  copper  and  zinc  concentrations  in  serum  of  female  rats  during 
various  physiological  states:   (1)  during  the  estrus  cycle, 

(2)  during  pregnancy,  (3)  during  pseudopregnancy,  (4)  following 
castration,  (5)  following  castration  and  hypophysectomy  and  during 
administration  of  the  pituitary  gonadotropins  LH  and  FSH,  and 

(6)  following  castration  and  during  administration  of  estrogen, 
progesterone  or  estrogen  and  progesterone  together.   Results  of 
these  studies  indicate  that  there  is  cyclic  pattern  of  change  of 
both  copper  and  zinc  during  the  estrus  cycle  with  serum  copper  and 
zinc  concentrations  both  reaching  a  peak  at  the  time  of  estrus 
coincident  with  ovulatbn.   During  pregnancy  serum  copper  concen- 
trations increase  until  day  15,  then  decrease  thereafter,  again 
following  the  pattern  of  estrogen  secretion.   Serum  zinc  does  not 
change  during  this  early  period,  but  falls  dramatically  during  the 
last  days  of  pregnancy.   Post-partum  serum  copper  concentrations 
fall  precepitously  to  values  below  those  of  castrated  animals, 
but  return  to  estrus  levels  within  5  days  of  parturition.   Zinc 
4!< "       concentrations  in  sertrni  increase  immediately  post-partum  to  estrus 

levels  and  remain  there  throughout  the  post-partum  period.   Following 
the  production  of  pseudopregnancy  by  means  of  vaginal  manipulation 
of  the  rat  at  an  appropriate  time  during  estrus  there  is  a  signi- 
ficant increase  in  serum  concentration  of  copper  and  of  zinc,  with 
the  zinc  increase  lagging  the  copper  increase  by  6  days.   This 
condition  demonstrates  that  changes  in  seriom  copper  and  zinc  con- 
centrations in  the  rat  are  significantly  influenced  by  endogenous 
progesterone  secretion  as  well  as  by  estrogen  secretion.   Following 
castration  there  is  a  significant  decrease  in  serum  copper  con- 
centration and  an  increase  in  serum  zinc  concentration.   This 
could  be  due  to  the  lack  of  estrogen  and  progesterone  or  to  the 
effects  of  LH  and  FSH.   To  evaluate  this  concent  overiectomized, 
hypophysectomized  rats  were  treated  with  LH  and  FSH.   There  was 
no  effect  of  either  hormone  alone  on  serum  copper  or  zinc  con- 
centrations in  these  animals.   Administration  of  estradiol  17  beta 
to  castrated  female  rats  resulted  in  a  dose-response  increase  in 
serum  copper  without  any  change  in  serum  zinc.   Administration  of 
progesterone  resulted  in  similar  increases  in  serum  copper  and 
decreases  in  serum  zinc  although  the  dose  response  relationships 

6  4VA 


:;J 


NHLI-lll(c) 
noted  with  estradiol  administration  did  not  occur.   Administration 
of  both  progesterone  and  estradiol  17  beta  produced  an  enhancement 
of  both  effects  with  a  significant  increase  in  serum  copper  con- 
centration and  a  significant  decrease  in  serum  zinc  concentration. 


2. 


These  results  demonstrate  that  copper  and  zinc  metabolism  in 
the  rat  are  in  part  controlled  by  estrogen  and  progesterone  and 
that  the  concentrations  of  these  metals  in  the  serum  change  in 
association  with  changes  in  either  or  both  of  these  hormones.   The 
role  of  these  metals  in  these  physiological  processes  are  at 
present  being  investigated  as  follows:   (1)  the  role  of  copper  is 
suggested  as  one  of  the  controlling  factors  initiating  ovulation 
in  the  rat,  (2)  estrogen  and  progesterone  can  produce  changes  in 
copper  and  zinc  metabolism  in  male  rats. 

6o-  Copper  and  zinc  metabolism  in  female  sheep  and  cows. 
In  a  study  similar  to  that  noted  above  in  the  rat,  but  much  less 
extensive  changes  in  serum  concentrations  of  copper  and  zinc  in 
sheep  and  cow  were  evaluated  during  pregnancy,  post-partum  and 
following  administration  of  estrogeno   Distinctly  different  from 
results  in  rat  or  man  no  changes  in  either  copper  or  zinc  metabo- 
lism were  observed  in  sheep  or  cow  during  pregnancy,  post-partum 
or  following  administration  of  estrogen.   These  studies  will  be 
extended  by  administration  of  progesterone  to  these  same  animals. 

7 .   Effects  of  metals  on  norepinephrine  (NE)  and  choline  (Ch) 
uptake  of  brain  synaptosomes  and  on  adenosinetriphosphatase 
activity.   Because  of  the  interrelationships  between  steroid 
synthesis  and  metal  cofactors  noted  above  and  because  of  inter- 
relationships between  this  system  and  neural  conduction  which  we 
have  suggested  we  investigated  the  interrelationships  between 
metals  and  uptake  of  NE  and  Ch  on  brain  synaptosomes  and  ATPase 
activity.   Using  established  techniques  the  following  effects  on 
activity  have  been  demonstrated  using  various  physiological  con- 
centrations of  metals: 


r 


3t¥3 


Ch 

NE 

ATPase 

20 

40 

10 

70 

80 

40 

80 

20 

50 

0 

0 

0 

0 

0 

0 

60 

120 

30 

110 

300 

150 

NHLI-1 11(c) 

Uptake  (%)*      Activity  {%)* 

METAL 
Zn 
Ne 
Co 
Cu 
Hg 
'|ii       Mn 

',!       *  Results  indicate  %   uptake  and  indicate  activity  remaining  at 
'         highest  concentration  of  metal  ion  used. 

These  results  demonstrate  that  most  metals  inhibit  both  Ch 
,„       and  NE  uptake  and  ATPase  activity  but  to  varying  degrees.   The 
'''!„,       manner  by  which  this  inhibition  occurs  is  being  investigated. 
i.''"|l       Two  metals  Cu  and  Hg  are  extremely  toxic  to  Ch  and  NE  uptake 
lljiii.       and  ATPase  activity  whereas  two  other  metals  Mn  and  Sn  appear  to 
enhance  the  uptake  of  NE  while  Sn  appears  to  enhance  ATPase  acti- 
vity.  The  results  of  Sn  in  this  system  are  specific  for  Na-K 
dependent  ATPase  activity  having  no  effect  on  Mg-activated  ATPase 
activity.   These  results  are  being  confirmed  in  further  experiments, 

8.   Effects  of  hormones  of  the  adrenal  cortex  on  metal  con- 
centration in  various  tissues  of  the  cat.   We  have  previously 
demonstrated  the  interrelationships  between  steroid  hormone  syn- 
thesis and  copper.   In  these  studies  we  have  looked  at  the  converse 
problem;  i.e^i    in  the  presenceof  too  little  or  too  much  secretion 
of  the  adrenal  cortex,  what  are  the  effects  on  metal  concentration 
in  several  tissues  of  the  cat?   Cats  were  adrenalectomized  and 
maintained  for  3-5  weeks  at  which  time  they  were  exsanguinated  and 
their  tissues  removed  using  special  techniqueSo   Control  cats 
were  sacrificed  in  parallel  with  the  adrenalectomized  cats.   Brain, 
spinal  cord,  sciatic  nerve,  liver  and  sciatic  muscle  mass  of  both 
groups  of  cats  were  analyzed  for  the  concentration  of  approximately 
25  metals  by  spark-source  mass  spectrometry.   Results  of  these 
studies  indicate  that  concentrations  of  most  trace  metals  increase 


d4¥ 


^ 


\'' 


NHLI-lll(c) 

in  the  liver  following  adrenalectomy  whereas  concentrations  of 
these  metals  in  brain  are  either  unchanged  or  decreased.   Changes 
in  spinal  cord,  sciatic  nerve  and  muscle  mass  generally  follow 
the  pattern  observed  in  liver.   Administration  of  excessive  doses 
of  both  carbohydrate-active  and  Na-K  active  adrenal  corticosteroids 
to  adrenalectomized  cats  demonstrated  that  metal  concentration  may 
be  decreased  significantly  in  liver,  spinal  cord,  sciatic  nerve 
and  muscle  mass.   These  studies  indicate  that  concentrations  of 
metals  in  several  tissues  are  dependent  upon  the  endogenous 
secretion  of  hormones  of  the  adrenal  cortex. 

9.   Uptake  of  Zn   by  various  tissues  of  the  rat.   Ten  u-c 
of  Zn"^  v;ere  injected  into  rats  and  its  distribution  evaluated 
in  terms  of  %   dose  found  in  physiological  fluids  and  tissues  from 
minutes  to  7  days  after  injection.   In  general,  blood  levels 
rapidly  fell  within  the  first  24  hours  after  injection  and  uptake 
was  highest  in  bone,  liver  and  tongue,  respectively.   Separating 
the  tongue  into  an  anterior  portion,  containing  fungiform  papillae 
and  taste  buds,  and  posterior  portion,  containing  foliate  and 
vallate  papillae  and  taste  buds,  indicated  that  the  posterior 
portion  contained  more  Zn""'  than  the  anterior  portion.   Many  more 
taste  buds  are  present  in  the  posterior  portion  than  in  the 
anterior  portion. 

Significance;   During  the  p  ast  year  we  have  systematically  inves- 
tigated several  aspects  of  the  physiology  and  biochemistry  of 
metal  metabolism  in  mammalian  systems. 

1.  Metals  and  steroidogenesis:   We  have  extended  our  work  of 
last  year  and  designated  that  the  A^-3  hydroxysteroid-dehydrogenase- 
isomerase  step  in  steroidogenesis  in  rat  and  cat  requires  copper. 
We  have  also  shown  that  the  concentrations  of  metals  in  several 
tissues  are  dependent  upon  the  presence  of  the  endogenous  secretion 
of  the  adrenal  cortex  and  that  these  effects  may  differ  among 
various  tissues.   The  liver  appears  to  be  an  important  source  of 
all  metals  and  that  these  metals  are  mobilized  by  increasing  the 
amount  of  circulating  adrenocorticosteroids .   Metal  concentration 
in  brain  is  not  controlled  in  the  same  manner.   These  studies 

have  great  importance  to  the  manner  by  which  tissues  take  up, 
store,  and  metabolize  metals. 

2,  Metal-protein  interactions:   We  have  demonstrated  that 
metals  in  urine  are  not  "free"  in  the  sense  of  appearing  in  urine 
as  ions  but  are  liganded  with  relatively  small  molecular  weight 
peptides  or  proteins.   Two  distinct  metal  containing  proteins 


J^i^ 


NHLI-lll(c) 

have  been  identified  in  dog  kidney.   One  is  of  MW  34,000,  contains 
equal  amounts  of  copper  and  zinc  and  is  probably  cytocuprein. 
The  other  is  of  MW  10,000,  corresponds  to  metal lothionein  in  size 
but  contains  only  copper,  without  zinc  or  cadmium.   These  latter 
studies  indicate  that  kidney  metallothionein  may  differ  signifi- 
cantly in  different  animal  species,  may  contain  different  m^etals 
and  may  not  contain  cadmium. 

3.  Metal-neurotransmitter  interactions:   We  have  initiated 
studies  in  this  area  and  v/e  have  demonstrated  that  m.etals,  in 
general,  are  inhibitory  for  the  uptake  of  NE  and  Ch  by  brain 
synaptosomes  and  for  the  activity  of  both  Na-K  activated  and  Mg 
activated  ATPase  activity.   The  specific  nature  of  this  inhibition 
has  been  systematically  investigated  and  related  to  the  nature 

of  the  metal-neurotransmitter  complex.   However,  two  metals  Mn 
and  Sn  appear  to  enhance  norepinephrine  uptake.   The  mechanisms 
by  which  this  enhancement  occurs  has  been  systematically  investi- 
gated.  Sn  acts  to  enhance  Na-K  ATPase  activity  specifically 
without  effect  on  Mg-ATPase  activity.   It  is  through  this  effect 
that  it  enhances  the  uptake  of  NE  by  brain  synaptosomes.   These 
studies  suggest  the  first  physiological  role  for  Sn  in  mammalian 
systems. 

4.  Pituitary-gonadal  regulation  of  copper  and  zinc  metabolism 
in  the  female  rat.   These  studies  have  demonstrated  that  serum 
concentrations  of  copper  and  zinc  are  influenced  primarily  by 

both  estrogen  and  progesterone,  not  by  LH  or  FSH.   The  relation- 
ship between  copper  and  the  initiation  of  ovulation  has  been 
investigated,,   The  role  of  copper  in  the  control  of  fertility  in 
man  is  of  particular  importance  since  intrauterine  devices  coated 
with  copper  are  more  effective  for  several  reasons  than  the  same 
devices  coated  with  Teflon.   The  mechanism  for  this  phenomenon 
is  not  yet  apparent. 

5.  Circadian  variation  of  copper  and  zinc  in  man.   We  have 
established  that  seriom  concentrations  of  copper  and  zinc  exhibit 
circadian  changes  in  man  and  that  these  changes  can  be  observed 
for  copper  in  urine  but  not  for  zinc.   A  circadian  variation  for 
ceruloplasmin  in  serum  has  also  been  observed  for  the  first  time 
and  this  may  relate  to  the  circadian  changes  observed  for  the 
first  time  and  this  may  relate  to  the  circadian  changes  observed 
in  serum  copper  concentrations.   These  changes  are  not  abolished 
following  blocking  of  the  endogenous  secretion  of  the  adrenal 
cortex  and  of  pituitary  ACTH  for  a  period  of  3  days. 


10  '^^ 


NHLI-1 11(c) 
6.   Role  of  metals  in  taste.   Injection  of  Zn^^  in  rats 
demonstrated  that  the  tongue  was  one  of  the  most  active  tissues 
in  acciomulating  Zn  after  bone  and  liver.   Correlating  these  studies 
with  those  previously  noted  in  which  zinc  was  found  in  the  epi- 
thelial layer  of  papillae  by  laser  microprobe  spectroscopsy 
suggest  that  taste  bud  bearing  papillae  and  perhaps  even  taste 
buds  themselves  may  take  up  zinc  avidly. 

Proposed  Course  of  Project;   Each  of  the  studies  outlined  will  be 
continued  in  the  specific  manner  indicated.   In  general,  we  will 
investigate  the  physiology  and  biochemistry  of  metal-protein 
complexes  in  several  physiological  fluids,  the  nature  of  the  role 
of  Sn  and  Mn  in  enhancement  of  NE  uptake  by  synaptosomes  and  the 
role  of  Sn  in  ATPase  activity.   We  will  continue  to  investigate 
the  role  which  metals  play  in  neural  activity.   These  studies 
will  fit  closely  with  the  role  which  metals  play  in  the  taste 
process.   In  addition,  the  role  of  taste  buds  and  other  tissues 
in  the  uptake  of  Zn°^  and  the  role  of  various  factors  which  in- 
fluence the  uptake,  metabolism  and  storage  of  this  metal  will  be 
evaluated  in  man  and  other  animals . 

Honors  and  Awards:   None 

Publications: 

Meret,  S.  and  Henkin,  R.I.:  Simultaneous  estimation  of 
copper  and  zinc  by  atomic  absorption  spectrophotometry. 
Clin.  Chem.  17:369-373,  1971. 

Lifshitz,  M.D.  and  Henkin,  R.I.:   Circadian  variation  in 
copper  and  zinc  in  man.   J.  Applied  Physiology.   In  press, 
1971. 

Henkin,  R.I.:   Newer  aspects  of  copper  and  zinc  metabolism. 
Book  chapter.  New  Trace  Metals.  Marcel  Dekker,    New  York. 
In  press,  1971. 

Henkin,  R.I.,  Marshall,  J.R.  and  Meret,  S.:   Maternal-fetal 
metabolism  of  copper  and  zinc  at  term.   Amer.  J.  of  Obstetrics 
and  Gynecology.  109:  131-134,  1971. 


^' 


11 


J^? 


Serial  No.  TxTHT  .T-ll  2fc> 

1 .  Experimental  Therapeutics  Branch 

2 .  Section  on  Neuroendocrinology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 


Project  Title:   Taste  and  Olfaction 

Previous  Serial  No.:   NHLI  -  167  (c) 

Principal  Investigator:   R.  I.  Henkin,  M,D.,  Ph.  D. 

Other  Investigators:   Paul  J,  Schechter,  M.D.,  Ph.D., 

Frank  Catalanotto,  D.D.S. 
Eugene  L.  Giroux,  Ph.  D. 
Naoki  Sato,  M.D. 


Cooperating  Units: 


University  of  Buffalo,  N.Y.,  Cornell 
University,  N.Y.,  University  of  Iowa  School 
of  Medicine,  Iowa  City,  Iowa,  Presbyterian 
Hospital,  N.Y.,  N.Y.,  City  of  Hope,  Duarte, 
California,  Campbell  Institute  for  Food 
Research,  Camden,  N.J.,  Jarrell  Ash,  Division 
of  Fischer  Scientific,  Boston,  Mass.,  Gedlco 
Boston,  Mass.,  NIAID:LCI, NEI,  NCI:SURG, 
NIDRrOMS  and  NIAMD:  A  &  R. 


Project  Description: 

Objectives;  To  investigate,  in  a  systematic  manner,  the  ana- 
tomical, physiological,  pharmacological  and  pathological  corre- 
lates of  taste  and  olfaction. 

Major  Findings:   Taste 

Anatomy .  In  order  to  understand  the  physiology  and  biochemistry 
of  taste,  the  taste  receptors  must  be  identified  and  their  function 
specified.   The  correlation  of  form  and  function  in  taste  buds  of 
any  vertebrate  or  invertebrate  system  at  present  is  rudimentary. 
In  order  to  identify  and  specify  the  taste  receptors  we  have 
undertaken  the  following  projects: 


A*^ 


I 


Serial  No.  NHLI-112(c) 

1.   Do  taste  buds  in  different  papillae  in  man  and  animals 
differ  in  form  or  function?   In  man,  in  order  to  answer  this 
basic  question  taste  buds  from  fungiform,  circumvallate  and  pa- 
latal papillae  in  normal  man  were  studied  systematically  by 
light  and  electron  microscopy.   Because  of  the  size  and  location 
of  the  buds  in  papillae  their  orientation  is  difficult.   Know- 
ledge of  taste  acuity  in  those  subjects  or  animals  in  whom  taste 
buds  are  studied  anatomically  is  essential  for  evaluation  of 
normality.   These  two  aspects  of  anatomical  studies  of  taste  buds 
have  been  carried  out. 

Systematic  evaluation  of  taste  buds  in  man  has  been  made  in 
patients  who  have  had  their  taste  acuity  tested  and  found  to  be 
normal.   These  patients  are  usually  to  undergo  a  surgical  pro- 
cedure in  the  National  Cancer  Institute.   With  their  informed 
consent,  at  the  time  of  surgery,  fungiform,  circumvallate  and 
palatal  papillae  are  excised,  immediately  placed  in  glutaralde- 
hyde  and  embedded  in  plastic  and  fixed  for  sectioning.   Thick 
sections  are  cut  (less  than  1|J.)  and  first  evaluated  by  light 
microscopy  to  insure  the  presence  of  buds  and  their  proper  orien- 
tation .   This  has  saved  weeks  of  time  previously  spent  hunting  for 
buds  through  the  use  of  traditional  thin  sectioning  techniques. 
After  the  bud  is  properly  oriented  thin  sections  are  made  and  the 
bud  evaluated  in  detail.   In  this  manner  buds  from  approximately 
five  circumvallate  papillae  have  been  studied  in  detail  and  they 
confirm  our  earlier  observations  of  the  anatomical  organization 
of  the  bud;  i.e.,  from  the  pore  inward  there  are  8  levels  of  or- 
ganization; (1)  a  pore  filled  with  granules  and  finger-like  proc- 
esses of  Type  I  cells,  (2)  the  cell  process  layer,  (3)  the  dense 
extracellular  layer,  (4)  the  layer  of  neurosecretory  granules, 
(5)  the  kinetosomal  layer,  (6)  the  nuclear  layer,  (7)  the  synaptic 
vesicle  layer  and  (8)  the  axonal  layer.   There  are  4  anatomically 
distinct  types  of  cells  identifiable  in  this  taste  bud  as  follows: 
Type  I  cells —  these  comprise  at  least  80%  of  the  cells  of  the  bud, 
have  processes  which  extend  out  of  the  bud  into  the  open  pore 
have  paired  kinetosomes,  have  neurosecretory  granules,  have  their 
synapses  such  that  they  appear  to  be  afferent  to  the  central  ner- 
vous system  and  appear  to  be  the  receptor  cells  of  the  taste  bud; 
Type  II  cells — these  comprise  about  10-15%  of  the  cells  of  the 
bud,  have  processes  which  extend  near  the  pore  but  not  necessarily 
into  the  pore,  have  centrioles  adjacent  to  the  cell  nuclei  but 
not  kinetosomes,  have  vacuolated  cytoplasm,  do  not  have  neuro- 
secretory granules,  have  their  synapses  such  that  they  appear  to 
efferent  from  the  CNS,  have  large  fiber  bundles  which  run  from 


9fff 


Serial  No.  NHLI-1 12(c) 

below  the  nuclear  area  to  the  infra-pore  area  and  appear  to  be 
the  effector  cell  which  participate  in  the  closure  of  the  bud; 
Type  III  cells — these  comprise  1-5%  of  the  cells  of  the  bud, 
have  processes  which  extend  into  the  pore  but  other  characteristics 
including  their  possible  function  are  not  well  established;  Type 
IV — these  cells  have  been  seen  only  occasionally  in  the  bud,  are 
located  near  the  base  of  the  bud  and  their  anatomy  and  function 
are  not  well  evaluated.   In  a  more  preliminary  m.anner,  two  buds 
from  fungiform  papillae  have  been  evaluated.   Only  two  cell  types. 
Type  I  and  Type  II  cells^have  been  observed  in  them.   They  differ 
in  anatomy  from  the  buds  of  the  circumvallate  papillae  due  to 
their  long  necks  and  their  opening  directly  into  the  oral  cavity. 
The  buds  are  located  on  the  lingual  surface  of  the  papillae  as 
>'        opposed  to  the  position  of  the  buds  of  the  circumvallate  papillae 

which  are  in  crypts  beneath  the  lingual  surface.   Palatal  papillae 
i|        possess  buds  which  are  more  like  buds  from  fungiform  than  circum- 
'I        vallate  papillae  since  they  open  into  the  oral  cavity  being 
'        positioned  on  the  palatal  surface  of  the  papilla. 

In  the  rat,  this  same  anatomical  framework  has  been  observed 
in  the  vallate  papillae  containing  buds  similar  to  the  circum- 
vallate papillae  in  man.   Similar  placement  of  buds  are  in  fungi- 
j^,i.        form  and  palatal  papillae  containing  equivalent  structures. 


.  I* 


It  is  of  interest  that  no  blood  vessels  or  lymphatic  channels 
were  observed  in  any  taste  bud.   Similarly,  no  mitotic  figures 
were  clearly  identified  which  raises  the  questions  of  how  the 
taste  bud  maintains  itself,  from  which  tissues  do  taste  cells 
form,  in  what  manner  do  taste  cells  degenerate  and  how  does  the 
bud  handle  degenerating  material.   These  questions  have  not  yet 
been  approached  systematically  although  estimates  suggest  that 
the  cells  of  the  rat  taste  bud  regenerate  over  a  period  of  3-11 
days  . 

2,   What  specialized  sensory  receptors,  if  any,  exist  in  the 
papillae  themselves  and,  if  they  exist,  can  they  be  related  to  the 
taste  process  in  any  manner?   In  order  to  ansv/er  this  question 
a  systematic  evaluation  of  the  anatomy  of  the  fungiform,  circum- 
vallate and  palatal  papillae  in  man  and  also  the  valate  papilla 
in  rat  has  been  undertaken  independent  of  the  study  of  the  taste 
buds  .   These  studies  produced  results  not  previously  obvious  due 
to  the  technique  by  which  they  were  carried  out.   Since  all  tissues 
were  fixed  in  glutaraldehyde,  little  artifactual  damage  to  the 
tissue  was  made  in  comparison  to  usual  techniques  of  fixing  in 

3  JSP 


Serial  No.  NHLI-112(c) 

formalin  and  embedding  in  parafin  as  earlier  anatomists  had  done. 
Sections  from  the  tissues  used  in  the  present  study  were  stained 
with  osmium.   In  these  tissues  myelinated  fibers  could  be  demon- 
strated extending  into  the  epithelial  layer  of  the  papillae  ad- 
jacent to  the  taste  bud  in  circumvallate  papillae  of  man  and  valate 
papillae  of  rat.   These  fibers,  not  previously  described,  may 
subserve  touch,  temperature  and  pain  in  the  papilla.   In  addition, 
Paccinian  corpuscles  (specialized  pressure  receptors)  were  demon- 
strated for  the  first  time.   Meissner's  corpuscles  and  other 
specialized  sensory  receptors  and  nerve  endings  were  also  observed . 
These  structures  indicate  that  the  papillae  of  the  tongue  contain 
the  same  sensory  receptors  as  does  the  epithelial  layer  of  the 
skin.   In  addition,  striated  muscle  has  been  observed  to  be 
present  at  the  base  of  each  papilla  and  fibers  from  major  muscle 
bundles  of  the  lingual  muscles  appear  to  branch  off  to  each 
papilla.   No  specific   connections  between  the  striated  muscle 
fibers  and  the  Type  II  cells  have  yet  been  clearly  observed . 

3.   What  differences,  if  any,  exist  between  the  taste  buds 
of  patients  or  animals  with  altered  taste  acuity  and  those  with 
normal  taste  acuity?   The  taste  receptor  is  the  only  receptor 
(other  than  touch)  that  can  be  anatomically  evaluated  serially 
in  patients  with  a  sensory  abnormality  without  limitation  to 
subsequent  sensory  function.   For  this  reason  taste  buds  from 
circumvallate  papillae  in  patients  with  idiopathic  hypogeusia, 
aglycogeusia  and  known  vitamin  A  deficiencies  have  been  evaluated 
and  compared  to  taste  buds  from  individuals  with  normal  taste 
acuity.   The  changes  observed  in  buds  from  patients  with  idio- 
pathic hypogeusia  previously  described  were  confirmed  to  be 
localized  mainly  in  the  pore  region  of  the  bud  with  disruption 
of  the  normal  organization,  as  noted  in  this  report  of  1970. 

Preliminary  results  from  the  bud  of  the  one  patient  with 
aglycogeusia  studied  demonstrated  marked  degeneration  of  the 
Type  II  cells  with  a  perinuclear  hyalinized  appearance.   No 
processes  of  Type  I  cells  could  be  observed  in  the  pore  region 
but  rather  club-shaped  clumps  of  cytoplasm. 

Taste  buds  from  circumvallate  papillae  from  prisoners  before, 
during  and  after  feeding  a  diet  deficient  in  vitamin  A  were  ex- 
cised and  studied  in  detail.   Taste  and  smell  acuity  was  also 
measured  before,  during  and  after  feeding  the  diet  at  the  time  of 
the  biopsies.   Tissue  was  handled  as  nored  previously.   When 
these  men  were  vitamin  A  deficient  they  exhibited  several  ab- 


3iS'f 


Serial  No.  NHLI-112(c) 

normalities  of  vitamin  A  metabolism:   skin  abnormalities,  abnormal 
dark  adaptation  curves  and  abnormal  electroretinograms .   Buds 
from  men  who  were  vitamin  A  deficient  showed  disorganization  of 
the  pore  area  with  absence  or  marked  decreases  in  the  finger- 
like processes  of  the  Type  I  and  II  cells,  marked  decreases  of 
the  dense  extranuciear  material  and  the  presence  of  large  "lipid 
inclusion"  bodies  which  have  not  been  previously  observed  in  any 
bud  .   Although  these  patients  exhibited  marked  hyperparakeratosis 
of  their  skin,  the  papillae  samples  were  not  particularly  kerati- 
nized nor  were  the  pores  of  the  taste  buds  covered  by  keratin. 
In  fact,  the  pore  areas  were  clearly  open  to  the  crypt  of  the 
papilla.   The  number  of  taste  buds  present  in  the  papillae,  how- 
ever, were  estimated  to  be  decreased  below  normal  by  more  than 
1''       a  factor  of  2  .   Kinetosomes  were  present  in  Type  I  cells  as  were 

neurosecretory  granules;  the  latter  were  of  normal  number  and 
,|        stratification.   Type  II  cells  were  normal  in  number  and  appearance 

without  any  hyalinized  perinuclear  area.   The  abnormalities  noted 
;        appear  to  be  different  from  any  we  have  yet  seen.   In  following 
the  anatomy  of  the  buds  after  vitamin  A  replacement  using  each 
patient  as  his  own  control,  the  abnormalities  noted  during  the 
. ' "'       depleted  state  disappear  and  the  buds  cannot  be  distinguished 
^  ,i.       from  those  of  other  normal  subjects.   Taste  acuity  was  also 
jj  "•■       measured  in  these  patients  after  repletion  with  vitamin  A  and 
"''.*  found  to  be  normal  as  were  other  function  of  vitamin  A  metabolism 

}1I< '        including  skin  appearance,  grossly  and  by  histological  examination 
and  visual  function. 

4  .   Is  it  possible  to  find  an  animal  whose  taste  receptors 
respond  to  only  one  taste  quality  and  yet  are  simple  enough  to 
study  in  detail  by  present  day  anatomical  techniques?  We  believe 
that  in  the  blowfly,  Phormia  regina,  we  have  found  a  system  v;hich 
responds  primarily  to  only  one  taste  quality  which  is  simple  enough 
to  be  studied  anatomically  and  biochemically  and  which  is  similar 
enough  anatomically  to  the  mammalian  system  that  information  gained 
through  study  of  its  taste  responses  will  enable  us  to  generalize 
to  higher  organisms.   The  gustatory  system  of  this  fly  consists 
of  five  cells,  only  two  of  which  are  involved  with  taste.   One 
cell  responds  electrophysiologically  and  behaviorally  only  to 
sugars  of  various  types,  the  other  in  identifiable  manner  to  all 
other  taste  stimuli.   In  this  sense  this  system  can  be  identified 
as  responsive  to  one  taste  quality  provided  the  appropriate  stim- 
uli are  applied  and  responses  measured.   The  gross  anatomy  of  this 
system  consists  of  a  hair  cell  with  a  pore  on  one  end  and  two 
large  fibers,  within  a  chitinous  sheath,  which  lead  to  the  five 

5  ^ra 


Serial  No.   NHLI-112(c) 

cells  noted  above.   Only  rudimentary  electron  microscope  pictures 
of  the  anatomy  of  this  system  had  been  previously  carried  out. 
We  have  carried  out  preliminary  work  in  this  insect  and  have 
shown  that  there  are,  in  the  one  cell  which  presumably  responds 
to  sugar,  paired  kinetosomes  which  can  be  traced  by  means  of 
their  rootlets  to  the  fibers  which  enter  the  chitinous  sheath. 
Each  fiber  is  made  of  two  structures,  one  set  larger,  the  other, 
smaller.   Each  set  or  unit  is  filled  with  microtubules.   These 
microtubules  run  the  length  of  the  sheath  and  end  at  the  pore 
surrounded  by  granules  which  initially  appear  to  resemble  those 
seen  at  the  pore  of  the  human  taste  bud .   This  system  appears 
to  offer  much  promise  for  future  anatomical  and  biochemical  study, 


Physiology .  1.   Saliva.   Salivary  volume  and  composition,  as 
well  as  taste  acuity,  has  been  studied  in  rats  in  whom  all  major 
salivary  glands  had  been  excised  and  who  were  treated  with  pilo- 
carpine.  Analysis  of  saliva  has  not  been  carried  out  in  detail. 
Taste  acuity,  as  noted  by  others,  is  markedly  impaired.   Salivary 
composition  has  also  been  studied  in  patients  with  cystic  fibrosis 
of  the  pancreas .   These  studies  confirm  our  earlier  observations 
that  the  zinc  concentration  in  the  saliva  of  these  patients  was 
excessively  high  in  comparison  with  normal  subjects  but  that 
copper  concentration  was  within  normal  limits.   This  finding  may 
aid  in  the  diagnosis  of  this  condition  due  to  the  ease  with  which 
saliva  can  be  obtained  and  metals  measured,  even  in  infants. 
Saliva  has  also  been  collected  in  patients  with  various  taste 
abnormalities  and  is  being  evaluated  with  respect  to  its  metal 
concentration . 


2 .   Metals .   Metal  metabolism  in  some  manner  appears  to  be 
involved  with  the  control  of  taste  acuity  in  man  and  animals . 
In  order  to  specify  some  aspects  of  this  interaction  it  is 
necessary  to  know  whether  or  not  metal  ions  are  present  in  the 
taste  bud  or  the  taste  bud  bearing  papilla  and  whether  or  not 
this  is  a  general  or  specific  phenomena.   This  problem  has  been 
approached  from  three  directions.   First,  whole  tongues  and 
valate  papillae  from  normal  rats  were  excised  and  either  immedi- 
ately frozen  or  fixed  in  glutaraldehyde  and  studied  for  their 
metal  concentration  by  laser  microprobe  mass  spectrometer  tech- 
niques.  Serial  anatomical  analysis  of  the  whole  tongue  revealed 
that  zinc  was  found  only  in  the  area  defined  by  the  valate  papilla 
coincident  with  the  finding  of  barium  and  strontium.   Cross 
sectional  analysis  of  the  valate  papilla  itself  revealed  zinc  to 
be  present  only  in  the  epithelial  layer  of  the  papilla,  that 


-a53 


Serial  No.   i>iHLI-112(c) 

layer  containing  taste  buds  and  other  sensory  receptors,  while 
none  was  found  in  the  muscularis  or  adventitial  layers.   Similar 
analysis  of  circumvallate  papillae  was  carried  out  in  man,  in 
patients  with  normal  taste  acuity  and  in  patients  with  idiopathic 
hypogeusia.   Zinc,  barium  and  strontium  was  present  in  papillae 
from  patients  with  normal  taste  acuity  while  zinc  could  not  be 
clearly  demonstrated  in  papillae  from  one  patient  with  idiopathic 
hypogeusia.   Technical  problems  with  this  technique  severely 
limit  interpretation  of  these  results  due  to  the  inadequate 
resolution  of  the  instrument  used  (about  25  m.icrons)  .   However, 
the  results  suggest  that  zinc  is  present  in  the  taste  bud  bearing 
papillae  in  amounts  adequate  to  measure  but  not  in  surrounding 
portions  of  the  tongue  and  that  zinc  could  not  be  measured  by 
''"        the  techniques  used  in  the  papillae  of  one  patient  with  idiopathic 
y,  hypogeusia.   Taste  thresholds  of  this  patient  returned  to  normal 

,  i|; '        after  treatment  with  zinc  ion. 

;;  Secondly,  rat  tongue  and  valate  papillae  were  studied  by 

electron  microprobe  microscopy  but  the  results  could  not  be  in- 
terpreted due  to  technical  difficulties  with  sample  preparation. 

.,< '•■  Thirdly,  Zn°^,  10  M-c,  were  injected  intravenously  into  rats 

J' II'         and  the  tracer  located  in  various  tissues  at  various  time  periods 
i!!j  <•         following  injection.  With  the  exception  of  bone  and  kidney,  no 
tf"'         other  tissue  contained  as  much  Zn^^  per  gm  wet  weight  of  tissue 
as  did  the  tongue.   Gross  fractionation  into  posterior  portion, 
containing  the  valate  papilla,  and  the  anterior  portion,  con- 
taining only  fungiform  papillae,  was  consistent  with  finding 
more  Zn°^  in  the  posterior  rather  than  in  the  anterior  portion 
of  the  tongue.   These  studies  suggest  that  zinc  may  play  a  role 
in  the  papilla  bearing  taste  buds. 

3  .   Thiol-containing  drugs  and  amino  acids  and  their  relation- 
ship to  taste  acuity.   We  have  previously  suggested  that  thiols 
are  important  as  inhibitors  in  the  taste  process  in  man.   To 
demonstrate  this  suggestion  experimentally  rats  were  fed  diets 
with  added  D-penicillamine,  cysteine  or  methionine,  and  their 
taste  acuity  was  measured  by  the  twenty-four  hour,  two  bottle, 
free  choice  technique.   These  studies  demonstrated  that  D-peni- 
cillamine produced  decreases  in  taste  acuity  of  great  magnitude 
and  significant  differences  in  preference  for  NaCl  between  normal 
and  D-penicillamine  fed  rats  could  be  observed  during  presentation 
of  solutions  of  NaCl  as  aversive  as  0.75  M.   Similar  results  were 
also  shown  for  cysteine,  a  diet  containing  2  gm  cysteine  per 

7  Asy- 


1 

00 

o 

o 

3- 

n> 

3_ 

in 

r+ 

-s 

< 

— »  • 

Serial  No.  NHLI-1 12(c) 

100  gm  mixed  feed  (Purina  chow  and  dextrose)  being  more  effective 
in  reducing  taste  acuity  than  a  diet  containing  1  gm  cysteine  per 
100  gm  mixed  feed  .   Methionine  also  reduced  taste  acuity  but  not 
to  the  degree  noted  with  cysteine  or  D-penicillamine ,   Studies  of 
copper  and  zinc  metabolism  in  the  rats  fed  cysteine  demonstrated 
that  no  depletion  of  copper  or  zinc  had  occurred  in  blood  or 
urine  during  the  study  which  suggests  that  the  effect  on  taste 
acuity  was  due  to  feeding  the  amino  acid.   The  dose-response 
relationship  suggests  that  the  effect  on  taste  can  be  increased 
by  feeding  more  of  this  amino  acid . 

4.   Taste  Modifiers  and  Enhancers. 

a.   Miracle-fruit  protein (MFP) .   The  glycoprotein  of 
the  miracle-fruit  berry  has  been  known  to  alter  the  taste  of  sour 
substances  to  sweet.   Its  amino  acid  and  sugar  composition  has 
been  studied  and  some  disagreement  about  its  composition  has  been      '     H  ^ 
indicated  although  its  molecular  weight  of  44,000  is  well  docu-        '' 
mented . 

We  have  investigated  the  biochemical  and  physiological 
characteristics  of  the  berry.   Initially  a  new  purification 

technique  was  developed  which  differs  significantly  from  that  of  i  [ 

others.   Berries,  pulp  and  skin,  are  stirred  several  hours  at 
0-4°C  in  a  suspension  of  1/5  part  insoluble  polyvinyl  pyrrolidone 
(PVP)  in  10  parts  0.1  M  NaC03  buffer,  pH  10.5.   The  green-brown 
homogenate  is  filtered  in  the  cold.   The  supernatant  contains  the 
active  principle  as  determined  physiologically  by  bioassay.   A 
typical  yield  is  40  mg  of  TCA-precipitable  nitrogen  per  100  g  wet 
weight  of  berry.   The  supernatant  material  from  A  is  made  0.1  M 
in  e  amino  caproic  acid  and  insoluble  PVP  is  added .   Over  an 
one-half  hour  period  glacial  HAc  is  added  dropwise  to  the  stirred 
suspension  at  0-4°  C  to  lower  the  pH  to  6.0-5.5.   A  solution  con- 
taining the  active  principle  is  recovered  therefrom  by  filtration . 
Typical  yield  is  20  mg  of  TCA-precipitable  nitrogen  per  100  gm 
wet  weight  of  berry.   The  active  principle  is  then  adsorbed  onto 
a  short  column  of  BioGel-CM,  equilibrated  with  0.1  M  NaP04  buffer, 
pH  6.0,  at  0-4°  C.   The  column  is  then  washed  with  0.1  M  buffer, 
pH  6.5,  then  the  active  principle  is  eluted  stepwise  with  0.1  M 
Na2P04 .   Typical  yield  is  2  mg  of  TCA-precipitable  nitrogen  per 
100  gm  wet  berry  weight.   The  active  principle,  called  MFP  (miracle 
fruit  protein)  is  adjusted  to  pH  6.0-6.5  and  chromatographed  on 
a  column  of  carboxymethyl  polyacrylamide  gel.   A  shallow  pH 
gradient  in  NaP04  buffer  elutes  the  MFP  over  the  pH  range  7 .0- 
7.3.   Typical  yield  is  2  mg  TCA-precipitable  nitrogen  per  100  gm 

8  5rr 


Serial  No.  NHLI-112(c) 


wet  weight  of  berry . 


f;'teps  A,B  and  C  removed  condensed  tannins  which  otherwise 
interfere  with  the  stability  of  the  MFP .   Commercial  preparations 
are  unstable  with  respect  to  this  factor  and  do  not  store  well. 
A  papain-like  protease  was  also  removed  by  these  steps  and  this 
also  reduced  the  yield  of  MFP  reported  by  other  investigators  and 
contributed  to  the  instability  of  the  product.   By  this  process, 
MFP  recovery  is  at  least  two-fold  greater  than  yields  reported 
by  other  investigators  and  the  scheme  is  significantly  simpler. 
Elution  diagrams  of  the  step  D  chromatography  reveal  MFP  peaks 
which  vary  from  preparation  to  preparation.   MFP  is  easily 
aggregated  so  that  these  fractions  may  represent  various  aggregates 
or  compos it ionally  different  proteins.   These  potential  differences 
are  currently  under  investigation. 

Gel  filtration  data  have  confirmed  the  apparent  molecular 
weight  of  MFP  as  44,000,  as  noted  by  other  investigators.   Gel 
filtration  m  a  dissociating  solvent,  however,  indicates  that 
this  value  is  a  monomeric  molecular  weight.   Disc  electrophoresis 
studies  are  in  progress  as  are  studies  on  amino  acid  and  carbo- 
hydrate composition.   Limited  study  of  primary  sequence  is  con- 
■  II        templated,  as  are  experiments  aimed  at  removing  carbohydrate 
■y  components  by  enzymatic  and  chemical  means.   Effects  of  MFP  on 

"'        normal  subjects  have  been  carried  out  and  indicate  that  the  effects 
of  our  preparation  are  approximately  5  to  10  times,  mg  for  mg, 
those  of  other  investigators  for  altering  the  taste  of  sour  to 
sweet.   Utilization  of  MFP  in  patients  with  aglycogeusia  demon- 
strate that  there  is  no  effect  suggesting  that  an  intact  system 
for  the  sweet  taste  quality  is  required. 

b.   Protease  activity.   Anatomical  and  physiological  studies 
carried  out  in  the  past  year  suggest  that  the  taste  receptors  of 
the  bud  are  exposed  to  the  oral  environment.   We  have  also  sugges- 
ted that  the  membrane  of  the  Type  I  cells  are  these  taste  recep- 
tors and  that  these  are  the  receptors  exposed  to  the  oral  en- 
vironment .   If  these  hypotheses  are  correct  then  placement  of 
proteases  into  the  oral  environment  should  alter  taste  acuity  in 
the  direction  predicted;  i.e.,  disruption  of  secondary  or  tertiary 
structure  of  the  protein  of  the  receptor  membranes  with  resulting 
interference  with  the  pre-neural  events  of  taste.   Similarly  each 
taste  quality  should  be  affected  and  the  detection/recognition 
ratio  should  not  be  altered,  each  threshold  increasing  proportion- 
ately.  These  hypotheses  were  confirmed  in  experiments  carried  out 

9  JSg 


Serial  No.  NHLI-1 12(c) 


in  subjects  vjith  normal  taste  acuity  o   Several  proteases  were 
introduced  into  the  oral  cavity  (pronase,  trypsin,  papain)  and 
each  produced  an  iinmediate  reduction  in  taste  acuity  coincident 
with  slight  burning  sensations  in  the  areas  of  the  mouth  where 
taste  buds  are  located.   Pronase, a  relatively  non-specific 
protease,  was  more  effective  in  lowering  taste  acuity  than  was 
either  trypsin  or  papain.   The  pronase  effect  lasted  as  long 
as  18  hours  for  the  taste  of  bitter.   Administration  of  proteases 
resulted  in  the  sudden  onset  of  hypogeusia  for  all  taste  qualities 
Return  of  taste  acuity  to  normal  was  slow  and  followed  a  con- 
sistent pattern.   Initially,  sour  thresholds  returned  to  normal, 
followed  by  salt  and  sweet  thresholds,  and  lastly,  thresholds 
for  bitter.   Introduction  of  lipases  or  amylases  into  the  oral 
cavity  had  no  effect  on  taste  acuity.   Similarly  introduction  of 
chymotrypsin  or  pepsin  into  the  oral  cavity  did  not  alter  taste 
acuity.   Initial  histological  examination  of  valate  papillae  of 
rats  treated  with  pronase  suggest  no  anatomical  changes  in  the 
bud  were  produced  by  introduction  of  this  enzyme.   These  studies 
indicate  that  a  protein  containing  receptor,  subserving  four 
taste  qualities,  is  exposed  to  the  oral  environment  and  subject 
to  the  influence  of  proteases , 


>rr 


c.   Taste  modifiers.   Monosodium  glutamate,  various  nucleo- 
tides and  peptides  appear  to  modify  taste  acuity  in  such  a  way 
that  placement  of  them  in  food  makes  the  food  more  "flavorful". 
Studies  of  the  effects  of  these  substances  on  taste  acuity  in 
man  are  non-existant .   Drugs  and  amino  acids  which  affect  metal 
metabolism  also  affect  taste  acuity  when  given  systemically . 
To  evaluate  these  various  substances  we  have  studied  the  effects 
of  various  nucleotides,  peptides,  membrane  and  ATPase  modifiers 
and  metal  chelators  on  taste  acuity  in  normal  subjects  in  a 
manner  similar  to  that  carried  out  with  proteases.   These  studies 
are  currently  in  progress.   Initial  results  indicate  that  metal 
chelators  alter  taste  acuity  in  a  manner  similar  to  that  noted 
for  proteases.   Initial  results  also  indicate  that  nucleotides 
which  influence  taste  acuity  appear  to  do  so  by  inhibiting  only 
one  taste  quality,  that  for  bitter,  leaving  other  taste  qualities 
intact .   This  latter  result  could  explain  the  extremely  common 
use  of  these  substances  in  Japan  where  diets  consist  prominently 
of  soy  products.   These  products  which  are  intensely  bitter,  can 
be  made  to  taste  quite  palatable  through  the  utilization  of 
nucleotides  and  may  represent  a  specific  alteration  in  one  taste 
quality  similar  to  that  observed  with  the  utilization  of  miracle 


10 


^5-7 


Serial  No.  NHLI-1 12(c) 

fruit  berries  by  African  natives  to  alter  the  sour  quality  of 
their  wheat  products . 

5.   Role  of  vitamin  A.   Nine  prisoners,  housed  on  the  metab- 
olic unit  of  the  Department  of  Medicine,  University  of  Iowa 
School  of  Medicine,  have  been  taking  a  diet  deficient  in  vitamin 
A  for  1  1/2  years.   Serial  studies  of  their  vitamin  A  metabolism 
have  been  made  including  serum  levels  of  the  vitamin  and  its 
carrying  protein  (retinol  binding  protein) ,  clinical  and  histolog- 
ical changes  of  skin,  and  changes  in  visual  function.   Prior  to 
the  measurement  of  any  changes  in  vitamin  A  metabolism  taste 
and  smell  acuity  was  measured  and  biopsies  of  circumvallate 
papillae  taken  .   Patients  were  then  depleted  of  vitamin  A  and 
thresholds  again  measured  and  biopsies  taken.   Patients  were  sub- 
sequently repleted  with  vitamin  A  and  thresholds  were  measured 
again  and  biopsies  taken.   These  studies  indicate  that  when 
depleted  of  vitamin  A  thresholds  for  each  of  four  taste  qualities 
were  significantly  elevated  and  they  returned  to  normal  following 
vitamin  A  repletion.   Changes  in  the  taste  buds  of  these  patients 
have  been  detailed  above.   These  studies  demonstrate  that  vitamin 
A  is  required  for  normal  taste  acuity  although  the  mechanism  for 
this  is  not  knowii , 

Pathology .  1.   Idiopathic  hypogeusia  with  dysgeusia,  hyposmia 
and  dysosmia .   This  new  disease  was  described  in  detail.   Although 
previously  mentioned  in  this  report  of  1970  the  number  of  patients 
who  suffer  with  this  abnormality  was  not  fully  appreciated .   We 
have  received  requests  for  assistance  for  treatment  from  over 
3,000  patients  who  appear  to  suffer  with  this  illness.   Etiolog- 
ically,  the  disease  generally  occurs  following  an  attack  of  in- 
fluenza but  may  occur  following  surgical  procedures  or  without 
known  cause.   Pathologically  there  is  a  characteristic  lesion 
of  the  taste  bud  which  accompanies  those  cases  studied.   Treat- 
ment of  52  patients  in  a  single  blind  study  with  zinc  sulfate, 
in  doses  of  25,  50  or  100  mg  orally,  daily,  as  zinc  ion,  has  in- 
dicated that  thresholds  for  four  taste  qualities  return  to  normal 
in  25%-67%  of  the  patients  treated.   This  disease,  although  not 
usually  life  threatening  can  be  severely  discomforting-   The 
dysgeusic  and  dysosmic  components  of  the  disease  allow  the  suff- 
erer little  or  no  pleasure  from  the  intake  of  food.   We  are  at 
present  studying  the  efficacy  of  treatment  of  this  disease  with 
zinc  sulfate  in  a  double-blind  design  which  will  entail  the 
eventual  treatment  of  108  patients  in  a  modified  crossover,  block 
design  . 

11  5Jff 


UfttJb-     .  '■■U.LIL^.J!^. 


Serial  No.   NHLI-112(c) 


\^. 


2.      Sjogren's  Syndrome.   Described  in  this  report  in  1970 
this  study  has  been  continued  and  expanded  such  that  30  patients 
with  this  abnormality  have  been  carefully  evaluated  and  their 
loss  of  taste  acuity  documented.   Median  detection  and  recognition 
thresholds  for  each  taste  quality  except  for  sweet,  which  is  at 
the  upper  limit  of  normal,  were  elevated  above  normal  in  these 
patients.   Treatment  with  Cytoxan  or  other  agents  designed  to 
correct  their  underlying  abnormality  has  not  been  successful  in 
returning  their  taste  acuity  to  normal  except  in  patients  in  whom 
salivary  flow  has  returned  significantly.   Treatment  of  a  limited 
number  of  patients  with  zinc  ion  was  also  unsuccessful  in  either 
returning  salivary  flow  or  in  returning  taste  acuity  to  or  toward 
normal  in  any  patient,  although  equivalent  treatment  in  patients 
with  idiopathic  hypogeusia  was  associated  with  significant  bene- 
ficial effect. 


^1 


3.  Wegener's  Granulomatosis  and  Midline  Granuloma.   Described 
in  this  report  in  1970  this  study  has  been  continued  and  expanded 
such  that  15  patients  with  these  abnormalities  have  been  carefully 
evaluated  and  their  loss  of  taste  acuity  documented.   Treatment 

of  patients  with  Wegener's  granulomatosis  with  Cytoxan  and  carbo- 
hydrate-active steroids  which  produced  a  remission  of  their  disease 
also  produced  a  spontaneous  return  of  taste  acuity  to  normal. 
Treatment  of  a  small  number  of  patients  with  zinc  ion,  independent 
of  treatment  with  Cytoxan  or  steroids,  also  produced  a  return  of 
taste  acuity  to  normal  in  each  patient  so  treated.   Treatment  of 
patients  with  midline  granuloma  with  X-irradiation  which  produced 
a  remission  of  their  disease  also  produced  some  return  of  taste 
acuity  toward  normal.   Virulent  and  aggressive  infections  in  the 
nasal  area  were  not  associated  with  loss  of  taste.   These  findings 
may  be  useful  in  the  oftimes  difficult  diagnosis  of  these  rare 
diseases .   Similarly,  the  loss  of  taste  appears  to  be  highly 
correlated  with  a  recurrence  of  these  diseases  .   The  mechanism 
by  which  these  changes  occur  is  not  known. 

4.  Type  III  familial  dysautonomia .  Two  patients  with  diabetes 
mellitus,  optic  atrophy,  neurogenic  bladder,  and  neurosensory 
hearing  loss  with  hyposmia,  hypogeusia,  hyperalanineuria,  abnormal 
heat  intolerance  and  other  autonomic  dysfunctions  were  studied  in 
two  sibs,  aged  16  and  18. 

The  diabetes  was  insulin  dependent,  but  resistant  to  ketosis. 
Plasma  growth  hormone  and  Cortisol  were  normal,  but  neither  re- 
sponded to  intravenous  piromen .   Arginine  produced  markedly 


12 


AS-f 


£: 


Serial  No.  NHLI-1 12(c) 

elevated  and  prolonged  increases  in  iitununoreactive  plasma  growth 
hormone .   Metapyrone  and  ACTH  produced  normal  responses  .   Oral 
papillae  and  taste  buds  v/ere  present.   Nerve  conduction  veloc- 
ities were  normal .   Both  shov/ed  normal  responses  to  intradermal 
histamine  and  pilocarpine,  but  abnormal  cold  pressor  tests.   Both 
had  miosis  after  conjunctival  placement  of  2.5%  methacholine . 
In  a  constant  environment  at  120 °F  with  12%  relative  humidity  for 
45  minutes  their  v;eight  losses  were  significantly  less  than  nor- 
mal (80  gm) ,  demonstrating  the  absence  of  sweating,  while  their 
body  temperatures  rose  to  40°C  with  an  abnormal  temperature  curve. 
Nevertheless,  pilocarpine  iontophoresis  of  the  skin  of  the  fore- 
arm produced  normal  sweat  volumes.   Abnormal  glucose  tolerance 
curves  were  demonstrated  in  the  father  and  paternal  uncle.   The 
mother  demonstrated  miosis  after  conjunctival  placement  of  2  .5% 
methacholine.   Both  parents  showed  hypogeusia  and  abnormal  cold 
pressor  tests  but  normal  responses  to  intradermal  pilocarpine . 

These  findings  suggest  that  this  syndrome  belongs  in  the 
general  category  of  familial  dysautonomia .   In  contrast  to  Types  I 
and  II  familial  dysautonomia,  this  syndrome  appears  to  have  little 
peripheral  component  and  to  be  due  primarily  to  central  autonomic 
dysfunction  as  opposed  to  the  predominant  peripheral  autonomic 
dysfunction  in  Type  II,  and  the  central  and  peripheral  autonomic 
dysfunction  in  Type  I  familial  dysautonomia. 

5  .   Aqlycoqeusia  and  Hypoglycoqeusia .   Inability  to  recognize 
the  taste  of  any  sweet  substance,  aglycogeusia   was  described  in 
patients  with  congenital  idiopathic  hypoparathyroidism  in  this 
report  in  1970.   During  this  past  year  we  have  observed  the 
presence  of  this  defect  and  a  partial  defect,  hypoglycogeusia, 
a  quantitative  decrease  in  the  recognition  of  the  taste  of  some 
sweet  substances,  in  patients  with  Hand-Schuller-Christian 
disease,  and  in  the  mothers  of  these  patients.   This  latter  ab- 
normality has  also  been  observed  in  some  unaffected  siblings  of 
the  patients  with  this  disease.   These  preliminary  observations 
suggest  that  the  ability  to  taste  sweet  may  be  controlled  in  some 
specific  manner  perhaps  similar  to  that  noted  for  the  bitter  taste 
of  phenylthiocarbamide .   These  studies  are  also  important  in 
suggesting  that  a  protein  abnormality  of  an  as  yet  unspecified 
type  or  location  could  be  responsible  for  this  defect  and  may 
offer  us  an  important  clue  to  the  understanding  of  the  basic 
mechanism  of  taste,  particularly  the  taste  of  sweet. 


13  Ji^o 


Serial  No.  NHLI-112(c) 

6.   Disseminated  carcinoma  of  various  types.   Patients  with 
carcinomatosis  either  untreated  or  treated  with  various  toxic 
agents  have  developed  anorexia  and  taste  loss .   These  symptoms 
have  not  been  susceptible  to  standard  forms  of  therapy.   We  have 
been  informed  of  patients  with  carcinoma  of  the  pancreas,  liver, 
stomach  or  lymphoma  who  have  anorexia  and  loss  of  taste  as  their 
primary  complaint  subsequent  to  treatment  of  their  underlying 
condition.   Although  the  complaints  of  these  patients  are  ob- 
viously complex  and  determined  by  many  factors  we  have  treated 
several  of  these  patients  with  zinc  sulfate  in  a  single  blind 
study  to  observe  the  effects  of  zinc  ion  on  the  anorexia  and 
taste  loss  of  these  patients .   These  studies  have  been  carried 
out  through  the  cooperation  of  physicians  at  the  City  of  Hope, 
Duarte,  California  and  others  throughout  the  U.S.   Serum  and 
urine  were  collected  in  metal  free  containers  and  zinc  sulfate 
in  the  amount  of  100  mg  daily,  as  zinc  ion  has  been  administered. 
To  date,  studies  on  5  patients  have  been  completed.   In  two 
patients  with  carcinoma  of  the  liver  administration  of  zinc  ion 
reversed  the  subjective  complaints  of  anorexia  and  taste  loss  in 
both  patients;  in  one  patient  with  disseminated  lymphoma  change 
in  appetite  was  evaluated  subjectively  while  taste  loss  was 
evaluated  objectively  before,  during  and  after  treatment  with 
zinc.   Taste  acuity  in  this  latter  patient  returned  to  near  normal 
levels  and  the  anorexia  disappeared  during  therapy.   Each  of  these 
3  patients  had  significant  elevations  of  their  serum  zinc  concen- 
trations while  taking  the  metal.   Each  has  subsecjuently  died  of 
their  underlying  disease.   One  patient  with  carcinoma  of  the 
pancreas  and  one  with  carcinoma  of  the  stomach  were  also  studied. 
Their  zinc  levels  in  serum  were  normal  prior  to  treatment  and 
following  treatment  for  one  month  there  were  no  subjective  changes 
in  anorexia  or  taste  acuity  although  serum  zinc  concentrations 
increased  significantly.   These  studies  suggest  that  the  anorexia 
observed  with  carcinomatosis  may  be  aided  in  some  patients  with 
the  administration  of  zinc  ion. 


3. 


7.   Hypertension  and  taste.   Since  1906  the  specific  interre- 
lationships between  salt,  fluid  intake  and  hypertension  have  been 
well  documented.   Treatment  of  essential  hypertension  with  salt 
and  fluid  restriction  has  been  an  accepted  mode  of  therapy  since 
that  time.   Many  attempts  have  been  made  to  link  salt  intake  to 
hypertension,  and  while  generally  successful  clinically,  the 
correlation  between  the  two  phenomena  has  not  been  specifically 
convincing  with  respect  to  a  model  for  possible  mechanisms  under- 
lying hypertension.   In  an  effort  to  investigate  the  possible  role 


14 


3L^f 


Serial  No.   NHLI-li2(c) 

of  the  appreciation  of  salt  as  an  etiological  factor  in  hyper- 
tension systematic  studies  in  man  and  animals  were  undertaken. 
In  man,  thresholds  for  the  taste  of  each  of  four  cjualities  were 
investigated  in  patients  with  hypertension  of  several  different 
etiologies.   Untreated,  with  significant  systolic  and  diastolic 
hypertension,  or  treated  with  drugs  or  surgery  which  lov/ered  the 
blood  pressure  to  the  normal  range,  detection  or  recognition 
thresholds  for  the  population  of  patients  with  hypertension 
studied  did  not  differ  from  that  of  normal  subjects.   These  re- 
sults differed  from  those  of  other  investigators  who  measured 
an  aspect  of  taste  acuity  which  is  neither  detection  nor  recog- 
nition of  salt  and  found  to  be  abnormal.   The  meaning  of  these 
latter  relationships  were  not  clear  but  this  unresolved  incon- 
gruity prompted  us  to  measure  the  preference  of  a  genetically 
hypertensive  strain  of  rats  (SHR)  for  NaCl  in  comparison  with 
normal  rats.   These  studies,  briefly  commented  upon  in  this 
report  in  1970,  have  been  carried  out  in  detail  in  SHRs  using 
the  twenty-four  hour,  two  bottle,  forced  choice  technique,   SHRs 
show  a  significant  decrease  in  their  acuity  for  NaCl  as  indicated 
by  their  continued  acceptance  of  solutions  of  NaCl  normally  re- 
jected by  other  rats.   This  extends  even  to  solutions  as  concen- 
trated as  0.45  M  NaCl.   However,  their  preferences  for  other  taste 
','.•  stimuli  which  are  normally  aversive  such  as  quinine  sulfate  and 

'J  HCl  are  not  different  from  normal.   Curiously,  the  intake  of 

4" "  ... 

excessive  quantities  of  NaCl  m  the  SHR  is  not  accompanied  by 

excessive  intake  of  fluid,  something  previously  seen  in  each 
animal  in  previous  experiments  in  whom  hypogeusia  occurred  when 
produced  by  other  means  (e.g.,  feeding  D-penicillamine,  cysteine 
etc.) .   In  order  to  document  this  finding  we  have  repeated  it  on 
four  occasions,  each  time  with  the  same  results .   Given  a  choice 
between  KCl  and  water  or  NaHC03  and  water  the  differences  between 
the  SHRs  and  normal  rats  were  not  clear  and  these  experiments  will 
be  repeated  before  any  definitive  interpretation  will  be  made . 
Interpretation  of  the  results  of  previous  experiments  also  sugges- 
ted that  as  the  SHRs  increased  in  age  there  was  a  decrease  in 
their  choice  for  NaCl  over  water  such  that  the  differences  seen 
early  in  their  life  could  be  demonstrated  but  not  as  clearly  as 
they  were  previously. 

In  an  effort  to  clarify  some  of  the  variables  in  these  ex- 
periments taste  preference  for  various  solutions  were  also 
studied  in  rats  made  hypertensive  by  the  adrenal  regeneration 
technique.   Results  of  these  experiments  showed  that  taste 
preferences  in  rats  made  hypertensive  by  this  technique  did  not 

15  M% 


9 


Serial  No.   NHLI-112(c) 

differ  from  those  of  control  rats  treated  in  a  manner  similar  to 
the  adrenal  regeneration  rats  but  without  significant  hypertension 
Studies  carried  out  by  other  investigators  in  rats  made  hyper- 
tensive by  renal  latex  encapsulation  or  by  deoxycorticosterone 
administration  report  that  these  animals  show  an  aversion  for 
NaCl  and  a  polydipsia,  exactly  the  opposite  phenomena  observed 
in  the  SHRs . 


aTT  ■  -j 


These  conflicting  data  may  allow  the  design  of  experiments 
which  may  elucidate  the  etiology  of  hypertension.   Either  the 
salt  preference  in  SHRs  may  be  related  to  their  genetic  diff- 
erences from  other  rats  or  this  preference  which  decreases 
positively  with  increasing  age  may  be  related  to  biochemical 
changes.   This  latter  hypothesis  raises  the  possibility  that 
since  renin  production  in  the  SHRs  may  decrease  with  increasing 
age  there  may  be  a  correlation  between  these  two  events .   Current 
studies  are  being  undertaken  to  evaluate  this  possibility. 
Studies  are  also  being  undertaken  to  measure  taste  preference  in 
the  SHRs  following  correction  of  their  hypertension  with  antihy- 
pertensive drugs  and  after  treatment  with  various  antiadrenergic, 
anticholinergic  and  antiserotinergic  drugs. 


Studies  in  patients  with  hypertension  have  taken  a  form 
similar  to  that  described  for  rats .   Patients  are  given  a  con- 
stant, dry  9  Meq  NaCl  diet  and  all  fluid  imbibed  taken  from 
either  of  two  bottles,  one  containing  distilled  water,  the  other 
150  mM  NaCl.   The  subjects  are  given  free  access  to  these  fluids 
over  a  24  hour  period.   Total  fluid  imbibed,  percent  of  NaCl 
imbibed  and  Na  and  K  in  the  serum  and  urine  are  measured.   Similar 
studies  have  also  been  carried  out  in  two  other  groups  of  subjects; 
normal  volunteers  with  normal  blood  pressure  and  without  any 
family  history  of  hypertension  or  cardiac  disease  and  normal 
volunteers  with  normal  blood  pressure  and  significant  family 
histories  of  hypertension  or  cardiac  disease.   Taste  thresholds 
for  4  taste  qualities  are  measured  in  each  subject  prior  to  the 
start  of  the  test.   Results  of  studies  in  patients  with  hyper- 
tension indicate  that  they  prefer  20-60%  of  their  daily  fluid 
as  150  mM  NaCl  whereas  normal  volunteers  without  any  family  his- 
tory of  hypertension  or  cardiac  disease  imbibe  less  than  10% 
of  their  total  daily  fluid  as  150  mM  NaCl.   These  studies  have 
lasted  from  5  to  8  days ,   Preference  for  NaCl  in  normal  volunteers 
with  family  histories  of  hypertension  or  cardiac  disease  differ 
from  that  of  the  other  normal  volunteers  in  that  they  usually 
begin  taking  in  less  than  10%  NaCl  but  end  the  period  of  the 


i 


I 


16 


J£Z 


Serial  No.   NHLI-112(c) 

study  imbibing  more  than  20%  NaCl.   These  provocative  preliminary 
results  obtained  from  5  patients  with  hypertension  and  6  normal 
volunteers  will  be  expanded  during  the  next  year  by  the  continued 
study  of  normal  subjects  and  patients  with  hypertension. 

Pharmacology .   Many  drugs  appear  to  affect  taste  acuity  in 
an  adverse  manner.   These  drugs  have  been  brought  to  our  attention 
by  reports  made  to  medical  journals  throughout  the  world  and 
sent  along  to  our  unit  for  comment.   These  drugs  have  caused 
subjective  abnormalities  of  taste  acuity  in  patients  and  were 
made  known  to  the  physician  administering  the  drug  by  the  patient's 
spontaneous  complaints.   In  general,  these  reports  have  not  been 
followed  up  systematically  with  quantitative  testing  of  detection 
y*  or  recognition  thresholds.   These  drugs  include  D-penicillamine, 

■j[l;'',        lincomycin,  5-mercaptopyridoxal,  6  axauridine  triacetate,  acetyl 
,(['        sulfosalicylic  acid,  griseofulvin,  reserpine,  ildamen,  chlofi- 
,. "        brate,  valium  and  other  tranquilizers  and  phenindione.   Some  of 
'!,  these  drugs  have  similar  actions  and  chemical  structures  and 

thereby  may  provide  important  clues  to  the  mechanisms  by  which 
they  act  on  taste  acuity.   For  example,  drugs  which  deplete  the 
body  stores  of  norepinephrine  and  which  may  alter  taste  acuity 
,.4'"        suggest  that  catecholamines  may  play  some  role  in  taste.   Indeed, 
.i' ;;'        granules  of  at  least  two  types  are  present  in  and  around  the 
'.'■■}*  taste  bud  and  their  composition  is  unknown.   Similarly,  the 

i^"'  manner  by  which  the  taste  information  is  transduced  at  the  re- 

ceptor in  terms  of  a  neurotransmitter  agent  is  unknown .   We 
therefore  have  undertaken  a  systematic  evaluation  of  drugs  which 
alter  possible  neurotransmitter  agents  in  the  rat  in  an  effort 
to  identify  the  manner  by  which  this  transmission  may  occur. 

Education  of  physicians  of  taste  abnormalities.   Because  of 
the  prevalence  of  taste  abnormalities  in  the  population  and  the 
lack  of  knowledge  of  this  subject  by  physicians  who  see  patients 
with  these  abnormalities  some  method  for  supplying  them  with 
this  information  and  techniques  by  which  they  can  document  and 
record  these  abnormalities  must  be  made  available.   The  technique 
used  in  our  laboratory  is  cumbersome  and  time  consuming.   There-    I 
fore,  a  simplified  modification  of  our  technique  has  been  devised 
and  reduced  to  practice  in  the  form  of  a  Taste  Testing  Kit.   With 
the  aid  of  the  Campbell  Institute  for  Food  Research  this  kit  has 
been  assembled  and  reduced  to  practice.   Instructions  for  its  use 
and  Taste  Record  Cards  by  which  results  can  be  formally  recorded 
have  been  made  and  will  be  available  for  distribution  to  interested; 
physicians  who  wish  to  use  this  kit  within  the  next  4  months .       | 

17  Ji^^/ 


Serial  No.  NHLI-112(c) 


vl 


As  of  this  date  over  500  physicians  in  the  U .S 
to  obtain  such  a  kit . 


have  requested 


Major  Findings:   Olfaction 

Physiology .   1.   Vitamin  A  metabolism.   In  the  study  previ- 
ously noted  with  prisoners  at  the  University  of  Iowa  olfactory 
acuity  for  several  vapors  were  measured  b.efore,  during  and 
after  depletion  of  vitamin  A  by  feeding  a  diet  deficient  in 
vitamin  A  and  then  readding  vitamin  A  to  the  diet .   Patients 
developed  hyposmia  while  on  the  vitamin  A  deficient  diet .   This 
confirms  the  widely  held  belief  that  vitamin  A  plays  some  role 
in  maintaining  normal  olfactory  acuity.   However,  the  role  which 
vitamin  A  plays  in  olfaction  is  not  clear.   Studies  in  patients 
with  acute  viral  hepatitis  have  demonstrated  that  olfactory  acuity 
is  impaired  during  the  acute  phase  of  the  disease  and  it  returns 
to  normal  as  the  disease  process  wanes .   Attempts  to  correlate 
this  hyposmia  with  changes  in  serum  concentrations  of  vitamin  A 
were  unsuccessful.   However,  there  was  a  significant  inverse 
correlation  between  hyposmia  and  levels  of  bilirubin  during  the 
disease  and  a  significant  positive  correlation  between  hyposmia 
and  retinol  binding  protein  (RBP) ,  the  major  transport  protein 
for  vitamin  A  alcohol  in  serum.   The  correlation  between  vitamin 
A  metabolism  and  olfaction  suggests  that  a  relationship  between 
olfaction  and  vitamin  A  alcohol  exists  rather  than  between  ol- 
faction and  vitamin  A  itself. 

Attempts  to  correlate  abnormalities  of  dark  adaptation  and 
hyposmia  with  vitamin  A  and  RBP  metabolism  in  patients  with 
acute  and  chronic  hepatitis  were  carried  out  in  12  patients 
studied  at  Harlem  Hospital,  New  York  City.   Dark  adaptation  was 
normal  in  each  patient  although  hyposmia  and  abnormalities  of 
both  serum  vitamin  A  and  RBP  were  present  in  most.   The  interre- 
lationships between  vision,  olfaction  and  vitamin  A  metabolism, 
although  provocative  from  the  point  of  view  of  mechanism,  are 
not  yet  clear . 


2.   Hypogonadism  and  olfaction.   As  noted  in  this  report  in 
1970,  a  systematic  evaluation  of  the  effects  of  ablation  of  the 
olfactory  bulb  in  female  rats  has  been  undertaken.   These  studies 
have  been  continued  and  appropriate  control  studies  carried  out. 
Ablation  of  the  olfactory  bulbs  reduces  the  intake  of  food  in 
female  rats  such  that  their  body  weights  are  significantly  de- 
creased from  appropriate  control  animals.   Paired  feeding  of  rats 


18 


«3^r 


,^r 


Serial  No.  NHLI-112(c) 

showed  that  starvation  was  a  significant  factor  in  the  delay  of 
sexual  maturation  but  could  not  account  for  the  profound  delay 
in  sexual  maturation  noted  after  ablation  of  the  olfactory  bulbs. 
FSH  and  LH  measurements  are  at  present  being  undertaken  in  these 
rats  by  Dr.  Charles  Barraclough,  Department  of  Physiology, 
University  of  Maryland  School  of  Medicine. 

In  man,  the  relationships  between  hypogonadism  and  olfaction 
has  been  carried  out  in  a  prospective  manner.   Qsing  the  data 
collected  over  the  past  4  years  in  females  with  amenorrhea  it  is 
possible  to  predict,  on  a  statistical  basis,  the  association  of 
Type  II  hyposmia  with  patients  with  gonadal  dysgenesis  and  streak 
ovaries  as  opposed  to  the  association  of  Type  I  hyposmia  or 
'I'"        anosmia  with  patients  with  hypogonadotrophic  hypogonadism  and 
;'l;'^        unstimulated  ovaries.   Further  studies  are  in  progress  at  the 
ll'        present  time. 
.  * 
' ;; '  3.   Mechanism  of  hyposm.ia  following  laryngectomy.   In  this 

.'        report  in  1970  it  was  possible  to  state  that  bilateral  surgical 
interruption  of  the  9th  and  10th  nerves  was  associated  with  the 
production  of  hyposmia  as  opposed  to  alterations  in  air  flow 
^.1         which  did  not  produce  hyposmia.   In  patients  in  whom  various  sur- 
,(*  •'        gical  procedures  involving  the  larynx  have  been  carried  out  it 
|!j  f         has  been  possible  to  demonstrate  that  bilateral  interruption  of 
iU'  only  the  motor  nerves  to  the  larynx,  i.e.,  the  recurrent  laryngeal 

nerves,  was  associated  with  hyposmia.   The  mechanism  underlying 
this  phenomenon  is  not  clearly  understood  since  sensory  fibers 
involved  in  several  reflex  actions  of  the  larynx  accompany  these 
motor  nerves  to  the  larynx .   The  feedback  between  nerves  9  and  10 
and  neural  interconnections  in  the  limbic  cortex,  as  observed  by 
Dell,  may  be  ultimately  responsible  for  this  interaction.   Negus' 
work,  which  demonstrates  the  role  of  larynx  in  olfaction  in  lower 
mammals  and  vertebrates,  supports  this  entire  concept  on  a  be- 
havioral level. 

4.   Nosology.   The  characterization  of  "primary"  olfactory 
stimuli  to  correspond  to  the  "primary"  taste  qualities  of  salt, 
sour,  bitter  and  sweet  has  produced  much  confusion.   In  general, 
volatile,  low  molecular  weight  substances  (  <300)  have  an  odor 
but  their  definitive  grouping  into  qualities  is  not  clear.   In 
our  studies  of  patients  with  aglycogeusia  and  with  various  forms 
of  hyposmia  it  has  been  possible  to  isolate  a  specific  "olfactory 
primary";  i.e.,  sweet.   Patients  with  aglycogeusia  can  detect 


19  jia4 


Serial  No.  NHLI-112(c) 


i. 


gustatory  and  olfactory  stimuli  such  as  sucrose  or  chloroform, 
respectively,  but  cannot  recognize  them  as  sweet;  indeed, 
all  haloforms  are  detected  by  taste  and  smell  but  not  recognized 
as  sweet .   This  is  in  contrast  to  the  ability  of  patients  with 
anosmia  who  can  taste  all  haloforms  as  sweet,  but  cannot  detect 
or  recognize  the  vapor.   These  studies  demonstrate  that  sweet  is 
a  smell  quality  as  well  as  a  taste  quality. 

Pathology .  1.  Sjogren's  syndrome.   Each  of  the  30  patients 
with  Sjogren's  syndrome  studied  demonstrated  significant  hyposmia 
of  varying  degree.   As  with  taste  acuity,  no  treatment  schedule 
returned  olfactory  acuity  to  normal  in  any  patient  unless  sig- 
nificant olfactory  and  nasal  mucous  was  formed.   Since  these 
patients  exhibit  dry  nasal  mucous  membranes  due  to  the  lack  of 
nasal  and  olfactory  mucous  it  is  not  surprising  that  olfactory 
acuity  would  diminish  as  suggested  by  the  electrophysiological 
studies  of  Shibuya . 


2.  Wegener's  granulomatosis  and  Midline  granuloma.   As 
noted  previously  patients  with  these  two  diseases  suffer  from 
hyposmia  of  varying  degree.   Of  the  15  patients  studied,  those 
treated  and  in  remission  exhibited  a  return  to  or  toward  normal 
olfactory  acuity.   The  mechanism  underlying  this  change  is  un- 
known.  However,  in  spite  of  the  rarity  of  this  disease,  any 
patient  with  a  history  of  hyposmia  should  be  carefully  evaluated 
to  rule  out  either  of  these  two  diseases  since  early  treatment 
can  result  in  remission. 

3.  Idiopathic  hyposmia.   We  have  studied  patients  with 
idiopathic  hyposmia  over  the  past  4  years .   They  fall  into  two 
categories,  congenital  hyposmia,  usually  Type  I,  and  acquired 
hyposmia,  which  can  be  either  Type  I  or  Type  II.   We  have  treated 
patients  with  both  varieties  with  aqueous  vitamin  A,  50,000  units 
daily  for  up  to  three  years  .   The  hyposmia  was  returned  to  or 
toward  normal  in  some  patients  with  both  congenital  and  acquired 
hyposmia  but  not  in  others  .   In  an  effort  to  document  these  changes 
we  have  undertaken  a  small  double  blind  study  in  which  a  number 

of  patients  will  be  investigated,  some  receiving  placebo  for  6 
months,  some  receiving  aquasol  A,  50,000  units  daily,  in  an  effort 
to  evaluate  the  efficacy  of  vitamin  A  in  the  treatment  of  these 
ill  defined  conditions.   Mr.  M.  Raff  and  Dr .  W .  Friedewald  will 
assist  us  in  the  designing  of  this  project. 


i 


20 


3^7 


Serial  No.  NHLI-112(c) 

4.  Retinitis  pigmentosa  (RP) .   Patients  with  this  abnormality 
represent  a  variety  of  retinal  defects.   In  a  single  blind  study, 
22  patients  with  this  abnormality  have  been  studied  over  a  period 
of  6  months .   Documentation  of  their  retinal  defects  were  made 
independent  of  measurement  of  their  taste,  smell  or  auditory 
acuity.   Results  demonstrate  that  patients  with  RP  fall  into 
three  categories  of  sensory  abnormalities.   The  10  patients 
studied  with  "typical  RP"  exhibited  a  demonstrable  loss  of  taste 
and  smell  acuity.   Auditory  acuity  was  significantly  decreased 

in  approximately  45%.   The  14  patients  studied  with  "atypical  RP" , 
with  pigmentary  changes  of  the  retina,  exhibited  some  decreased 
taste  acuity  but  normal  olfactory  acuity.   Auditory  acuity  was 
also  significantly  decreased  in  about  40%.   Only  four  patients 
]i"  with  other  retinal  degenerations  without  pigmentary  changes  were 

', '  studied  .   In  general  they  exhibited  hyposmia  but  normal  taste 

|. '       acuity.   None  had  decreases  in  auditory  acuity.   Too  few  patients 
";        in  this  latter  category  have  been  studied  to  consider  them  an 
[.',<•  adequate  control  group.   At  the  present  time  measurements  of 

' '  vitamin  A  and  RBP  levels  in  serum  do  not  clearly  differentiate 

patients  with  RP .   Hovv^ever,  systematic  studies  of  vitamin  A  and 
of  RBP  are  at  present  underway  to  establish  the  presence  or 
absence  of  any  correlative  biochemical  changes. 

''%  These  results  suggest  that  patients  with  RP  may  have  abnor- 

jjl. "        malities  of  several  sensory  systems.   Since  RP  is  inherited  in 
a  dominant  or  an  autosomal  manner  and  since  geneologies  have 
been  carefully  documented  by  members  of  the  NEI,  patients  with 
these  abnormalities  will  be  studied  in  a  prospective  manner  such 
that  the  hypotheses  made  from  results  of  the  single  blind  study 
may  be  tested  and  evaluated.   If  these  abnormalities  do  define 
patients  with  RP  evaluation  of  receptor  changes  will  be  carried 
out . 

5.  The  Molecular  Basis  of  Olfaction.   A  report  on  the 
molecular  basis  of  olfaction  has  been  prepared  for  the  Office 
of  Naval  Research  during  this  past  year.   The  purpose  of  the 
report  was  to  review  the  state  of  the  science  of  the  sense  of 
smell.   In  this  report  all  major  molecular  theories  of  olfaction 
were  evaluated  and  critically  analyzed  with  respect  to  pre- 
vailing scientific  thought  and  information.   Over  5000  papers 
were  reviewed,  approximately  500  in  detail  and  these  were  criti- 
cally analyzed.   Based  upon  these  findings  the  requirements  which 
an  adequate  molecular  theory  of  olfaction  must  satisfy  have  been 
established  . 

21  j^e 


J2 


Serial  No.  NHLI-1 12(c) 

Sicrnif  icance:  Taste .   We  have  operationally  divided  the  taste 
system  into  5  functional,  component  parts:  (1)  pre-neural  events, 
(2)  transduction  events,  (3)  neural  events,  (4)  CNS-feedback 
events  on  the  bud  and  (5)  humoral  influences  on  the  entire  system. 
The  major  contribution  which  we  have  made  is  primarily  limited 
to  knowledge  of  the  pre-neural  events  and  to  the  definition  of 
the  transduction  events . 

We  have  clearly  shown  that  there  are  at  least  two  types  of 
pre-neural  events  in  taste.   We  have  hypothesized  that  some  form 
of  chemical  sieving  controls  the  non-specific  portion  of  the  pre- 
neural  events .   Experiments  carried  out  which  demonstrate  the 
inhibitory  effects  of  thiol  containing  drugs  and  amino  acids 
on  taste  support  this  concept.   Similarly  supportive  are  data 
demonstrating  oral  placement  of  proteases  affect  all  taste 
qualities.   These  results  suggest  that  proteases  are  effective 
on  protein  of  the  taste  bud  exposed  to  the  oral  environment.   We 
suppose  this  protein  is  part  of  the  membrane  of  the  receptor 
cells  of  the  taste  bud .   Our  anatomical  observations  suggest  that 
this  is  the  Type  I  cell  of  the  taste  bud  which  comprise  80% 
of  the  cells  of  the  bud  . 

We  have  also  demonstrated  that  there  is  a  specific  portion 
of  the  pre-neural  events  of  taste  which  relate  to  each  taste 
quality.   We  have  demonstrated  specific  pathophysiological 
abnormalities  for  the  taste  of  sweet  and  also  have  isolated, 
in  a  purer  form  than  have  others,  a  substance  which  specifically 
alters  the  taste  of  sour  to  sweet.   The  specific  taste  events 
are  most  probably  involved  with  the  binding  of  tastant  to  the 
receptor  membrane.   Our  anatomical  investigations  of  the  receptor 
of  Phormia  regina  and  of  taste  buds  from  patients  with  aglycogeusia 
have  given  us  information  of  the  anatomical  receptor  configuration 
related  to  appreciation  of  sweetness. 

We  have  placed  the  entire  problem  of  taste  acuity  within  the 
framework  of  medical  practice.   The  prevalence  of  the  disease 
Idiopathic  Hypogeusia  which  we  have  recently  described  indicates 
the  need  for  an  awareness  and  understanding  of  the  loss  of  taste. 
The  results  of  our  single  blind  study  indicates  that  zinc  is 
beneficial  in  the  treatment  of  patients  with  this  disorder,  al- 
though the  mechanism  is  not  yet  known.   With  the  discovery  of 
this  disease  we  have  also  demonstrated  the  first  known  pathology 
of  the  taste  bud  and  we  have  published  the  first  high  power 
electron  microscopic  pictures  of  the  normal  histology  and  pathology 


22 


cS^f 


Serial  No.  NHLI-1 12(c) 


of  the  human  taste  bud 


We  have  also  devised  a  test  system  by  which  a  model  for  the 
role  of  salt  intake  in  hypertension  may  be  possible  in  rat  and 
in  man , 

Olfaction .   We  have  limited  the  role  of  vitamin  A  in  olfaction 
to  one  which  involves  RBP  and  vitamin  A  alcohol.   We  have  sep- 
arated the  abnormalities  of  vision  from  olfaction  in  hepatitis 
and  this  may  lead  to  a  clearer  understanding  of  the  role  which 
vitamin  A  alcohol  plays  in  olfaction.   We  have  also  clarified 
the  interrelationship  between  olfactory  function  and  gonadal 
function  in  lower  mammals.   We  have  identified  sweet  as  a  pri- 
mary olfactory  stimulus. 

Proposed  Course  of  Project:   1.   To  clearly  establish  whether  oir 
not  zinc  is  efficacious  in  the  treatment  of  patients  with  idio- 
pathic hypogeusia  through  the  completion  of  a  double  blind  study 
of  108  patients  with  this  disease. 


i5' 


2  .   To  establish  whether  or  not  there  is  an  etiological  role 
for  taste  acuity  for  sodium  in  essential  hypertension  in  man  or 
in  SHRs . 

3  .   To  clearly  identify  the  anatomical  characteristics  of 
taste  buds  in  fungiform,  circumvallate  and  palatal  papillae  in 
man  and  in  vallate  papillae  in  rat . 


4.   To  specify  the  anatomical  abnormalities  of  taste  buds  in 
patients  with  aglycogeusia  and  otpathological  abnormalities  of 
taste . 

5  .   To  identify  anatomically  and  physiologically  the  taste 
receptor  in  Phormia  regina  and  to  perform  initial  biochemical 
studies  of  specific  binding  of  radioactive  sugars  to  the  receptor. 

6.  To  define  the  interrelationship  between  RP  and  taste  and 
smell  abnormalities.   If  these  do  exist  we  will  undertake  an 
anatomical  analysis  of  the  taste  receptor  in  patients  with  typical 
and  atypical  RP . 

7.  To  complete  the  distribution  of  Taste  Testing  Kits  to  those 
physicians  in  the  U.S.  who  have  requested  them. 


23 


3l70 


Serial  No.  NHLI-112(c) 

Honors  and  Awards:   None 

Publications: 

Hoye,  R.C.,  Ketcham,  A.S.,  and  Henkin,  R.I.:  Hyposmia  after 
paranasal  sinus  exenteration  or  laryngectomy.  Am .  J .  Surg . 
120:  485-491,  1970. 

Henkin,  R.I.  and  Bradley,  D.F.:  Report  on  the  molecular 
basis  of  olfaction.  Office  of  Naval  Research,  1970. 

Henkin,  R.  I.  and  Shallenberger ,  R.S.:  Haloforms:  Sweet 
taste  or  smell.   Experientia,  27:  154-155,  1971. 

Giroux,  E.L.  and  Henkin,  R.  I.:  Oral  effects  of  hydrolytic 
enzymes  on  taste  acuity  in  man.   Life  Sci .  10:  351-3  70,  1971, 

Henkin,  R.I.,  Schechter,  P. J.,  Hoye,  R.C.  and  Mattern, C .F .T . : 
Idiopathic  hypogeusia  with  dysgeusia,  hyposmia  and  dysosmia: 
A  new  syndrome.   JAMA,  In  press,  1971. 

Henkin,  R.I.  and  Smith,  F.R.:  Hyposmia  in  acute  viral  hepatitis 
Lancet,  In  press,  1971. 

Marshall,  J.R.  and  Henkin,  R.I.:  Olfactory  acuity,  menstrual 
abnormalities  and  oocyte  status .  Annals  of  Int.  Med.,  In 
press,  1971. 

Henkin,  R.I.:  Molecular  Basis  of  Odor.   New  Eng .  J .  Med . , 
284:  560,  1971.  (Book  Review) 

Henkin,  R.  I.:  Molecular  Basis  of  Odor.  Annals  of  Int .  Med . 
74:  460,  1971.  (Book  Review) 

Henkin,  R.I.  :  Griseofulvin  and  dysgeusia:  implications? 
Annals  of  Int.  Med.  74:  795-796,  1971.  (Letter  to  the  Editor) 

Henkin,  R.I.:  Idiopathic  hypogeusia — A  new  disease.  JAMA,  In 
press,  1971.  (Editorial) 

Henkin,  R.I.:  Idiopathic  hypogeusia — A  new  disease.  JAMA,  In 
press,  1971.  (Letter  to  the  Editor) 


24  <3L1/ 


Serial  No.  NHLI-112(c) 

Patents 

Henkin,  R.  I.:  Diagnostic  device  and  method  of  treatment. 
U.S.  Patent  Application  Serial  No.  107,279,  January  13,  1971 


i:i: 


25  ^7A 


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ANNUAL  REPORT  OF  THE 
LABORATORY  OF  KIDNEY  AND  ELECTROLYTE  METABOLISM 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1970  through  June  30,  1971 

The  Laboratory  of  Kidney  and  Electrolyte  Metabolism  is  studying  the 
mechanism  of  water  and  electrolyte  transport  in  the  following  tissues: 
mammalian  nephron,  amphibian  bladder,  and  the  avian  erythrocyte.   In  addition 
experiments  directed  at  elucidating  the  rate -controlling  steps  in  cardiac 
contractility  and  the  cause  of  a  hereditary  cardiomyopathy  in  hamsters  also 
are  in  progress.  This  report  will  be  concerned  with  the  kidney,  toad  bladder 
and  cardiac  muscle  studies.  Detailed  summaries  of  the  erythrocyte  experiments 
are  included  in  the  appended  individual  reports. 

Mammalian  nephron; 

A  method  for  the  perfusion  of  isolated  segments  of  the  rabbit  nephron  in 
vitro  was  developed  in  this  laboratory  several  years  ago.  This  method  permits 
direct  measurements  of  transport  under  rigidly  controlled  conditions  across  the 
individual  barriers  of  those  epithelial  cells  responsible  for  the  maintenance 
of  water  and  electrolyte  balance  in  the  living  animal.   The  in  vitro  prepara- 
tion has  a  number  of  advantages  over  current  in  vivo  techniques.   Difficulties 
of  interpretation  of  data  attendant  upon  effects  of  the  surrounding  interstitial 
space,  unstirred  layers,  inadequate  control  of  the  composition  of  the  extra- 
cellular bathing  fluid,  etc.  are  avoided.   The  technique  has  proved  useful 
in  elucidating  the  mechanism  of  action  of  antidiuretic  hormone  and  its  inter- 
cellular mediator,  cyclic  3' ,5 '-monophosphate  in  the  cortical  collecting 
tubule  of  the  rabbit.   In  addition  it  has  been  demonstrated  that  the  path  of 
bulk  water  flow  along  an  osmotic  gradient  in  this  tissue  is  both  through  and 
between  the  cells.  Antidiuretic  hormone  increases  water  permeability  in  the 
nephron  in  a  manner  analogous  to  that  in  amphibian  structures  such  as  frog 
skin  and  toad  bladder.   In  the  latter  tissues  the  hormone  increases  net  sodium 
transport,  an  effect  presumed  to  be  secondary  to  a  change  in  the  passive 
permeability  of  a  limiting  cell  membrane  to  sodium,  rather  than  a  direct 
stimulation  of  the  transport  pump.   It  has  not  been  established  that  sodium 
transport  is  accelerated  by  ADH  in  the  kidney.  Results  based  on  in  vivo 
studies  in  the  rat  and  other  mammals  are  conflicting.  We  have  noted  that  the 
hormone  increases  the  potential  difference  across  the  cortical  collecting 
tubule  without  altering  transtubular  electrical  resistance.  Although  the 
results  may  indicate  that  vasopressin  accelerates  net  Na  transport  in  the 
kidney  by  a  direct  effect  on  the  sodium  pump,  the  conclusion  cannot  be 
accepted  as  definitive  without  concurrent  measurements  of  sodium  flux.  These 
are  at  present  in  progress.   In  preliminary  studies  employing  ^^Na  as  a  marker 
the  unidirectional  flux  of  Na  from  lumen  to  bath  rose  10-20%  following 
addition  of  25  jiU/ml  of  ADH  to  the  bathing  medium.   Current  studies  are 
directed  at  measuring  backflux,  i.e.,  from  the  bathing  medium  to  lumen  under 
these  circumstances  to  substantiate  the  tentative  conclusions  alluded  to  above. 

Active  transport  of  potassium  from  bath  to  lumen  in  exchange  for  reabsorbed 
sodium  occurs  in  the  cortical  collecting  tubule.   It  is  probable,  though  not 
established,  that  the  individual  ions  are  transported  through  rather  than 
between  the  cells.  The  mass  of  transported  ions  contained  in  the  cells  in  the 


A73 


course  of  transport  is  defined  as  the  "transport  pool."  From  ito  size, 
turnover  rate  and  specific  activity,  the  sites  of  ion  transport  and  the  trans- 
port properties  of  the  individual  cell  membranes  can  be  defined  definitively. 
A  method  previously  developed  in  this  laboratory  for  the  estimate  of  cellular 
pools  of  paraminohippurate  and  glucose  in  the  proximal  tubule  has  been  adapted 
for  the  cortical  collecting  tubule  to  measure  the  K  transport  pool.  Cellular 
radioactivity  is  measured  following  addition  of  an  appropriate  isotope  to 
either  side  of  the  epithelium.   In  order  to  identify  the  fraction  of  the 
intracellular  radioactive  IC""  contained  in  the  transport  pool  as  opposed  to 
that  in  other  non-transport  pools  in  the  tissue,  the  tubule  is  first 
perfused  with  a  potassium- free  solution.  After  the  rate  of  net  K  secretion 
reaches  a  steady  state,  isotope  is  added  to  the  bathing  fluid.  The  tubule 
fluid  is  then  sampled  at  frequent  intervals  until  specific  activity  equilibrium 
obtains.  The  tubule  may  be  removed  during  the  equilibration  period  at 
specified  times  and  the  specific  activity  of  the  cells  compared  to  the  simul- 
taneous specific  activity  of  the  secreted  fluid  in  the  lumen.   If  the  specific 
activity  of  cell  and  luminal  fluid  potassium  is  identical,  all  of  the  cellular 
potassium  must  be  resident  in  a  transport  pool.   If  not,  other  nontransport 
pools  may  be  present.  These  studies  are  currentlj^  in  progress  as  x^ell  as 
others  which  involve  examination  of  the  effect  o 0  a  variety  of  diuretic  agents 
on  the  transport  processes  in  this  portion  of  the  nephron.   With  respect  to 
the  latter,  it  has  been  established  that  acetazolamide,  ethacrynic  acid  and 
amiloride  all  alter  the  potential  difference  and  electrical  resistance  and/or 
the  sodium  flux  in  this  tissue . 

The  proximal  tubule  of  the  mammalian  nephron  reabsorbs  70-80%  of  the 
glomerular  filtrate  under  normal  circumstances.  This  process  is  obviously  of 
considerable  importance  in  the  maintenance  of  hemostasis.   Its  mechanism  is 
the  subject  of  intensive  investigation  in  laboratories  m  this  country  and 
abroad.   It  had  generally  been  assumed  that  sodium  is  actively  reabsorbed  from 
the  lumen  creating  a  favorable  electrical  gradient  for  the  passive  movement  of 
attendant  anion,  primarily  chloride,  out  of  the  tubule  fluid.  A  resultant 
increase  in  osmotic  pressure  in  an  inaccessible  third  compartment  either  bet-ween 
or  closely  adjacent  to  the  blood  surface  of  the  epithelial  cells  is  purported 
to  provide  sufficient  driving  force  for  reabsorption  of  water.  The  isolated 
proximal  nephron  transports  an  isosmotic  salt  solution  from  lumen  to  bath  at 
a  rate  similar  to  that  reported  in  in  vivo  studies.  The  sodium  concentration 
of  luminal  fluid  and  that  of  the  bulk  bathing  solution  is  unchanged  during 
this  process  as  it  is  in  vivo.  Furthermore,  as  in  the  intact  nephron, 
interference  with  net  water  reabsorption  by  introduction  of  a  poorly  absorbable 
osmotically  active  solute  (raffinose)  lowers  luminal  sodium  concentration  to  a 
limiting  value  approximately  30  mEq/L  lower  than  its  concentration  in  the 
bathing  solution,  without  changing  the  osmolality  of  either  solution.  Though 
these  results  are  consistent  with  tlie  view  that  the  primary  driving  force  for 
the  reabsorptive  process  is  active  transport  of  sodium,  this  has  been  questioned 
on  the  basis  of  in  vivo  results  elsewhere.  Crucial  to  an  understanding  of  the 
process  are  precise  estimates  of  the  electrochemical  driving  forces  for  the 
individual  ions,  the  individual  conductivities  and  the  permeability  of  the 
cell  membrane  to  water.   Since  no  measurable  chemical  gradient  for  sodium 
exists  across  the  tubule  wall  under  normal  circumstances  active  sodium 
transport  coupled  with  passive  chloride  reabsorption  requires  an  appropriately 
oriented  electrical  gradient  across  the  tissue.  Initially  a  potential 

2  ^>y 


difference  (20  mV  lumen  negative)  was  reported  in  vivo.  In  recent  years, 
however,  this  has  been  questioned  and  the  observed  potential  attributed  to 
artifacts  in  part  attendant  upon  incorrect  positioning  of  the  exploring  elec- 
trode in  the  tissue  in  vivo.  The  isolated  tubule  afforded  a  unique  opportunity 
to  re-examine  the  question  since  it  is  unnecessary  to  introduce  the  exploring 
electrode  into  the  lumen  by  puncturing  the  epithelial  wall.   In  contrast  to 
the  in  vivo  studies  the  electrode  may  either  be  positioned  directly  in  the 
lumen  through  the  open  end  of  the  tubule  or  placed  in  an  appropriate  bridge 
in  direct  communication  with  lumen  fluid.  Furthermore  virtually  total 
electrical  insulation  of  both  ends  of  the  tubule  has  been  achieved  by  a 
technique  developed  in  this  laboratory  which  employs  a  liquid  dielectric, 
Sylgard  184;,  applied  to  the  tubule  ends.  Using  this  technique  it  has  been 
established  that  a  potential  difference  of  approximately  4.5  millivolts 
(lumen  negative)  is  established  during  sodium  chloride  transport,  unequivocal 
evidence  of  active  sodium  transport.  Although  the  electrical  gradient  on  the 
basis  of  preliminary  calculations  and  studies  appears  sufficient  to  account 
for  passive  coupled  chloride  absorption  further  experiments  are  in  progress 
to  establish  this  with  certainty.  These  involve  direct  measurements  of 
chloride  flux,  potential  difference  and  conductance  under  appropriate  cir- 
cumstances as  well  as  transepithelial  resistance. 

Estimates  of  transepithelial  resistance  are  also  of  considerable  impor- 
tance in  determining  the  anatomical  pathway  for  electrolyte  movement 
across  the  tissue,  that  is,  whether  it  occurs  through  or  between  the  cells  as 
is  considered  by  many.  The  latter  path  would  be  the  case  were  the  trans- 
epithelial resistance  higher  than  the  sum  of  the  individual  transmembrane 
resistances  (i.e.  the  transcellular  resistance).  Measurements  of  resistance 
are  extremely  difficult  in  the  intact  nephron  and  interpretation  of  the 
results  requires  multiple  assumptions  concerning  the  geometry  of  the  tissue, 
the  properties  of  the  individual  barriers,  all  of  which  are  subject  to 
considerable  uncertainty.  Many  of  these  problems  are  obviated  in  the  in  vitro 
preparation  and  thus  far  it  has  been  established  that  the  transepithelial 
resistance  is  extremely  low,  consistent  with  the  high  permeability  of  the 
tubule  to  sodium  and  chloride.  Methods  for  measuring  longitudinal  resistance 
of  the  epithelial  cell  as  xrell  as  transcellular  resistance  of  the  serosal 
membrane  are  presently  being  developed. 

Toad  Bladder; 

The  toad  bladder  is  a  particularly  useful  model  for  examining  the  effects 
of  hormones  and  other  agents  on  water  and  electrolyte  transport  across  oriented 
epithelial  cells.   It  has  been  established  in  this  laboratory  that  vasopressin 
induces  an  increase  in  water  permeability  and  sodium  transport  v^ich  involves 
the  intermediacy  of  the  intracellular  nucleotide,  cyclic-AMP.   The  mechanism 
of  action  of  cyclic-AMP  on  the  processes  of  water  and  sodium  transport  is 
unknown.   Since  the  nucleotide  has  recently  been  shown  to  stimulate  the 
activity  of  a  kinase  which  catalyzes  the  transfer  of  a  labelled  phosphate 
from  ATP  to  an  appropriate  protein  substrate  in  a  number  of  tissues  including 
toad  bladder,  it  is  likely  that  the  isolation  and  characterization  of  a 
membrane  protein,  the  phosphorylation  of  which  is  influenced  by  cyclic-AMP 
and/or  hormone  will  afford  important  information  with  respect  to  the  mechanism 
of  action  of  the  hormone  on  water  and  electrolyte  transport.  Thus  far  we  have 


\ 


confirtned  that  the  toad  bladder  does  in  fact  contain  a  cyclic-AMP  sensitive 
protein  kinase  which  catalyzes  the  phosphorylation  of  histone  but  not  of 
protamine  or  phosvitin.   No  effect  on  the  activity  of  the  kinase  by  anti- 
diuretic hormone  has  been  observed,  however.   We  are  currently  developing 
methods  for  the  isolation  of  toad  bladder  membrane  fractions,  separation  of 
pbosphorylated  constituents  within  the  membrane,  in  preparation  for  the  studies 
outlined  above.   In  addition  a  method  for  the  estimation  of  the  intracellular 
concentration  of  cyclic-AMP  based  on  the  binding  of  the  nucleotide  to  a  muscle 
kinase,  a  method  developed  in  the  Laboratory  of  Biochemical  Genetics,  has 
been  successfully  adapted  for  use  in  the  toad  bladder. 

The  current  studies  differ  from  earlier  attempts  in  this  laboratory  and 
elsewhere  in  which  intact  tissue  had  been  employed  in  an  attempt  to  elucidate 
the  mechanism  of  action  of  hormones.   Pure  epithelial  cells  devoid  of  sur- 
rounding interstitial  tissue  and  muscle,  both  of  which  complicate  inter- 
pretation of  data,  are  now  used.   The  cells  are  scraped  from  intact  tissue 
previously  incubated  in  collagenase  for  a  required  period  of  time.   These 
cells  respond  raetabolically  to  vasopressin,  aldosterone  and  other  agents  as 
does  the  intact  tissue.  Thus  ADH,  for  example,  increases  the  oxidation  of 
I'^C  labelled  glucose  and  pyruvate  in  both  preparations,  evidence  of  the 
viability  of  the  tissue  cells  and  their  potential  for  experimental  manipula- 
tions.  The  preparation  thus  far  has  pemiitted  a  clear  definition  of  the 
mechanism  of  action  of  vasopressin,  aldosterone,  ouabain,  an  inhibitor  of 
sodium  transport,  and  amiloride,  a  diuretic  agent,  on  sodium  transport  in  the 
toad  bladder.  Cells  and/or  intact  tissue  are  incubated  with  one  of  these 
agents  under  conditions  in  which  net  sodium  transport  is  altered.  At  appro- 
priate intervals  pure  epithelial  cell  sheets  are  removed  and  analyzed  for  their 
sodium  content.   In  order  to  define  the  rate  limiting  step  in  transport 
affected  by  the  agent  the  following  simplifying  assumptions  have  been  made: 
the  toad  bladder  epithelial  cell  is  considered  to  be  a  three  compartment 
system;  a  mucosal  solution  from  which  sodium  is  transported  into  the  cell, 
a  cell  transport  pool  of  sodium,  and  a  serosal  solution  into  which  sodium  is 
transported.  Changes  in  the  size  of  the  transport  pool  within  the  cell 
coincident  wich  alterations  in  transport  rate  may  be  interpreted  in  terms  of 
a  primary  effect  on  sodium  movement  from  mucosal  solution  to  cell  or  from  cell 
to  serosal  solution.   Thus  if  sodium  transport  is  accelerated  by  a  hormone  as 
a  result  of  stimulation  of  a  specific  sodium  pump  on  the  blood  surface  of  the 
cell  this  will  be  associated  with  a  decrease  in  the  sodium  content  of  the 
tissue.  Conversely,  were  transport  to  be  accelerated  primarily  by  increasing 
the  entry  of  sodium  across  the  mucosal  permeability  barrier,  cell  sodium 
content  should  increase.   Analogous  studies  employing  the  intact  tissue  in 
this  laboratory  and  elsewhere  have  repeatedly  been  negative,  presumably  a 
consequence  of  complications  introduced  by  heterogeneity  of  the  tissue.   In 
the  present  study,  however,  in  vii  ich  pure  epithelial  cells  were  employed  and 
analyzed,  both  aldosterone  and  vasopressin  significantly  increased  the  sodium 
content  of  the  tissue,  indicating  that  they  must  have  induced  a  clvange  in  the 
permeability  of  the  mucosal  barrier  which  facilitated  entry  of  Na  into  the 
cell.  Ouabain,  on  the  other  hand,  which  decreases  sodium  transport  in  many 
tissues  by  interfering  with  the  putative  sodium  pump  resulted  in  an  expected 
rise  in  cell  sodium  content.   Finally,  amiloride,  a  diuretic  agent  which 
decreases  sodium  reabsorption  in  the  intact  kidney  and  lovrers   net  sodium 
transport  in  the  toad  bladder,  induced  a  significant  fall  in  the  sodium  content 

4  JL76 


clearly  indicative  of  an  effect  of  the  agent  on  the  permeability  of  the  luminal 
surface  to  sodium  rather  than  to  inhibition  of  sodium  pump.  Additional 
studies  directed  at  measuring  changes  in  high  energy  phosphate  donors  and 
acceptors  in  the  cell  were  also  performed.  Although  neither  aldosterone  nor 
vasopressin  altered  the  concentration  of  ATP  in  the  epithelial  cells  at  a 
t-ime  when  sodium  transport  was  accelerated,  both  agents  resulted  in  a  signifi- 
cant decrease  in  the  concentration  of  creatine  phosphate  and  a  rise  in  the 
concentration  of  creatine.   It  has  been  concluded  that  acceleration  of  sodium 
transport  is  the  primary  event  and  the  changes  in  metabolism  secondary,  rather 
than  the  converse  as  certain  other  investigators  have  suggested. 

Other  studies  in  this  laboratory  have  defined  the  defect  in  cardiac 
muscle  function  in  hereditary  cardiomyopathy  of  hamsters.  These  animals, 
though  devoid  of  evidence  of  heart  disease  at  birth  ultimately  develop  fatal 
congestive  heart  failure  characterized  by  peripheral  edema  and  pulmonary  and 
hepatic  congestion.   Cardiac  contractility  in  normal  hamsters  as  in  many 
other  species  is  dependent  upon  two  complementary  phenomena.  The  first,  the 
Bowditch  phenomenon,  is  characterized  by  an  augmentation  in  contractility  at 
high  rates  of  stimulation  (or  heart  rate);  the  other,  the  Woodworth,  by 
augmented  contractility  at  low  rates.  Right  ventricular  muscle  from  hearts 
of  hamsters  in  frank  failure  do  not  possess  the  Bowditch  phenomenon  whereas 
that  from  normal  animals  or  those  hamsters  with  the  trait  prior  to  development 
of  failure  do.  The  absence  of  the  Bowditch  phenomenon  is  a  consequence  of  a 
disease-induced  increase  in  the  uptake  of  calcium  from  the  bathing  medium. 


^-77 


Serial  No.    NHLI-113 

1.  Kidney  &  Electrolyte  Metabolism 

2.  Renal  Mechanisms 

3.  Bethesda,  Maryland 

PHS  -  NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Mechanism  of  salt  and  water  transport  by  proximal 
renal  tubules. 

Previous  Serial  No.:   NHLI-168  and  NHLI-169 

Principal  Investigators:   Maurice  B.  Burg,  M.D. 

Jack  Orloff,  M.D. 
Michael  Lutz,  M.D. 
Dennis  Waring,  Ph.D. 
Michael  Horster,  M.D. 
Jean  Cardinal,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   We  devised  a  method  for  dissecting  individual 
renal  tubules  from  rabbit  kidneys,  keeping  them  alive  in  vitro, 
and  measuring  their  function.   This  is  an  important  advance  over 
previous  methods  in  renal  physiology  (such  as  clearance  and 
micropuncture)  since  it  permits  the  study  of  transport  mechanisms 
in  a  way  not  previously  possible.   At  present  we  are  using  this 
method  to  investigate  the  mechanism  of  salt  and  water  transport 
in  the  proximal  renal  tubule,  a  problem  which  had  not  been 
elucidated  by  previous  techniques  despite  considerable  effort. 

In  our  initial  studies  we  found  that  the  isolated  perfused 
proximal  convoluted  tubules  continued  to  absorb  fluid  from  the 
lumen  at  an  apparently  normal  rate,  when  bathed  in  rabbit  seriom 
and  perfused  with  an  ultrafiltrate  of  the  serum  (Burg,  M.B.  and 
Orloff,  J.,  Amer.  J.  Physiol.  47:2016,  1968).   Na  concentration 
in  the  tubule  fluid  did  not  change  during  reabsorption  under 
these  conditions,  indicating  isotonic  transport,  but  did  decrease 

(by  approximately  20%)  when  raffinose  was  included  in  the  perfusate 
to  limit  water  absorption.   The  transport  mechanism  was  not  able 
to  lower  Na  concentration  further  because  of  the  high  Na  perme- 
ability (10~4  cm  sec~l  from  isotope  flux  measurements)  and 
correspondingly  low  NaCl  reflection  coefficient  (c^NaCl  ~  '^"^^ 

(Kokko,  J.,  Burg,  M.B.,  and  Orloff,  J.,  J.  Clin.  Invest.  50:69, 
1971)  . 


Ay  9 


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3 

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Serial  No.    NHLI-113 

The  major  ions  reabsorbed  from  the  proximal  tubule  lumen  are 
Na  and  CI.   In  order  to  determine  whether  they  are  actively 
transported  it  is  necessary  to  measure  the  electrical  P.D.   The 
results  given  above  concerning  Na  concentration  and  permeability 
are  in  general  agreement  with  those  of  micropuncture .   This  is 
not  true,  however,  of  the  measurement  of  P.D.   Previous  measure- 
ments of  electrical  P.D.  across  the  proximal  tubule  by 
micropuncture  v/ere  conflicting.   Values  of  both  -20  and  zero  mV 
were  reported  by  different  workers,  and  the  conclusion  regarding 
ion  transport  mechanism  depended  on  which  number  was  chosen. 
Across  isolated  perfused  rabbit  proximal  tubules  we  measured  the 
electrical  P.D.  to  be  -4  mV  (lumen  negative)  (Burg,  M.B.,  and 
Orloff,  J.,  Amer.  J.  Physiol.  219:1714,  1970).   When  active  ion 
transport  was  eliminated  by  addition  of  ouabain  the  P.D.  fell 
promptly  to  zero. 

Considering  that  the  P.D.  is  negative  in  the  lumen  and  that 
Na  reabsorption  can  take  place  both  against  a  concentration  and 
electrical  gradient,  Na  transport  m.ust  be  active.   CI  transport 
could  be  passive,  driven  by  the  negative  luminal  P.D.   Evidence 
concerning  the  mechanism  of  chloride  transport  in  proximal  renal 
tubules,  however,  is  also  conflicting.   Various  investigators 
have  considered  that  chloride  transport  may  involve  (1)  passive 
reabsorption,  (2)  active  reabsorption  by  a  neutral  pump  mechanism 
together  with  Na,  and/or  (3)  active  secretion  as  HCl .   Preliminary 
evidence  from  our  laboratory  favors  (1) ,  but  the  further  studies 
listed  below  are  required  for  proof. 

These  new  experimental  techniques  are  also  being  used  to 
investigate  the  following  additional  problems: 

1.  The  pathway  for  salt  transport  through  the  proximal 
tubule  epithelium.   At  present  the  low  electrical  resistance  and 
high  salt  permeability  of  the  tubule  are  generally  attributed  to 
shunting  of  ions  between  the  cells,  although  the  supporting 
evidence  is  meager  (a  preliminary  report  in  Necturus  tubules 
several  years  ago) .   We  intend  to  determine  the  pathways  in 
rabbit  proximal  tubules  by  measuring  transtubular  and  trans- 
cellular  electrical  resistances. 

2.  Cell  communications.   It  seems  likely  that  the  contents 
of  adjacent  tubule  cells  may  be  in  direct  content  through  com- 
munications between  the  cells  (gap  junctions) ,   This  will  be 
tested  directly  by  measuring  the  longitudinal  electrical 
resistance  of  the  tubule  wall. 

3.  The  mechanism  of  action  of  diuretic  drugs.   It  has  been 
difficult,  using  standard  micropuncture  and  clearance  methods,  to 
determine  the  mechanism  of  action  of  the  various  diuretics  on 


^^C 


Serial  No.   NHLI-113 


specific  segments  of  the  kidney  tubule.   Indeed,  it  is  still 
unclear  on  which  tubule  segments  some  of  the  diuretics  act.   The 
direct  and  controlled  study  of  individual  tubule  segments  by  in 
vitro  perfusion  should  help  answer  these  questions.   Electrical 
methods  have  been  chosen  for  initial  screening  since  they  provide 
information  conveniently  and  rapidly  as  evidenced  by  their 
successful  application  to  other  epithelial  tissues.   Ouabain,  the 
one  drug  previously  tested,  caused  a  rapid  depolarization  of  the 
P.D.  in  proximal  tubules.   The  agents  to  be  tested  on  the  proximal 
tubule  are  acetazolamide,  theophylline,  cyclic-AMP,  ethacrynic 
acid,  furosemide,  amiloride,  mersalyl  and  hydrochlorothiazide. 

4.  Permeability  to  macromolecules .   It  has  been  reported 
that  relatively  large  molecules  (MW  >  500)  permeate  the  proximal 
tubules  of  rats  and  Necturi.   We  wish  to  see  if  this  occurs  in 
isolated  perfused  rabbit  proximal  tubules,  and  if  so,  to  define 
the  transport  mechanism.   This  question  is  important  since 
simple  passive  permeability  to  such  large  molecules  requires  much 
larger  openings  through  the  epithelium  than  had  been  previously 
envisioned  and  would  require  re-evaluation  of  our  concepts  of  the 
structure  of  the  epithelium.   Permeability  to  macromoles  (e.g. 
proteins,  dextrans ,  and  other  polysacchrides)  will  be  measured 
from  radioactive  isotope  flux  or  relative  osmotic  efficiency 
(reflection  coefficient  of  Staveman) . 

5.  Effect  of  protein  on  electrolyte  transport  in  proximal 
tubules.   Peritubular  protein  concentration  is  purported  to  be  a 
major  factor  controlling  the  rate  of  electrolyte  transport  in 
proximal  tubules,  and  has  been  reported  (by  others)  to  affect  the 
rate  of  fluid  absorption  in  isolated  perfused  rabbit  proximal 
convoluted  tubules.   We  wish  to  confirm  the  latter  observation 
and  to  determine  the  mechanism  involved.   The  effect  of  protein 
in  the  bath  on  electrolyte  transport  will  be  determined  by  remov- 
ing protein  from  serum  (ultrafiltration) ,  replacing  protein  with 
other  colloids,  or  adding  protein  to  a  high  concentration. 

6.  Measurement  of  electrochemical  potential  difference  in 
kidney  tubules.   Evaluation  of  the  mechanism  of  ion  transport  in 
epithelia  requires  precise  measurement  of  both  the  flows  (net  ion 
and  water  movement)  and  external  forces  (electrochemical  P.D.'s). 
When  identical  solutions  bathe  the  two  sides  of  the  epithelivim 
and  there  is  no  hydrostatic  pressure  difference,  the  external 
force  is  in  general  equal  to  the  electrical  P.D.  and  is  readily 
measured.   It  is  usually  desirable,  however,  to  vary  the  flows  by 
using  non-identical  solutions  on  the  two  sides  of  the  epithelium, 
which  complicates  the  measurement  of  electrochemical  P.D.   Under 
these  conditions  the  electrical  potential  across  the  epitheliiom 
cannot  be  evaluated  without  either  eliminating  or  measuring  the 
liquid  junction  P.D.'s  between  non-identical  solutions  which  are 
in  contact.   The  liquid  junction  P.D.  can  be  more  or  less  elimi- 
nated by  the  use  of  bridges  filled  with  concentrated  KCl,  and 

3  SlS( 


Serial  No.    NHLI-113 

the  residual  uncertainty  of  a  few  millivolts  is  unimportant  in 
such  tissues  as  nerve  and  muscle  where  the  transmembrane  P.D.  and 
resistance  are  high.   In  the  proximal  kidney  tubule,  hov/ever, 
where  the  measui-ed  P.D.'s  are  only  a  few  mV  this  system  is 
unsatisfactory.   The  objective  of  the  study  is  to  develop  satis- 
factory methods  for  evaluating  electrochemical  P.D.  across 
proximal  kidney  tubules  when  non-identical  solutions  are  used. 

In  order  to  resolve  this  pi-oblem  we  are  attempting  to  evaluate 
the  liquid  junction  potentials  more  accurately.   A  more  promising 
approach,  however,  is  to  avoid  the  problem  of  liquid  junction 
potential  entirely  by  the  use  of  the  equivalent  of  the  physical 
chemists'  "cells  without  transference."   This  involves  the  pre- 
paration of  microelectrodes  reversible  to  the  specific  ions 
studied  and  the  direct  reading  of  the  electrochemical  P.D.  between 
two  such  electrodes,  one  in  the  tubule  lumen  and  one  in  the  bath. 
Such  electrodes  reversible  to  CI  and  to  K  have  been  built  and 
tested  by  others  using  liquid  ion  exchangers.   We  are  constructing 
similar  electrodes  for  use  in  isolated  tubules. 

Methods:   The  major  methodological  advances  have  been  in 
electrical  measurements.   Electrical  methods  are  a  powerful  tool 
for  study  of  ion  transport  and  widely  used  in  muscle  and  nerve 
physiology  and  for  studying  such  epithelia  as  frog  skin  and  toad 
bladder.   They  have  been  relatively  little  used  in  kidney 
physiology,  however,  because  of  the  difficulty  of  applying  them 
in  micropuncture .   When  we  discovered  that  Sylgard  184  liquid 
dielectric  will  electrically  seal  the  ends  of  fragments  of  kidney 
tubules,  detailed  electrical  study  of  isolated  kidney  tubules 
became  possible.   The  initial  studies  were  of  electrical  P.D. 

(Burg,  M.B.,  et  al  Amer.  J.  Physiol.  215:788,  1968  and  Burg,  M.B. 
and  Orloff,  J.  Amer.  J.  Physiol.  219:1714,  1970).   Electrical 
resistance  was  first  measured  in  the  cortical  collecting  tubule 

(Helman,  S.,  et  al  Amer.  J.  Physiol.,  in  press).   Measurement  of 
transepithelial  electrical  resistance  in  proximal  tubules  had 
been  a  more  difficult  problem  because  of  their  fragility  and 
because  their  low  resistance  and  short  space  constant  made  it 
necessary  to  isolate  extremely  short  lengths  (approximately  200y) . 
In  the  new  method  which  we  have  developed  the  electrical  resistance 
is  measured  via  the  perfusion  pipet  in  the  tubule  lumen,  passing 
direct  current  through  a  platinum  coating  on  the  surface  of  the 
pipet  and  recording  P.D.  through  the  lumen  of  the  pipet.   Sylgard 
184  is  used  to  insulate  the  ends  of  the  short  length  of  tubule. 
The  P.D.  is  also  measured  at  the  other  end  of  the  tubule.   The 
resistance  is  calculated  using  cable  theory,  from  the  increment 
in  electrical  P.D.  at  the  ends  of  the  tubule  associated  with  the 
electrical  current. 

In  order  to  determine  electrically  whether  there  are  ion 
shunts  between  the  cells  it  is  necessar^^  also  to  measure  the 


Aga. 


Serial  No.   NHLI~113 


transcellular  resistance  in  order  to  see  if  this  is  higher  than 
the  transepithelial  resistance,  as  it  would  be  if  there  is  an 
electrical  leak  between  the  cells.   Transcellular  P.D.  and 
resistance  can  be  measured  using  a  single  micropipet  inserted 
into  a  cell  from  its  luminal  surface.   This  pipet  is  advanced 
into  the  tubule  lumen  by  mounting  it  coaxially  within  the  per- 
fusion pipet.   In  principle  P.D.  and  resistance  can  be  measured 
simultaneously  by  using  a  bridge  circuit  to  pass  current  through 
the  single  pipet.   In  a  preliminary  study  a  stable  transcellular 
P.D.  (approximately  -50  mV)  was  found  using  this  method,  which 
indicates  its  feasibility. 

Longitudinal  resistance  of  the  tubule  wall  was  determined  in 
non-perfused  tubules .   Direct  current  was  passed  through  a  segment 
of  tubule  immersed  in  either  Sylgard  184  (liquid  dielectric  for 
electrical  insulation)  or  seriom  and  the  resistance  measured  using 
a  bridge  circuit. 

Major  Findings: 

1.  Transepithelial  resistance  in  the  proximal  tubule  was 
approximately  7  ohm  cm^,  a  low  value  consistent  with  the  high 
permeability  of  the  tubule.   Changes  in  NaCl  concentration  in  the 
bath  (replacement  with  non-electrolyte)  caused  a  proportionate 
change  in  transepithelial  conductance,  indicating  that  much  or 
all  of  the  electrical  current  is  carried  through  the  tubule  wall 
by  Na  and  CI. 

2.  The  relative  conductance  of  CI  vs.  Na  (i.e.  its  transport 
or  transference  number  across  the  membrane)  was  determined 
potentiometrically .   When  NaCl  concentration  in  the  lumen  was 
lowered  by  replacement  with  raffinose,  the  diffusion  potential 
which  developed  was  small,  indicating  approximately  equal  Na  and 
CI  conductance . 

3.  The  absolute  CI  conductance  (CI  transport  number  divided 
by  total  electrical  resistance)  is  sufficiently  large  so  that 
the  CI  absorption  can  be  accounted  for  by  passive  transport  along 
the  measured  electrical  gradient  (-4  mV) .   However,  the  data  are 
not  sufficiently  precise  to  rule  out  some  active  CI  transport. 

In  order  to  investigate  this  possibility  chloride  transport  will 
be  measured  while  varying  the  chloride  concentration  difference 
across  the  tubule  such  that  both  secretion  and  absorption  occur 
in  different  experiments.   From  the  measured  chloride  electro- 
chemical P.D.  under  these  conditions  it  will  be  possible  to 
calculate  an  independent  value  for  chloride  conductance  and  also 
to  determine  more  precisely  whether  there  is  active  chloride 
transport  or  not. 

4.  Longitudinal  resistance  of  the  tubule  epithelium  of 

5  'Sin 


Serial  No.   NHLI-113 


tubules  itumersed  in  Sylgard  was  approximately  10  ^  ohm  cm   and 
was  identical  whether  or  not  the  tubule  liomen  was  filled  with 
Svlgard  indicating  that  the  current  was  passing  through  the 
tissue  itself.  V!he.n   the  ends  of  the  tubule  were  in  Sylgard  and 
a  short  central  segment  (approximately  lOOy)  was  in  serum  (which 
was  electrically  grounded)  there  was  no  transmission  of  the  P.D. 
from  one  Sylgard  enclosed  end  to  the  other  through  the  portion 
immersed  in  serum..   This  indicates  a  relatively  low  electrical 
resistance  at  the  boundaries  of  the  epithelium,  possibly  corres- 
ponding to  the  low  transepithelial  resistance.   Further  studies, 
including  m.easurement  of  transcellular  resistance,  are  required 
to  distinguish  v;hether  the  low  boundary  resistance  is  in  the  cell 
memibranes  or  between  cells. 

Proposed  Course  of  Project:   Listed  above  in  reference  to  the 
different  areas  studied. 

Honors  and  Awards:   None 

Publications:   Burg,  M.B.  and  Orloff,  J.  Electrical  potential 

difference  across  proximal  convoluted  tubules.  Ari, 
J.  Physiol.  219:1714-1716,  1970. 

Kokko,  J. P.,  Burg,  M.E.  and  Orloff,  J.   Charac- 
teristics of  NaCl  and  water  transport  in  the  renal 
proximal  tubule.   J.  Clin.  Invest.  50:69-76,  1971. 


*a«^ 


Serial  No.  NHLI-114 _ 

1 .  Kidney   &   Electrolyte  "Metabolism 

2.  Renal  Mechanisms 

3.  Bethesda,   Maryland 

PHS    -   NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Study  of  ion  transport  in  renal  cortical 
collecting  tubules. 

Previous  Serial  No.:   NHI-192  and  NHI-193  (Annual  Report  dated 

July  1,  1968  through  June  30,  1969) 

Principal  Investigators:   Maurice  B.  Burg,  M.D. 

Jack  Orloff,  M.D. 
Gustavo  Frindt,  M.D. 
Larry  Stoner,  Ph.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives: 

1.   The  technique  of  perfusion  of  isolated  renal  tubules 
in  vitro,  which  we  developed,  has  proved  especially  useful  for 
elucidating  the  mechanism  of  action  of  the  antidiuretic  hormone 
(ADH)  in  rabbit  cortical  collecting  tubules  (Grantham  et  al , 
Am.  J.  Physiol.  211:255,  1966;  Ganote ,  et  al ,  J.  Cell  Biol.  36: 
355,  1968;  Grantham,  J.J.  and  Orloff,  J.,  J.  Clin.  Invest. 
47:1154,  1968;  Grantham  et  al,  J.  Cell  Biol.  41:562,  1969). 
Most  of  these  studies  were  concerned  with  the  mechanism  by  which 
ADH  increases  water  permeability  in  the  kidney  tissue.   ADH  also 
has  a  second  effect  in  frog  skin  and  toad  bladder,  namely  it 
increases  Na  transport.   Whether  it  has  a  similar  effect  in  kidney 
t\abules  is  uncertain.   Conflicting  results  have  been  reported  in 
clearance  studies.   In  the  single  micropuncture  study  reported, 
ADH  increased  Na  permeability  in  the  papillary  collecting  duct, 
but  had  had  no  effect  on  Na  transport  per  se. 

Previous  results  in  this  laboratory  indicated  that  ADH 
transiently  increased  electrical  P.D.  in  cortical  collecting 
tubules  (Burg  et  al ,  Am.  J.  Physiol.  215:788,  1968)  without  any 
change  in  electrical  resistance  (Helman,  et  al ,  Am.  J.  Physiol., 
in  press) .   This  is  consistent  with  an  increase  of  active  Na 
transport,  but  provides  only  indirect  evidence.   One  objective  of 
the  present  project  is  to  test  directly  the  effect  of  vasopressin 
on  Na  transport  in  the  cortical  collecting  tubule. 


A^C 


Serial  No.   NHLI-114 


2.  We  previously  measured  Na  and  K  transport  in  cortical 
collecting  tubules  and  established  that  Na  is  absorbed  from  the 
lumen  and  K  secreted  into  the  lumen,  both  by  an  active  transport 
process  (Grantham  et  al ,  J.  Clin.  Invest.  48:1915,  1970).   These 
ions  are  most  likely  transported  through  the  tubule  cells.   The 
mass  of  transported  ion  transiently  contained  in  the  cells  in 
the  course  of  transport  is  the  "transport  pool."   From  the  size, 
turnover  rate,  and  specific  activity  of  the  pool  the  cellular 
sites  of  ion  transport  can  be  identified,  the  transport  properties 
of  the  individual  cell  membranes  defined,  and  the  effects  of  drugs 
and  ho2rmones  on  the  latter  elucidated.   We  are  now  attempting  to 
measure  ion  transport  pools,  beginning  with  the  K  transport  pool 
in  cortical  collecting  tubule  cells. 

3.  We  are  also  studying  the  effect  of  various  diuretic 
drugs  on  ion  transport  in  this  segment,  since  the  site  and 
mechanism  of  their  action  was  previously  poorly  understood. 

Methods:   ADH  effect:   electrical  P.D.  was  measured  in  order 
to  detect  whether  added  vasopressin  v/as  having  an  effect.   Na 
transport  was  estimated  from  the  unidirectional  flux  of  ^^Na  from 
lumen  to  bath.   Albumin--'-2^I  flux  from  lumen  to  bath  was  also 
measured,  in  order  to  detect  damaged  tubules  with  non-specific 
leaks . 

Transport  pools:   The  method  previously  developed  in  this 
laboratory  for  measurement  of  cellular  transport  pools  of 
p-aminohippurate  (Tune,  B.M.  and  Burg,  M.B.,  Am.  J.  Physiol.  217: 
1057,  1969),  and  glucose   (Tune,  B.M.  and  Burg,  M.B.,  Am.  J. 
Physiol.,  submitted  for  publication)  in  proximal  tubules  will  be 
adapted  to  study  ions  in  collecting  tubules.   In  this  method 
cellular  radioactivity  (e.g.  '^^K)  is  measured  following  addition 
of  radioisotopes  of  the  transported  siobstances  to  either  side  of 
the  epithelium.   The  principal  problem,  initially,  is  to  identify 
what  fraction  of  the  ^2k  radioactivity  is  contained  in  the 
transport  pool,  as  opposed  to  possible  other  non-transport  pools 
in  the  tissue.   In  order  to  identify  the  transport  pool  the 
tubule  is  perfused  with  a  K-free  solution.   After  a  steady  state 
of  K  secretion  into  the  lumen  is  reached,  42k  is  added  to  the 
bath  and  tubule  fluid  sampled  at  1-2  minute  intervals,       ing 
the  increase  of  K  specific  activity.   The  tubule  is  removed 
during  the  transient  and  specific  activity  of  K  in  the  cells  com- 
pared to  simultaneous  luminal  K  specific  activity.   If  the 
specific  activity  of  cell  and  tubule  fluid  K  is  identical,  all 
of  the  cell  K  is  in  the  transport  pool.   If  not,  other  non- 
transport  K  pools  are  present. 

Major  Findings:  ^^^ 

1.   Na  isotope  flux  from  lumen  to  bath  and  K  isotope  flux 


dS£ 


Serial  No.    NHLI-114 


-12      -2 
from  bath  to  lumen  are  low  (approximately  500  x  10    M  cm 

sec~l)  consistent  with  the  low  rates  of  net  Na  and  K  transport 

previously  measured  and  the  high  electrical  resistance  of  this 

tissue . 

2.  In  most  experiments  addition  of  ADH  (25  yU/ml)  caused  a 
small  (approximately  10  to  20%)  increase  in  sodium  flux  from 
lumen  to  bath,  consistent  with  an  increase  in  active  Na  transport. 
We  are  presently  measuring  the  effect  of  ADH  on  the  back  flux  of 
Na  (bath  to  lumen)  in  order  to  substantiate  this  conclusion. 

3.  Acetazolomide ,  ethacrynic  acid  and  amiloride,  the  first 
drugs  tested  in  collecting  tubules,  all  caused  changes  in  P.D., 
electrical  resistance,  and/or  Na  flux,  indicating  that  it  will  be 
possible  to  determine  the  mechanism  of  their  action  in  this  tissue, 

42 

4.  When   K  was  added  to  the  bath  during  the  steady  state  of 

K  secretion,  there  was  a  gradual  increase  in  specific  activity  of 
K  in  the  lumen  over  several  minutes.   The  delay  in  achievement  of 
specific  activity  equilibrium  is  due  to  mixing  with  the  cell  K 
transport  pool  and  indicates  the  feasibility  of  measuring  the 
transport  pool  with  this  technique. 

Proposed  Course  of  Project: 

1.  To  measure  Na  as  well  as  K  transport  pool  in  this  and 
other  tubule  segments . 

2 .  To  pretreat  rabbits  with  high  and  low  Na  and  K  diets  and 
with  DOCA  prior  to  study  in  order  to  investigate  the  phenomenon 
of  "adaptation"  to  K  (i.e.  why  more  of  a  K  load  is  excreted  by 
the  kidney  when  K  intake  is  high)  and  the  mechanism  of  action  of 
the  adrenal  mineralocorticoids  on  the  renal  tubule . 

3.  To  voltage  clamp  collecting  tubules  at  different  P.D.'s 
in  order  to  determine  the  relationship  between  P.D.  and  Na  and  K 
transport. 

Honors  and  Awards :   None 

Publications:   Grantham,  J.J.,  Maurice  B.  Burg  and  Jack  Orloff. 
The  nature  of  transtubular  Na  and  K  transport  in 
isolated  rabbit  renal  collecting  tubules.   J.  Clin. 
Invest.  49:1815-1826,  1970. 

Helman,  S.I.,  J.  J.  Grantham,  and  M.  B.  Burg. 
Effect  of  vasopressin  on  electrical  resistance  of 
renal  cortical  collecting  tubules.   Am.  J.  Physiol. 
(In  press) . 

3  Mr 


Serial  No.    NHLI-115 


1.  Lab.    Kidney  &  Elec .   Metabolism 

2.  Electrolyte  Transport 

3.  Bethesda,    Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   A  study  of  the  mechanism  of  stimulation  of  sodium  transport 

by  vasopressin  and  by  aldosterone  by  determining  their  effect 
on  the  electrolyte  content  and  energy  metabolism  of  the 
epithelial  cells  of  the  toad  urinary  bladder 

Previous  Serial  Number:  None 

Principal  Investigators:   Joseph  Handler,  M.D. 

Jack  Orlcff,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Vasopressin  and  aldosterone  increase  the  rate  of  active 
sodium  transport  by  the  toad  urinary  bladder.  Experimental  studies  intended 
to  define  the  rate  limiting  step  in  transport  that  is  accelerated  by  each 
hormone  have  usually  made  the  reasonable  and  simplifying  assumption  that 
the  toad  bladder  epithelial  cells  can  be  considered  as  a  three  compartment 
system,  a  mucosal  solution  from  which  sodium  is  transported,  a  cell  transport 
pool  of  sodium,  and  a  serosal  solution  into  which  sodium  is  transported. 
Changes  in  the  size  of  the  cell  transport  pool  coincident  with  changes  in 
the  transport  rate  can  be  interpreted  in  terms  of  an  effect  on  sodium 
movement  from  mucosal  solution  to  cell  or  from  cell  to  serosal  solution. 
Although  numerous  experiments  have  been  performed,  none  has  been  successful. 
In  large  part,  earlier  experimental  design  and  interpretation  have  been 
handicapped  by  the  heterogeneous  nature  of  the  toad  bladder,  which  consists 
of  a  single  layer  of  transporting  cells  supported  on  a  much  thicker  layer 
of  connective  tissue,  blood  vessels  and  smooth  muscle  bundles.   In  this  study, 
a  technique  for  the  preparation  of  epithelial  cells  free  of  supporting 
tissue  is  developed  and  used  to  identify  the  cell  surface  at  which  the  rate 
limiting  step  affected  by  aldosterone  and  vasopressin  is  located.   In  addition, 
the  transporting  cells  are  examined  for  an  effect  of  the  hormones  on  the 
concentration  of  certain  high  energy  compounds  in  order  to  clarify  the 
relationship  between  metabolic  energy  utilization  and  sodium  transport. 

Methods  Employed:   In  paired  experiments  epithelial  cells  are  scraped 
off  intact  collagenase  treated  bladders  and  incubated  with  no  addition 
(control)  or  with  vasopressin  or  aldosterone  or  an  inhibitor  of  transport, 
amiloride,  or  ouabain.  The  viability  of  the  cells  and  their  responsiveness 
to  the  hormones  or  inhibitors  is  verified  by  measuring  the  rate  of  oxidation 

1  ^8 


Serial  No.   NHLI-115 


of  ^"^C  labelled  glucose  or  pyruvate.   Cells  are  collected  for  measurement  of 

electrolytes  by  centrifuging  the  cells  at  a  time  corresponding  to  a  steady 

state  stimulation  or  inhibition  of  sodium  transport.   ^^C-inulin  is  used  to 

estimate  extracellular  water.   Cells  are  collected  for  measurement  of  labile 

high  energy  compounds  by  freezing  in  liquid  nitrogen.   The  compounds  are  I 

extracted  into  perchloric  acid  and  assayed  using  highly  sensitive  fluorometric 

techniques • 

Major  Findings:   The  oxidative  metabolism  of  the  epithelial  cells  is 
affected  by  vasopressin,  aldosterone,  amiloride,  and  ouabain  as  expected  from 
the  effects  of  these  agents  on  sodium  transport  and  on  the  oxidative  metabolism 
of  the  intact  bladder.   Vasopressin  and  aldosterone,  at  a  time  when  they 
stimulate  sodium  transport,  each  increase  the  sodium  content  of  the  cells. 
At  a  time  when  they  inhibit  sodium  transport,  ouabain  causes  an  increase  in 
the  sodium  content  of  the  cells  and  amiloride  a  decrease.   These  results 
are  interpreted  as  indicating  that  each  hormone  stimulates  sodium  transport 
by  increasing  the  rate  at  which  sodium  enters  the  cell  from  the  mucosal 
solution.   Ouabain  inhibits  the  movement  of  sodium  from  the  cell  to  the 
serosal  solution,  and  amiloride  inhibits  the  movement  of  sodium  from  the 
mucosal  solution  into  the  cell.  Aldosterone  and  vasopressin  do  not  change 
the  ATP  level  in  cells  significantly,  but  each  hormone  causes  a  marked  fall 
in  the  concentration  of  phosphocreatine .   This  effect  is  interpreted  as 
indicating  that  each  hormone  primarily  stimulates  sodium  transport,  the 
increase  in  metabolism  occurring  secondarily. 

Proposed  Course  of  Project:   Project  is  completed. 

Honors  and  Awards:   None 

Publications:   None 


'A9f 


Serial  No.         NHLI-116 

1.  Lab.   Kidney  &  Elec .   MeLabolism 

2.  Electrolyte  Transport 

3.  Bethesda,   Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  A  study  of  the  effect  of  vasopressin  and  certain  other 

hormones  and  drugs  on  the  concentration  of  adenosine  3',5'- 
monophosphate  in  the  epithelial  cells  of  the  toad  urinary 
bladder 

Previous  Serial  Number: 

Principal  Investigators:   Jeffrey  Stoff,  M.D. 

Joseph  Handler,  M.D. 
Jack  Orloff,  M.D. 

Other  Investigators:  None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   A  considerable  amount  of  evidence  has  been  gathered  in  this 
laboratory  indicating  that  an  early  step  in  the  action  of  vasopressin  on 
mammalian  kidney  and  toad  urinary  bladder  is  stimulation  of  the  enzyme  adenyl 
cj'clase,  resulting  in  increased  production  of  adenosine  3' ,5 '-monophosphate 
(cyclic-AMP)  and  that  the  ensuing  accumulation  of  cyclic-AMP  within  the  cell 
elicits  the  permeability  and  transport  changes  characteristic  of  the  effect 
of  the  hormone.   In  previous  studies  the  concentration  of  cyclic-AMP  in  the 
toad  urinary  bladder  was  measured  in  the  intact  bladder  v^ich  is  a  hetero- 
geneous tissue  composed  of  smooth  muscle,  blood  vessels,  and  mesothelial 
cells,  as  well  as  the  epithelial  cells  that  determine  permeability  and 
respond  to  vasopressin.   In  this  study,  the  epithelial  cells  will  be  isolated 
and  their  content  of  cyclic-AMP  measured,  utilizing  a  new  and  sensitive 
technique.   In  addition,  the  effect  of  a  number  of  other  hormones  and  certain 
drugs  which  modify  the  response  to  vasopressin  will  be  examined  for  their 
effect  on  the  cyclic-AMP  content  of  the  epithelial  cells. 

Methods  Employed:   Preliminary  experiments  indicate  that  reproducible 
results  are  obtained  by  incubating  the  intact  bladder  with  vasopressin  and 
then  rapidly  scraping  off  the  mucosal  epithelial  cells  with  the  edge  of  a 
glass  microscope  slide.   The  cells  are  quickly  frozen  in  liquid  nitrogen  and 
extracted  in  trichloracetic  acid  containing  tracer  amounts  of  tritium  labelled 
cyclic-AMP  for  measurement  of  recovery  which  is  usually  60  to  757o .  The 
trichloroacetic  acid  is  removed  by  cation  exchange  chromatography  and  the 
samples  lyophilized  and  taken  up  in  a  small  volume  of  water.  C^yalic-AMP  is 
measured  using  the  method  of  Oilman,  which  depends  upon  displacement  of 
radioactive  cyclic-AMP  from  high  affinity  binding  sites  on  an  enzyme  isolated 

*  1  •  Af<3 


Serial  No.  NHLI-116 


from  beef  muscle.   The  specificity  and  sensitivity  of  the  assay  are  suitable 
for  the  purposes  of  this  study,  permitting  accurate  measurement  of  picogram 
quantities  of  cyclic -AMP. 

Major  Findings:   Depending  upon  conditions  prior  to  separation,  the 
epithelial  cells  contain  5  to  20  picomoles  per  milligram  of  protein  (about 
2  X  10"6  moles  per  liter  of  cell  water) •  Fifteen  minutes  of  incubation  with 
vasopressin  causes  an  increase  of  approximately  507=  over  the  concentration 
in  paired  control  tissue. 

Proposed  Course  of  Project:   The  time  course  for  the  effect  of  vasopressin 
will  be  established  as  well  as  a  concentration  that  elicits  a  reproducible 
submaximal  response.  Experiments  will  then  be  designed  to  establish  directly 
the  effects  on  cell  cyclic-AMP  levels  of  such  agents  as  prostaglandins, 
catecholamines,  adrenal  steroid  hormones,  and  chlorpropamide,  which  have  been 
shown  to  alter  the  permeability  and  transport  response  to  vasopressin. 

Honors  and  Awards:   None 

Publications:      None 


^f/ 


Serial  No.   NHLI-117 

1.  Lab.  Kidney  &  Elec .  Metabolism 

2 .  Electrolyte  Transport 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  A  study  of  cyclic-AMP  dependent  protein  kinase  in  the  toad 
urinary  bladder 

Previous  Serial  Number:   None 

Principal  Investigators:   Rodney  Omachi,  M.D. 

Joseph  Handler,  M.D. 
Jack  Orloff,  M.D. 

Other  Investigators:   None 

Cooperating  Units:  None 

Project  Description: 

Objectives:  Previous  v7ork  in  this  laboratory  has  established  the  thesis 
that  vasopressin  alters  the  permeability  and  transport  properties  of  the 
toad  urinary  bladder  by  stimulation  of  the  production,  and  thus  the  accumula- 
tion within  the  responsive  cells  of  adenosine  3' ,5 '-monophosphate  (cyclic-AMP), 
Recent  repbrts  have  described  the  presence  in  many  animal  tissues  of  a  cyclic- 
AMP  dependent  enzyme,  a  protein  kinase  that  catalyzes  the  phosphorylation  of 
certain  proteins,  using  the  7-phosphate  of  ATP  as  phosphate  donor.   In  some 
tissues  in  which  the  phosphorylated  protein  is  an  enzyme,  it  has  been  shown 
that  the  activity  of  the  enzyme  is  markedly  altered  by  phosphorylation  and 
that  this  change  in  activity  can  explain  some  of  the  physiological  changes 
that  are  evoked  in  the  tissue  by  the  hormone.   This  study  is  intended  to 
characterize  cyclic-AMP  dependent  protein  kinase  activity  in  the  toad  urinary 
bladder  so  that  its  activity  can  be  assayed  after  experimental  manipulation 
of  the  bladder,  and  to  identify  the  protein  substrate(s)  for  the  enzjmie  in 
the  tissue.  Presumably  the  protein  substrate(s)  for  the  enzyme  has  a  key  role 
in  the  permeability  and  transport  response  to  vasopressin. 

Methods  Employed:   Epithelial  cells  (the  vasopressin  sensitive  portion 
of  the  toad  urinary  bladder)  are  scraped  from  collagenase  treated  bladders. 
After  exposure  to  vasopressin  the  cells  and  paired  control  tissue  are  frozen 
and  ground  to  a  fine  powder  in  liquid  nitrogen,  transferred  to  phosphate 
or  Tris  buffered  extraction  solution  and  gently  homogenized  with  a  glass 
homogenizer.  Activity  is  assayed  in  various  centrifugal  fractions  by 
incubation  with  p32-y-ATP,  ±  protein  substrate,  ±  cyclic-AMP.   The  reaction 
is  terminated  by  adding  cold  87o  trichloroacetic  acid  and  the  phosphorylated 
protein  trapped  and  then  washed  on  a  millipore  filter.   P^^  on  the  filter 
is  determined  with  liquid  scintillation  counting. 


Jlfa 


Serial  No.  NHLI-117 


Major  Findings:  Protein  kinase  activity  is  present  in  the  20,000  x  G 
particulate  fraction  and  the  20,000  x  G  and  100,000  x  G  supernatant  fractions. 
Cyciic-Al'lP  stimulates  activity  two  to  four  times  when  histone  is  the  substrate, 
fhere  is  little  or  no  stimulation  by  cyclic-AMP  when  casein,  protamine,  or 
phosvitin  are  used  as  substrate,  although  these  proteins  are  phosphorylated . 
Although  cells  incubated  with  vasopressin  for  5  to  30  minutes  are  known  to 
respond  to  the  hormone,  extracts  from  these  cells  have  the  same  cyclic-AMP 
independent  and  dependent  protein  kinase  activity  as  extracts  from  paired 
control  cells.  Aldosterone  which  enhances  the  response  of  the  toad  bladder 
to  cyclic-AMP,  is  also  xd.thout  effect  on  cyclic-AMP  dependent  or  independent 
protein  kinase  activity. 

Proposed  Course  of  Project:   Recent  reports  indicate  that  the  effect  of 
cyclic-AMP  on  protein  kinase  activity  is  rapidly  reversible.   This  may  explain 
the  similarity  of  the  activity  found  in  extracts  prepared  from  vasopressin 
and  control  tissues,  and  this  approach  will  not  be  pursued  further.   Cyclic- 
AMP  dependent  protein  kinase  of  high  specific  activity  will  be  prepared  from 
toad  bladder  epithelial  cells  using  techniques  that  have  been  successful  in 
other  tissues.  Proteins  and  other  materials  from  the  epithelial  cells  that 
are  phosphorylated  by  the  enzyme  in  the  presence  of  cyclic-AMP  will  be 
isolated  and  characterized. 

Honors  and  Awards:   None 

Publications:   None 


SifS 


Serial  No.     NHLI-118 


1.  Lab.    Kidney  &  Elec  .   Metabolism 

2.  Membrane  Metabolism 

3.  Bethesda,   Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   A  study  of  the  effect  of  prostaglandin  E]^  and  7-oxa-13- 
prostynoic  acid  on  the  sodium  transport  and  permeability 
properties  of  the  toad  urinary  bladder 

Previous  Serial  Number:  None 

Principal  Investigators:   William  C.  Albert,  M.D. 

Joseph  S.  Handler,  M.D. 
Jack  Orloff,  M.D. 

Other  Investigators:  None 

Cooperating  Units:   None 

Project  Description: 

Objectives:  The   response  of  the  urinary  bladder  of  the  toad  to 
vasopressin  is  characterized  by  an  increase  in  the  rate  of  active  sodium 
transport  and  by  an  increase  in  permeability  to  water.  Vasopressin 
elicits  this  response  by  stimulating  the  formation  and  thus  the  intra- 
cellular accumulation  of  cyclic-AMP.  Prostaglandins  are  naturally 
occurring  fatty  acids  that  in  low  concentrations  have  been  shown  to  have 
a  variety  of  effects  in  many  tissues.   Previous  studies  in  this  laboratory 
indicated  that  prostaglandin  Ei  (PGEi)  inhibits  the  water  permeability 
response  of  the  toad  bladder  to  vasopressin  but  not  the  water  permeability 
response  to  cyclic-AMP,  an  effect  interpreted  as  inhibition  of  the  effect 
of  vasopressin  on  adenyl  cyclase,  the  enzyme  that  catalyzes  the  formation 
of  cyclic-AMP.   Other  laboratories  have  confirmed  these  observations  while 
observing  no  effect  of  PGEi  on  the  sodium  transport  response  to  vasopressin. 
This  XTOuld  imply  that  there  are  two  different  vasopressin-responsive 
adenyl  cyclase  systems  in  the  toad  bladder,  only  one  (that  associated  with 
the  water  permeability  response)  sensitive  to  the  inhibitory  effects  of 
PGEjL*   This  study  is  intended  to  explore  the  effect  of  prostaglandins 
on  the  stimulation  of  sodium  transport  by  the  bladder  and  to  test  the 
effect  of  7-oxa-13-prostyTioic  acid,  a  newly  synthesized  analog  of  prosta- 
glandin that  has  been  reported  to  be  a  competitive  inhibitor  of  PGE-j^  in  the 
ovary  and  in  smooth  muscle. 

Methods  Employed:   Sodium  transport  is  measured  by  the  short-circuit 
current  technique  and  water  permeability  by  measuring  the  rate  ^  water 
flow  from  a  dilute  solution  bathing  the  mucosal  surface  to  Ringer's 
solution  bathing  the  serosal  surface  of  the  bladder.  Experiments  are 
designed  to  use  paired  tissue  from  the  same  toad. 

1  Aji/. 


Serial  No.   NHLI-118 


Major  Findings:   10"°  M  PGEi  frequently  stimulatE  s  sodium  transport 
slightly.  Despite  this  stimulatory  effect,  PGE]^  inhibits  the  submaximal 
stimulation  of  sodium  transport  elicited  by  low  concentrations  of  vasopressin, 
and  has  no  effect  on  comparable  stimulation  of  sodium  transport  by  cyclic-AMP. 
A  similar  pattern  of  inhibition  of  the  submaximal  water  permeability  response 
to  vasopressin  and  no  inhibition  of  the  response  to  cyclic-AMP  was  again 
obtained.   Thus,  these  studies  do  not  support  the  suggestion  that  only  the 
adenyl  cyclase  mediating  the  water  permeability  response  to  vasopressin  is 
inhibited  by  PGE]^.   7-oxa-prostynoic  acid  inhibits  sodium  transport  in  the 
concentration  (10"^M)  used  by  others  to  study  its  effects  on  intact  tissues. 
8  X  10~7  M  7-oxa-13-prostynoic  acid,  a  concentration  that  does  not  alter  the 
sodium  transport  rate,  does  not  alter  the  inhibitory  effect  on  the 
sodium  transport  or  water  permeability  response  to  vasopressin  of  a 
300-fold  lower  concentration  of  PGE]^,  indicating  that  it  is  probably  not  a 
competitive  inhibitor  of  PGE^  in  this  preparation.  The  effect  of  higher 
concentrations  of  7-oxa-13-pro£tynoic  acid  on  sodium  transport  by  the 
bladder  raises  the  possibility  that  the  inhibitory  effect  on  the  response 
to  PGE^  observed  in  other  tissues  is  non-specific. 

Proposed  Course  of  Project:  The  effect  of  PGE]^  on  the  concentration  of 
cyclic-AMP  in  the  epithelial  cells  of  the  toad  bladder  will  be  measured  as 
well  as  its  effect  on  the  changes  in  cell  cyclic-AMP  levels  elicited  by 
vasopressin. 

Honors  and  Awards:   None 

Publications:   None 


Afr 


Serial  No  ■_.NHTJ-1 19 


1.  Kidney  &  Electrolyte  Metabolism 

2.  Electrolyte  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  Volume  regulation  in  duck  erythrocytes 

Previous  Serial  Number:  NHLI-176 

Principal  Investigator:   Floyd  M.  Kregenow  ,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Previous  studies  in  this  laboratory  have  shown  that  duck 
erythrocytes  are  capable  of  returning  to  their  original  volume  in  a  hjrpotonic 
medium  after  first  swelling.   This  capability  requires  that  duck  erythrocytes 
possess  a  "volume  controlling  mechanism"  that  is  sensitive  to  some  cellular 
parameter  associated  with  cell  size.   The  previous  annual  report  (1970)  also 
indicated  that  duck  erythrocytes  can  regulate  their  volume  in  hypertonic 
medium  after  first  shrinking.  Although  this  response  had  not  been  characterized 
completely,  it  was  apparent  that  it  also  required  a  "volume  controlling 
mechanism." 

(I)  The  cellular  changes  and  the  nature  of  the  membrane  events 
associated  with  the  volume  adjustment  in  hypertonic  media  were  first  conqiared 
to  those  in  hypotonic  media.   This  analysis  facilitated  a  comparison  of  the 
operation  of  the  "volume  controlling  mechanism"  in  two  separate  environments 
during  which  markedly  different  changes  in  cellular  content  developed.   The 
changes  in  cell  volume  in  an  isotonic  medium,  induced  by  the  addition  or 
removal  of  norepinephrine  (See  Annual  Report,  1969),  were  then  examined  to 
determine  whether  a  similar  mechanism  was  operative  here. 

(II)  This  comparison  involved  the  use  of  Ouabain,  a  cardiac  glycoside, 
which  specifically  inhibits  the  Na-K  exchange  pump  mechanism  in  mammalian 
erythrocytes  and  is  known  to  inhibit  the  active  transport  of  Na  and  K  in  duck 
erythrocytes.   This  agent  was  used  to  determine  whether  the  "volume  controlling 
mechanism"  and  the  classical  Na-K  exchange  pump  mechanism  were  interrelated. 

(III)  A  calculation  of  the  quantity  of  electrolytes  and  H2O  lost  during 
the  volume  regulatory  phase  (previously  called  the  osmoregulatory  phase)  in 
hypotonic  media  supported  the  concept  that  the  changes  in  cell  size  were 

the  result  ot  a  nearly  isosmotic  adjustment  in  cell  water.  A  similar  cal- 
culation during  the  hypertonic  volume  regulatory  phase,  however,  failed  to 
support  this  concept  as  the  quantity  of  electrolyte  gained  was  in  excess  of 

1  ^6 


Serial  No.    NHLI-119 

the  quantity  required  for  amount  of  water  accumulated.  A  direct  attempt  was 
therefore  made  to  establish  whether  the  changes  in  cell  size  brought  about 
by  the  "volume  controlling  mechanism"  were  examples  of  "isosmotic  intra- 
cellular regulation".   The  activity  of  water  in  the  intracellular  and  extra- 
cellular phase  was  compared  before,  during,  and  after  the  adjustments  in 
cell  size  by  noting  the  melting  point  of  a  microscopic  aliquot  of  cells  and 
medium. 

(IV)   Several  cellular  components  were  examined  during  the  adjustments  in 
cell  volume  to  establish  which  cellular  parameter  was  regulating  cell  size  by 
controlling  the  activity  of  the  "volume  controlling  mechanism," 

Major  Findings:   lA.  As  reported  previously  (Annual  Report,  1970)  the 
response  of  duck  erythrocytes  in  hj'pertonic  media  can  be  divided  into  two 
phases;  an  initial  rapid  phase  of  osmotic  shrinkage  and  a  more  prolonged 
volume  regulatory  phase  in  which  the  cells  swell  until  they  reach  a  new 
steady  state  volume.   The  hormone,  norepinephrine,  need  not  be  present  in  the 
bathing  medium,  as  was  originally  thought,  for  the  readjustment  in  cell 
volume  to  occur.   Under  optimal  conditions,  duck  erythrocytes  incubated  in  a 
hypertonic  solution  without  norepinephrine  and  with  10"^  M  propranolol,  a 
potent  inhibitor  of  the  norepinephrine  response,  swell  after  first  shrinking 
and  approach  their  lower  steady  state, isotonic  volume.   Optimal  conditions 
include  an  increase  in  the  [kJo  from  a  "normal"  value  of  2.5  mM/L  to  15  mM/L 
and  a  hypertonic  solution  whose  osmolality  does  not  exceed  the  osmolality 
of  an  isosmotic  solution  by  more  than  110  mosmoles .   In  contrast  there  are  no 
extracellular  limitations  to  volume  adjustment  in  hypotonic  media  other  than 
a  bathing  medium  so  dilute  that  hemolysis  occurs.   Provided  optimal 
conditions  are  present,  an  increase  in  the  tonicity  of  the  bathing  medium 
initiates  a  response  which  is  just  sufficient  to  return  the  cell  to  its 
original  volume.  At  a  given  tonicity  the  relationship  between  an  increase  in 
the  extracellular  potassium  concentration  and  the  increase  in  cell  volume  is 
a  gradual  one,  increasing  from  a  [k]o  of  2.5  in  mM/L  to  a  value  of  15  mM/L, 
where  a  maximal  response  is  obtained.   The  increase  in  cell  volume  is  always 
accompanied  by  a  gain  in  KCl  which  is  more  than  sufficient  to  explain  the 
changes  in  cell  volume  if  the  cells  remain  in  osmotic  equilibrium  with  their 
environment.   There  is  also  a  much  smaller  initial  gain  in  Na  content  followed 
by  a  decrease  but  not  to  original  levels.   The  changes  in  Na  and  K  are  initially 
associated  with  a  5-10-fold  increase  in  Na  and  K  influx  and  Na24  and  K'^2  loss 
from  the  cells.  The  fluxes  return  toward  normal  as  the  cells  swell  and  reach 
their  original  volume.   The  increase  in  Na  permeability  is  largely  ouabain 
insensitive  as  is  the  increase  in  K  permeability.   The  generalized  increase 
in  permeability  is  associated  with  the  active  accumulation  of  potassium  (the 
gain  in  potassium  occurs  against  an  electrochemical  gradient)  .   If  duck  red 
cells  are  incubated  in  a  hypertonic  solution  under  conditions  where  they 
remain  shrunken  (LK]o  =2.5  mM/L),  the  same  generalized  increase  in  perme- 
ability develops  which,  however,  persists  and  is  not  associated  with  the  active 
accumulation  of  potassium. 

The  marked  similarity  between  the  cellular  and  membrane  changes  reported 
here  and  those  seen  previously  when  cells  are  stimulated  in  an  isotonic  medium 

2  A<r7 


serial  No.    NHLI-119 

by  the  hormone  norepinephrine  (See  Annual  Report,  1970)  is  interpreted  as 
indicating  that  an  identical  "volume  controlling  mechanism"  produces  the  latter 
response.   Tiiis  is  taken  as  evidence  that  the  "volume  controlling  mechanism" 
can  also  regulate  cell  size  in  an  isotonic  medium  under  appropriate  circum- 
stances. The  similarities  between  the  action  of  norepinephrine  and 
hypertonicity  suggest  that  both  pertubations  produce  an  identical  cellular 
change  (the  norepinephrine  effect  is  presumably  mediated  through  cyclic-^^MF) 
which  in  turn  initiates  the  regulatory  mechanism.  An   laternative  explanation 
is  that  hypertonicity  can  regulate  adenyl  cyclase  activity.   The  marked 
dissimilarities  between  the  cellular  and  membrane  changes  during  the  hypertonic 
and  hypotonic  volume  regulatory  phase  is  interpreted  as  indicating  the  effector 
portion  of  the  "volume  controlling  mechanism"  is  different  in  the  two  responses. 
No  hypothesis  can  yet  be  presented  to  explain  all  of  the  changes  during  the 
hypertonic  volume  regulatory  phase.   There  are  numerous  parallels  however  on 
the  one  hand,  between  "facilitated  diffusion"  of  organic  solutes  and  the 
translocation  of  potassium  at  a  [KIq  of  2.5  mM/L  and  on  the  other  hand  besides 
the  obvious  idfficulty  of  applying  the  "pi.mip-leak"  hypothesis  to  each, 

IB.   Hie  response  of  enlarged  duck  erythrocytes  (which  had  been  incubated 
in  an  isotonic  bathing  medium  with  a  LK"'o  °^  ^^   niM/L  and  10"^  M/L  of 
norepinephrine)  was  followed  once  the  factors  responsible  for  the  cell 
enlargement  (an  elevated  [KIq  and  norepinephrine)  were  removed.   Experimentally 
this  was  accomplished  by  washing  norepinephrine  treated  enlarged  cells  in  an 
ice-cold,  norepinephrine -free  isotonic  solution  ([kIq  =2.5  mM/L)  and  then 
incubating  them  for  90  minutes  in  a  solution  identical  to  the  wash  solution, 
but  with  10"^  M  Inderol.  Cells  treated  in  this  fashion  shrunk  rapidly  and 
approached  the  lower  steady  state  isotonic  volume  within  90  minutes.  This 
response  is  identical  to  that  seen  during  the  I..3T)otonic  volume  regulatory 
phase  and  is  comparable  to  the  response  of  cells  in  a  hypotonic  solution  of 
approximately  240  mosmoles .  Potassium  and  chloride  are  the  major  electrolytes 
lost  during  the  response.   This  loss  is  sufficient  to  explain  the  changes  in 
cell  size  if  the  cells  remain  in  osmotic  equilibrium  with  their  environment. 
The  alteration  in  cellular  K  content  is  associated  with  a  large  transient 
increase  in  K  efflux  and  either  no  or  a  slight  change  in  K  influx.  Raising 
the  I  KHq  of  an  isotonic  solution  to  107  mM/L  prevents  the  cells  from  shrinking. 
Initially  prevention  is  associated  with  an  enormous  increase  in  K  influx  and 
either  no  or  a  slight  change  in  the  already  accentuated  increase  in  K  efflux. 

ITie  marked  similarity  between  the  cellular  and  membrane  changes  seen 
during  the  hypotonic  volume  regulatory  phase  and  those  reported  here  is 
interpreted  as  indicating  that  an  identical  "volume  controlling  mechanism" 
produces  the  latter  response.   This  is  taken  as  further  evidence  that  the 
"volume  controlling  mechanism"  can  regulate  cell  size  in  an  isotonic  medium. 
It  also  supports  the  concept  that  the  changes  in  cell  size  respond  to  differ- 
ences in  a  cellular  parameter  rather  than  an  extracellular  factor.   The  changes 
in  cell  potassium  and  permeability  reported  here,  like  those  that  occur  during 
the  hjrpotonic  volume  regulatory  phase  can  be  explained  best  by  jiostulating 
that  there  is  a  transient  increase  in  the  diffusional  pathways  for  potassium. 

II.  Ouabain  at  a  concentration  of  10 "-^  or  10"^+  does  not  significantly 

3  A9e 


Serial  No.  NHLI-119 

alter  the  volume  of  control  cells  nor  the  changes  in  cell  volume  that  develop 
during  either  the  hypotonic  (1)  or  hypertonic  (2)  volume  regulatory  phase  or 
upon  the  addition  (3)  or  removal  (4)  (See  Section  lA)  of  norepinephrine  from 
an  isotonic  medium.   During  the  90-minute  incubation  period,  during  which 
these  experiments  were  performed,  ouabain  does  alter  the  Na  and  K  content 
in  each  group  of  cells.   In  (1)  and  (4)  the  gain  in  Na  (-6  mM)  and  loss  of  K 
(-6  mM)  is  not  significantly  different  from  that  of  control  cells.   The 
ouabain  induced  changes  in  the  cation  content  of  norepinephrine  treated  cells 
(3)  has  been  reported  previously  (Annual  Report,  1970)  .   In  a  435  mosmolar 
hypertonic  solution  (4)  the  cells  gain  approximately  12  mM  Na  and  lose  approxi- 
mately 12  mM  K. 

These  findings  indicate  that  the  "volume  controlling  mechanism"  can 
operate  independently  of  the  classical  Na  and  K  exchange  pump  mechanism  -  at 
least  in  the  sense  that  the  changes  in  cell  volume  produced  by  the  former  are 
unimpaired  when  the  latter  is  inhibited.   The  changes  in  the  Na  and  K  content 
of  ouabain  treated  cells  in  (4)  indicate  that  in  this  condition  the  Na  and  K 
exchange  mechanism  operates  normally  more  rapid  than  usual  to  convert  a  gain 
in  cell  Na  to  one  in  potassium.   In  the  presence  of  ouabain,  the  "volume 
controlling  mechanism"  simply  utilizes  NaCl  instead  of  KCl  as  the  major 
intracellular  osmotic  substance. 

III.  The  melting  point  of  a  microscopic  aliquot  of  medium  and  hemolyzed 
frozen-thawed  cells  was  analyzed  on  a  nanoliter  osmometer.   Within  the  error 
of  the  measurement  (3-5  mosmoles) ,  the  osmolality  of  the  medium  and  cells 
remained  identical,  before,  during,  and  after  the  changes  in  cell  size  that 
develop  in  all  four  of  the  experimental  conditions  described  in  Section  II. 

These  findings  indicate  that  duck  erythrocytes  are  at  osmotic  equilibrium 
with  their  environment  normally  and  as  they  vary  their  volume  in  all  four 
experimental  conditions.   Volume  adjustments  under  these  circumstances  are 
therefore,  by  direct  analysis,  examples  of  "isosmotic  intracellular  regulation" 
and  comparable  to  the  kind  of  cellular  adaption  seen  more  commonly  in  inverte- 
brate animal  cells. 

IV.  Measurements  of  either  Na,  K  and  total  Na  and  K  content  and  concen- 
tration or  cellular  osmolality  as  the  cells  shrink  or  swell,  indicate  that  in 
the  four  experimental  conditions  described  in  Section  II,  none  of  these  factors 
could  serve  as  the  required  cellular  parameter.   They  either  fail  to  vary  or 
vary  so  much  during  the  changes  in  cell  size  that  they  lack  the  necessary  con- 
sistency required  of  this  factor. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Cell  size  is  an  intrinsic  property  of  all  animal  cells.   Since  it  has  been 
established  that  most  animal  cells  are  at  osmotic  equilibrium  with  their 
environment  and  contain  70-90%  water,  the  major  portion  of  cell  size  is  deter- 
mined by  factors  which  control  the  solute  content  of  the  cell.  The  extent  to 
which  a  mechanism  identical  to  the  one  in  the  duck  erythrocyte  controls  the 
solute  content  and  size  of  other  cells  will  determine  the  major  significance 
of  the  present  work.   If  an  identical  mechanism  is  present  in  other  cells, 
most  disciplines  in  biology  would  have  an  overlapping  interest  in  this 
mechanism. 

4  :>^rf 


Serial  No.   NHLI-U9 

Proposed  Course  of  Project: 

1.  To  establish  the  presence  of  the  volume  controlling  mechanism  in 
marimalian  and  non -mamma li an  erythrocyte  ghost  preparation. 

2.  To  attempt  to  isolate  the  membrane  elements  responsible  for  volume 
regulation  in  the  duck  erythrocyte.   This  study  would  require  the  use  of  an 
electron  microscope. 

3.  To  examine  the  relationship  between  extracellular  sodium  and  the 
cell  swelling  induced  by  a  hypertonic  solution  or  norepinephrine,  especially 
as  this  relationship  relates  to  amino  acid  transport. 

Honors  and  Awards:   None 

Publications:  Riddick,  D.  H.,  Kregenow,  F.  M.  and  Orloff,  J.:   The  effect 

of  norepinephrine  and  dibutyryl  cyclic-AMP  on  cation  transport 
in  duck  erythrocytes.   J.  Gen.  Physiol.  (In  press). 

Kregenow,  Floyd  M.:   The  response  of  duck  erythrocytes  to  non- 
hemolytic hypo -osmotic  media  -  evidence  for  a  volume -controlling 
mechanism.   J.  Gen.  Physiol.  (Accepted  for  publication) . 

Kregenow,  Floyd  M..:      The  response  of  duck  erythrocytes  to 
hj^jerosmotic  media  -  further  evidence  for  a  volume -controlling 
mechanism.   J.  Gen.  Physiol.  (Accepted  for  publication) . 


J«J 


Serial  No.  NHLI-120 


1.  Lab.  Kidney  &  Elec.  Metabolism 

2.  Exp.  Cardiovascular  Diseases 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Immunochemical  comparison  of  cardioglobulin  and  rat  muscle 
E-C  coupling  complex 

Previous  Serial  Number:  NHLI-177 

Principal  Investigator:   Stephen  Hajdu,  M.D. 

Other  Investigators:  Edward  J.  Leonard,  M.D. 

Cooperating  Units:  None 

Project  Description: 

Objectives:   To  determine  whether  there  is  any  chemical  relationship 
between  rat  cardioglobulin-C  and  rat  skeletal  muscle  excitation-contraction 
(E-C)  coupling  complex. 

Methods  Employed:   Isolated  frog  hearts  were  used  for  binding  and 
bioassay  of  rat  cardioglobulin-C.  These  hearts  were  also  used  as  a  specific 
immunoabsorbent  for  anti -cardioglobulin  C  antibody  (see  results) .   Standard 
techniques  were  used  for  antibody  production,  immune  fluorescence  microscopy 
and  separation  of  cardioglobulin-C  from  other  serum  proteins. 

Major  Findings:   A.  Development  of  an  immunochemical  assay  for  rat 
cardioglobulin-C.  A  rat  protein  fraction  containing  cardioglobulin-C 
activity  was  injected  with  complete  Freund's  adjuvant  into  rabbits.   The 
resulting  antiserum  had  anti -cardioglobulin-C  activity,  as  judged  by  binding 
to  surface -membrane  cardioglobulin-C  sites  of  a  BC  frog  heart,  and  inhibition 
of  the  biological  activity  of  cardioglobulin-C.   This  antiserum  makes  3 
precipitin  lines  when  reacted  in  gel  with  whole  rat  serum.  After  absorption 
by  passage  of  antiserum  through  a  series  of  BC  frog  hearts,  one  precipitin 
line,  called  precipitin  line  3,  is  no  longer  detected.   Our  tentative  con- 
clusion, therefore,  is  that  rat  cardioglobulin-C  is  in  precipitin  line  3. 
Thus,  we  at  least  have  an  immunochemical  marker  for  a  protein  which  up 
until  now  we  could  identify  only  by  its  biological  activity  on  the  frog 
heart . 

B.   Immunochemical  relationships  between  cardioglobulin-C  and  rat  E-C 
complex.  A  purified  fraction  from  rat  muscle,  containing  the  E-C  complex, 
was  set  up  in  gel  against  anti -cardioglobulin  antiserum.   Two  precipitin 
lines  formed,  which  made  lines  of  identity  with  rat  cardioglobulin-C 
fraction  antigens.   One  of  these  identity  lines  was  precipitin  line  3  which 
we  believe  represents  cardioglobulin-C.   Thus  there  appears  to  be  a  close 


3o/ 


Serial  No.   NHLI-120 


immunochemical  relationship  between  cardioglobulin-C  and  a  protein  in  the 
E-C  coupling  fraction.   Tlie  evidence  suggests  that  cardioglobulin-C  and  the 
muscle  E-C  complex  are  chemically  similar. 

Proposed  Course  of  Project:  Project  completed. 

Honors  and  Awards:   None 

Publications:   None 


O 


SdV 


Serial  No.   NHLI-121 


1.  Kidney  &  Electrolyte  Metabolism 

2.  Experimental  Cardiovascular  Diseases 

3.  Bethesda,  Maryland  20014 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Mechanism  of  cardiac  failure  in  hamsters  with 
hereditary  cardiomyopathy 

Previous  Serial  No.:   None 

Principal  Investigators:   Christian  J.  Posner,  M.D.,  Ph.D. 

Stephen  Hajdu,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  contractility  of  most  maimnalian  cardiac 
muscle  is  the  result  of  two  independent  phenomena  whose  contri- 
bution to  overall  contractility  varies  with  the  frequency  of 
stimulation.   At  high  frequencies  it  is  determined  by  the 
Bowditch  phenomenon  (BP) .   Contractile  tension  over  this  fre- 
quency range  is  supported  by  a  decrease  in  intracellular 
potassium  which  amplifies  the  effect  of  the  constant  amount  of 
free  extracellular  calcium  entering  the  fiber  at  every  frequency 
of  stimulation.   Over  the  low  frequency  range,  contractile 
tension  is  dependent  on  the  Woodworth  phenomenon  (WP)  which  is 
mediated  by  calcium  supplied  from  bound  sources.   The  amount  of 
this  available  calcium  increases  during  the  rest  intervals. 
Since  the  alkaloid  ryanodine  eliminates  only  the  WP  it  allows 
accurate  separation  of  the  contribution  of  WP  and  BP  to  overall 
contractility . 

When  it  was  reported  that  a  strain  of  hamsters  spontaneously 
develop  a  primary  cardiac  failure  we  were  able  to  study  the 
effect  of  the  disease  on  these  phenomena  (BP  and  WP) .   If  as  the 
result  of  the  disease  both  phenomena  are  equally  depressed,  it 
would  have  to  be  concluded  that  a  defect  in  the  contractile 
system  is  the  common  cause  of  the  depression  since  it  would  be 
unlikely  that  the  disease  would  affect  two  independent  functions 
equally.   On  the  other  hand  if  the  disease  is  restricted  to  only 
one  of  the  phenomena,  leaving  the  other  intact,  this  would 
indicate  a  normal  contractile  system  with  the  point  of  attack  on 
the  specific  mechanism  serving  only  the  affected  phenomenon. 

Methods:   The  hamsters  were  divided  into  3  groups.   Group  1 

1  3C3 


Serial  No.    NHLI-121 

contained  healthy  golden  hamsters,  group  2  cardiopathic  hamsters 
without  signs  of  cardiac  failure,  and  group  3  cardiomyopathic 
har.isters  with  severe  congestive  heart  failure.   Isometric  con- 
tractions were  recorded  from  right  ventricular  muscle 
preparations  suspended  in  Krebs  solution  vigorously  gassed  with  a 
mixture  of  95%  O2  and  5%  CO2.   For  experiments  involving  measure- 
ment of  ^^Ca  uptake  during  the  depolarized  state,  muscles  were 
suspended  in  Krebs  solution  in  which  all  sodium  was  replaced  with 
potassium  (Potassium  -  Krebs) .   The  control  muscle  in  each  pair 
remained  unstimulated  and  was  suspended  in  normal  Krebs  solution. 
Each  solution  contained  the  same  specific  activity  of  ^^Ca 
(equivalent  to  10^  cpm  ^^Ca/ml) .      At  the  end  of  the  incubation 
period  the  muscles  were  washed  in  ice-cold  Krebs  solution  every 
4  minutes  for  40  minutes.   At  the  end  of  the  washing  period,  the 
muscles  were  blotted  dry,  weighed  on  a  torsion  balance  and 
homogenized  in  Krebs  solution  in  a  glass  tissue  grinder.  Aliquots 
of  the  homogenate  were  dried  on  planchets  and  radioactivity 
counted  in  a  low-background  counter. 

Major  Findings:   The  interval-tension  curve  for  the  healthy 
golden  hamster  and  that  of  the  cardiomyopathic  hamsters  without 
evidence  of  cardiac  failure  is  similar,  with  tension  increasing 
at  both  high  and  low  frequencies  indicating  a  normal  BP  and  WP . 
The  interval-tension  curve  of  the  cardiomyopathic  hamsters  with 
severe  congestive  heart  failure  shows  a  parallel  course  with  the 
curves  obtained  from  the  first  two  groups  but  only  over  the  range 
of  the  WP  (that  is,  between  2-60  sec).   At  high  frequencies  when 
the  BP  begins  to  develop  in  the  normal  animals  (2  sec.  or  less) 
the  curve  of  the  diseased  animals  continues  to  decline  as  though 
the  BP  were  missing  in  these  animals. 

In  order  to  determine  whether  the  BP  is  absent  in  the  dis- 
eased hearts,  the  hearts  in  all  groups  were  treated  with 
ryanodine.   No  tension  was  recorded  in  the  WP  range  in  all  three 
groups  but  undiminished  contractility  was  observed  in  the  BP 
range  in  hearts  from  the  controls.   The  hearts  of  animals 
suffering  from  congestive  failure  developed  no  tension  over  the 
entire  frequency  range  showing  that  all  tension  exhibited  prior 
to  ryanodine  treatment  was  produced  by  the  WP .   A  normal  BP  is 
present  in  the  hearts  of  the  young  cardiomyopathic  animals  free 
of  the  signs  of  cardiac  incompetence  but  absent  by  the  time  that 
cardiac  failure  has  reached  its  final  stage. 

The  absence  of  BP  could  be  the  result  of  a  malfunction  in 
either  step  determining  the  BP,  that  is  failure  of  the  decrease 
in  intracellular  potassium  with  increasing  frequency  or  failure 
of  entry  of  free  extracellular  calcium  into  the  fiber.   The 
functional  state  of  the  potassium  mechanism  was  evaluated  by  use 
of  the  cardiac  glycosides.   While  addition  of  scilliroside  pro- 
duced a  shift  of  the  BP  toward  the  lower  frequency  range  in 


3o^ 


Serial  No. 


NHLI-121 


healthy  hamster  hearts,  it  did  not  increase  the  tension  over 
the  high  frequency  range  in  the  diseased  hearts  which  would  have 
indicated  return  of  the  BP .   A  large  dose  of  glycoside  eventually 
led  to  the  development  of  contracture  in  the  diseased  heart 
indicating  that  there  was  no  lack  of  response  to  this  inter- 
vention which  lowers  intracellular  potassium. 

The  probable  explanation  for  the  absence  of  increased 
contractility  over  the  BP  range  in  the  cardiomyopathic  hearts  is 
therefore  failure  of  extracellular  calcium  to  enter  during 
depolarization.   This  hypothesis  was  evaluated  by  measuring  ^^Ca 
uptake  of  heart  muscle  with  a  polarized  and  depolarized  membrane. 
Calcium  uptake,  expressed  as  cpm  ^^Ca/mq   muscle,  was  determined 
in  five  separate  muscles  for  both  normal  and  cardiomyopathic 
hamsters  after  incubation  times  of  20,  40  and  80  min . 

In  the  healthy  hamster  hearts,  the  ^^Ca  accumulation  during 
the  depolarized  state  continues  to  increase  above  that  in  the 
polarized  state  with  increasing  incubation  times.   In  the 
cardiomyopathic  hamster  hearts  there   is  no  significant  increase 
in  '^^Ca  accumulation  during  the  depolarized  state  compared  with 
that  in  the  polarized  state.   Thus  the  change  that  takes  place 
in  these  animals  later  in  life  and  leads  to  a  fatal  cardiac 
failure  is  an  impermeability  of  the  membrane  to  entrance  of  free 
extracellular  calcium  during  depolarization  of  the  membrane. 

Significance  to  Biomedical  Research  and  the  Program  of  the 
Institute:   This  represents  the  first  analysis  on  the  cellular 
level  of  the  mechanism  of  a  naturally  occurring  primary  cardiac 
failure.   This  study  shows  that  with  progression  of  the  disease, 
the  cardiac  muscle  fails  to  allow  entrance  of  free  extracellular 
calciimn  during  depolarization.   iVs  a  consequence  of  this,  the 
heart  is  deprived  of  normal  contractility  at  the  physiological 
heart  rates.   Due  to  this  lack  of  calcium  entrance  during 
depolarization  the  cardiac  glycosides  also  become  ineffective  and 
this  is  in  good  agreement  with  long  standing  clinical  observation 
(cardiac  failure  refractory  to  glycosides) .   This  study  delineates 
the  course  of  future  research  directed  toward  the  correction  of 
cardiac  failure. 

Proposed  Course  of  Project:   The  correction  of  the  defect 
leading  to  cardiac  incompetence  in  the  diseased  hamsters  can 
proceed  in  two  directions: 

1.   Restoration  of  calcium  entry:   This  approach  has  been 
explored  by  the  use  of  epinephrine,  the  most  potent  known  agent 
to  increase  permeability  of  calcium  during  the  depolarized  state. 
While  epinephrine  made  some  improvement  in  calcium  entry  in  some 
of  the  animals,  in  others  it  had  no  effect.   In  our  judgment 
this  indicates  that  when  the  disease  has  reached  its  final  state 


:J^r 


Serial  No.    NHLI-121 

there  is  no  practical  way  to  restore  calcium  entry.   However 
prevention  of  the  closing  of  the  membrane  to  calcium  entry  would 
be  correct  in  principle. 

2.   Improvement  in  the  Woodworth  phenomenon:   Extension  of 
the  WP  to  cover  the  high  frequency  range,  as  in  skeletal  muscle, 
would  restore  contractility  by  increasing  the  amount  of  calcium 
released  from  bound  sources.   Our  study  has  already  shown  that 
this  mechanism  of  compensation  is  present  to  a  small  degree  in 
the  diseased  hamster  hearts  but  this  is  not  adequate  to  maintain 
normal  cardiac  function.   Enough  evidence  has  been  collected 
which  suggests  that  the  WP  is  based  on  the  function  of  the  cardio- 
globulin  system.   Improvement  in  the  function  of  this  system 
seems  the  most  feasible  procedure  to  restore  contractility  in 
the  failing  heart. 

Honors  and  Awards :   None 

Publications:   Hajdu,  S . , and  Posner,  C.  J.:   Absence  of  Bowditch 
phenomenon  in  the  ventricular  muscle  of  hamsters 
with  hereditary  cardiomyopathy.   Am.  Heart  J. 
81:  June,  1971  (In  press) . 


i 

3oi 


:^ 


h 


■Pi 


ANNUAL  REPORT  OF  THE 

LABORATORY  OF  BIOCHEMICAL  GENETICS 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1970  through  June  30,  1971 

Three  aspects  of  molecular  biology  were  studied  during  the  past  year: 
Neurobiology,  mechanisms  of  protein  synthesis,  and  the  biochemistry  of  rare 
constituents  of  tRNA. 

A.   NEUROBIOLOGY 

1.   Neuroblastoma 

Clonal  lines  of  neuroblastoma  provide  an  unusual  opportunity  to  explore 
steps  in  neuron  differentiation  as  well  as  functional  characteristics  of 
mature  neurons.   The  cells  resemble  neuroblast  stem  cells  yet  are  capable  of 
multiplying  rapidly  in  vitro  and  give  rise  to  cells  exhibiting  many  properties 
characteristic  of  differentiated  neurons.   Many  new  clones  of  mouse  neuro- 
blastoma C1300  were  obtained  during  the  past  year  and  were  examined  for 
enzymes  catalyzing  the  synthesis  of  putative  neurotransmitters.   Three  types 
of  neuroblastoma  clones  with  respect  to  transmitter  synthesis  were  found: 
1)   Clones  with  no  apparent  transmitter;  2)  adrenergic  clones;  3)   cholin- 
ergic clones.   Two  varieties  of  human  neuroblastomas  are  known:  adrenergic 
and  non-adrenergic  tumors.   On  the  basis  of  studies  with  mouse  neuroblastoma 
a  third  class  of  human  tumors  may  be  expected:   i.e.,  tumors  of  cholinergic 
neuroblasts.   A  simple,  highly  sensitive  assay  for  acetylcholine  transferase 
was  developed  that  provides  a  ready  means  of  identifying  such  tumors. 

It  is  possible  that  clonal  differences  in  enzymes  for  neurohormone  syn- 
thesis reflect  genetic  heterogeneity;  alternatively,  the  differences  may  be 
a  function  of  the  developmental  potential  of  the  normal  neuroblast  precursor 
of  the  mouse  neuroblastoma. 

The  formation  of  synapses  between  the  various  neuroblastoma  clones  and 
dissociated  cells  from  cardiac  and  skeletal  muscle  was  investigated.   The 
rate  of  contraction  of  single  cardiac  cells  or  of  colonies  in  vitro  was 
determined  by  recording  pulsations  on  video-tape  and  displaying  the  output 
on  recorder  paper,  using  a  video-analogue  output.   Neuroblastoma  cells  were 
found  to  form  a  strong  physical  connection  with  muscle  cells.   Stimulation  of 
neuroblastoma  cells  altered  the  rate  of  contraction  of  muscle  cells.   These 
and  additional  results  suggest  that  neuroblastoma  cells  are  capable  of  syn- 
apsing  in  vitro;  however,  further  work  is  needed  to  substantiate  this  possi- 
bility. 

2.   Genes  for  neuronal  properties  expressed  in  neuroblastoma  x  L  cell  hybrids. 

Mutant  clones  of  mouse  neuroblastoma  were  selected  by  mutagenesis  and 
exposure  to  6-thioguanine.   The  electrically  excitable  neuroblastoma  cells 
were  fused  with  electrically  passive  L  cells  having  a  hitherto  undescribed 
electrical  marker.   Hybrid  clones,  examined  10-40  generations  after  fusion, 
were  found  to  be  electrically  excitable.   The  results  show  that  at  least  part 

1  2o7 


f 


of  the  genetic  information  from  neuron  differentiation  can  be  functionally 
expressed  in  neuroblastoma  x  L  cell  hybrids.   No  evidence  for  a  pleiotropic 
repressor  terminating  neuron  differentiation  was  found.   In  fact,  most  N  x  L 
cell  hybrids  were  more  active  electrically  than  the  parental  neuroblastoma 
line.   Somatic  cell  hybridization  applied  to  normal  neuroblasts  should  pro- 
^'ide  a  relatively  simple  means  of  establishing  clonal  lines  of  cells  derived 
from  different  types  of  neurons.   The  levels  of  acetylcholinesterase  of  N  x  L 
cell  hybrid  clones  were  determined  30-50  cell  generations  after  cells  were 
fused.   The  neuroblastoma  parent  and  all  N  x  L  cell  hybrids  contained  acetyl- 
cholinesterase; appreciable  amounts  of  enzyme  were  not  detected  in  the  I.  cell 
parent  or  in  L  x  L  hybrids.   Tlie  results  show  that .  neuroblastoma  genes  for 
acetylcholinesterase  continue  to  be  expressed  after  fusion. 

Results  obtained  with  neuroblastoma  clearly  show  that  rapidly  dividing 
cells  still  retain  the  ability  to  express  neuronal  functions  and  that  some 
neuronal  genes  remain  active  in  somatic  cell  hybrids.   The  possibility  that 
neurons  may  be  capable  of  initiating  a  program  of  neuron  differentiation  in 
recipient  cells  also  deserves  consideration. 

3.  Acetylcholinesterase 

We  have  previously  shown  that  neuroblastoma  cells  contain  acetylcholin- 
esterase and  both  excitatory  and  inhibitory  acetylcholine  receptors  and  that 
these  factors  respond  to  regulatory  mechanisms  in  vitro.   Thus,  purification 
and  characterization  of  the  factors  were  initiated  to  define  the  relationship 
between  acetylcholinesterase  and  acetylcholine  receptors  and  the  nature  of 
the  regulatory  mechanisms.   Acetylcholinesterase  was  fovind  to  be  associated 
with  membranes;  however,  the  enzyme  now  has  been  purified  extensively  and  is 
now  being  characterized. 

4.  Cyclic  AMP 

A  simple,  rapid  assay  for  adenosine  3',5'-cyclic  monophosphate  was 
developed  by  A.  Oilman  that  is  based  upon  competition  between  labeled  and  un- 
labeled cAMP  to  a  protein,  presumably  a  cAMP-dependent  protein  kinase.   The 
nucleotide-protein  complex  is  then  adsorbed  and  washed  on  a  cellulose  ester 
filter.   The  assay  is  sensitive  to  0.05-0.10  pmoles  of  cAMP,  and  is  being  used 
to  explore  cAMP  metabolism  in  neuroblastoma  and  other  cell  types  that  are 
grown  in  vitro. 

B.   MECHANISMS  OF  PROTEIN  SYNTHESIS 

1.   Mammalian  peptide  chain  termination 

Mammalian  peptide  chain  termination  has  been  studied  in  vitro  using  a 
modification  of  the  formyl  methionine  (fMet)  release  assay  developed  for 
bacterial  studies.   Release  of  fMet  from  rabbit  reticulocyte  fMet  tRNA^-ribo- 
some  intermediates  requires  a  protein  release  factor  (R) ,  terminator  codon 
template,  and  GTP.   Release  factor  has  been  purified  to  >95%  homogeneity 
prepared  from  rabbit  reticulocytes,  and  partially  purified  from  guinea  pig 
liver  and  Chinese  hamster  liver.   The  purified  reticulocyte  R  factor  has  a 


^00 


native  tnolecular  weight  by  G200  Sephadex  chromatography  of  -   255,000,  is 
composed  of  subunits,  has  a  ribosomal  dependent  GTPase  activity,  and  is 
active  in  the  fMet  release  assay  using  the  templates  UAAA,  UAGA,  or  UGAA. 
Multiple  release  factors  of  differing  codon  specificity  have  not  been  found. 
The  release  reaction  is  inhibited  by  GDPCP  which  is  consistent  with  the 
demonstrated  GTPase  activity  being  essential  for  peptide  chain  termination. 
The  release  reaction  is  also  inhibited  by  sparsomycin,  anisomycin  and  gou- 
gerotin,  inhibitors  of  the  mammalian  peptidyl  transferase.   This  extends  the 
correlation  found  in  bacterial  systems  implying  peptidyl  transferase  partici- 
pation in  peptide  chain  termination.   These  antibiotics  do  not  inhibit  R 
factor  ribosomal-dependent  GTPase  activity.   More,  recently,  we  have  studied 
the  formation  of  R* radioactive  terminator  codon 'ribosome  intermediates. 
GDPCP  and  ethanol  can  be  used  to  stabilize  such  intermediates  for  quantita- 
tion by  Millipore  filter  retention.   These  studies  suggest  that  recognition 
sites  for  all  three  terminator  codons  are  part  of  a  single  large  R  factor. 


C.   BIOCHEMISTRY  OF  RARE  CONSTITUENTS  OF  tRNA 

1.  The  effect  of  methylated  bases  on  the  biological  activity  of  Met-tRNA. 

The  properties  of  normal  and  methyl-poor  Met-tRNA  were  compared  in  a 
variety  of  test  systems.   The  results  show  the  following:   a)  of  the  two 
normally  occurring  isoacceptors  of  Met-tRNA,  the  nonformylatable  species  is 
acylated  more  quickly  than  the  formylatable  species,  and  both  of  these  are 
aminoacylated  by  purified  methionyl-tRNA  synthetase  more  rapidly  than  is 
methyl-poor  Met-tRNA;  b)  on  reverse-phase  chromatography,  methyl-poor  Met- 
tRNA,  Met-tRNAj^^  and  Met-tRNA^,  can  all  be  separated  from  each  other;  c)  methyl- 
poor  Met-tRNA  serves  as  a  substrate  for  the  trans formylating  enzyme,  is 
recognized  by  initiation  factors  and  is  excluded  from  recognition  by  elonga- 
tion factors  in  a  manner  indistinguishable  from  normally  methylated  Met-tRNA. 

2.  The  involvement  of  leucine  in  tRNA  modification. 


When  relaxed-rontrol  E.    coli  is  deprived  of  methionine,  new  species  of 
tRNA  are  formed.   Since  methionine  is  a  source  of  methyl  groups  of  tRNA,  the 
newly  formed  tRNA  is  methyl-deficient.   We  have  now  found  that  new  species 
of  tRNA  also  accumulate  when  relaxed-control  E.    coli  are  starved  of  leucine. 
New  RNA  synthesis  is  necessary  for  the  phenomenon  since  the  appearance  of 
new  species  is  prevented  by  leucine  starvation  of  stringent  control  cells 
or  uracil  starvation  of  relaxed-control  cells.   Leucine  starvation  leads  to 
formation  of  new  species  that  accept  the  amino  acids  leucine,  arginine  and 
histidine.   This  distribution  suggests  that  leucine  is  involved  in  a  modifi- 
cation reaction  specific  for  those  tRNA's  that  recognize  codons  beginning 
with  C.  This  is  in  keeping  with  previous  data  from  this  and  other  labora- 
tories showing  that  tRNA's  recognizing  codons  beginning  with  U  contain  iso- 
pentenyl  adenosine  and  those  recognizing  codons  beginning  with  A  contain 
N-(purin-6-ylcarbamoyl)-threonine.   Further  studies  on  these  new  species  of 
tRNA  will  continue. 


r 


3^ 


f 


3.   The  biochemistry  of  N-(purin-6-ylcarbamoyl)-threonine  in  tRNA. 

The  most  recently  discovered  minor  constituent  in  tRNA  is  N- (purin-6- 
carbamoyl)-threonine  (PCT) .   Since  it  has  been  found  in  the  tRNA  for  the 
amino  acids  isoleucine,  methionine,  serine  and  lysine,  it  has  been  suggested 
that  its  occurrence  will  be  restricted  to  those  tRNA's  whose  codons  begin 
with  A.   We  have  initiated  a  program  to  study  the  biosj'nthesis  and  distribu- 
tion of  PCT  in  tRNA.   By  exposure  of  a  "relaxed  control"  threonine  auxotroph 
of  E.    coli  to  threonine  under  conditions  where  RNA  synthesis  is  allowed  and 
protein  synthesis  is  blocked,  we  have  been  able  to  specifically  incorporate 
label  from  radioactive  threonine  into  PCT.   This  is  evidence  that  threonine 
is  at  least  a  partial  precursor  of  PCT.   Our  plan  for  the  immediate  future  is 
to  utilize  specifically  labeled  tRNA  to  deduce  which  species  of  tRNA  contain 
PCT.   This  should  allow  us  to  test  the  distribution  hypothesis  mentioned 
above. 


r^ 


3/a 


Serial  No.    NHLI-122 


1.  Biochemical  Genetics 

2.  Macromolecules 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   The  Initiation  of  Mammalian  Protein  Synthesis 

Previous  Serial  Number:   NHLI-339 

Principal  Investigator:   Daniel  Twardzik 

Other  Investigator:   Alan  Peterkofsky 

Cooperating  Unit:   Ettore  Appella,  National  Cancer  Institute 

Project  Description: 

The  project  described  last  year  is  progressing  as  anticipated.   Pyrro- 
lidone  carboxylic  acid  (PCA)  occurs  as  the  N-terminal  amino  acid  of  many 
mammalian  proteins.   Our  experiments  have  been  aimed  at  determining  the 
precursor  of  this  unique  amino  acid.   Using  cells  derived  from  a  mouse  plasma- 
cytoma, we  have  been  able  to  incorporate  label  from  either  radioactive  glu- 
tamic acid  or  glutamine  into  protein.   Degradative  analysis  of  labeled 
protein  originated  from  labeled  glutamic  acid  shows  label  in  only  PCA  and 
glutamic  acid.   On  the  other  hand,  if  label  originated  from  glutamine,  the 
labeled  protein  contains  label  in  PCA,  glutamic  acid  and  glutamine.   This 
finding  indicates  that  PCA  must  be  derived  more  directly  from  glutamic  acid 
than  from  glutamine,  a  finding  contrary  to  a  suggestion  in  the  literature. 
Tests  of  enzyme  activities  in  cell  extracts  support  our  data:  While  gluta- 
mine synthetase  is  absent,  glutaminase  activity  is  substantial.   This 
suggests  that  the  mechanism  whereby  glutamine  is  converted  to  PCA  involves 
a  prior  conversion  to  glutamic  acid.   These  observations  will  be  pursued 
with  the  intention  of  elucidating  the  mechanism  of  PCA  formation  from  glu- 
tamic acid. 

Hcnrs  and  Awards :   None 

Publications:   None. 


3// 


Serial  No.    NHLI-123 

1.  Biochemical  Genetics 

2.  Macromolecules 

3.  Bethesda,   Maryland 

PIIS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  Arainoacyl-tRNA  Synthetases 

Previous  Serial  Number:   NHLI-3A0 

Principal  Investigator:   Takis  Papas 

Other  Investigator:   Alan  Peterkofsky 

Cooperating  Units:   None 

Project  Description: 

The  mechanism  of  several  aminoacyl-tRNA  synthetases  has  been  studied  in 
this  and  other  laboratories.   While  each  aminoacyl-tRNA  synthetase  shows 
specificity  for  its  unique  amino  acid,  the  characteristics  of  the  reactions 
involved  are  generally  similar.   There  are,  however,  three  notable  exceptions 
to  this  similarity;  the  synthetases  for  arginine,  glutamine  and  glutamic 
acid  all  require  the  cognate  tRNA's  for  demonstration  of  the  ATP-PPi  exchange 
reaction,  while  all  the  other  synthetases  show  no  such  tRNA  requirement.   We 
have  initiated  a  study  of  the  mechanism  of  these  unique  aminoacyl-tRNA  syn- 
thetases with  a  view  to  exploring  these  unusual  properties. 

Honors  and  Awards :   None 

Publications:   None. 


3/x 


Serial  No.     NHLI-124 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Maryland 


s 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   The  Neuroblastoma  System  as  a  Model  for  Neuron  Differentia- 
tion. 

Previous  Serial  Number:   None 

Principal  Investigator:   Marshall  Nirenberg 

Other  Investigators:   Takehiko  Amano,  Elliott  Richelson  and  Edward  J. 
Thompson. 

Cooperating  Units :   None 

Project  Description: 

Clonal  lines  of  neuroblastoma  provide  an  unusual  opportunity  to  explore 
steps  in  neuron  differentiation  as  well  as  functional  characteristics  of 
mature  neurons.   The  cells  resemble  neuroblast  stem  cells  yet  are  capable 
of  multiplying  rapidly  in  vitro  and  give  rise  to  cells  exhibiting  many 
properties  characteristic  of  differentiated  neurons.   Many  new  clones  of 
mouse  neuroblastoma  C1300  were  obtained  during  the  past  year  and  were  exa- 
mined for  enzymes  catalyzing  the  synthesis  of  putative  neurotransmitters. 
Three  types  of  neuroblastoma  clones  with  respect  to  transmitter  synthesis 
were  found:   1)   Clones  with  no  apparent  transmitter;  2)  adrenergic  clones; 
3)   cholinergic  clones.   Two  varieties  of  human  neuroblastomas  are  known — 
adrenergic  and  non-adrenergic  tumors.   On  the  basis  of  studies  with  mouse 
neuroblastoma  a  third  class  of  human  tximors  may  be  expected:   i.e.,  tumors 
of  cholinergic  neuroblasts.   A  simple,  highly  sensitive  assay  for  acetyl- 
choline transferase  was  developed  that  provides  a  ready  means  of  identifying 
such  tumors . 

It  is  possible  that  clonal  differences  in  enzymes  for  neurohormone 
synthesis  reflect  genetic  heterogeneity;  alternatively,  the  differences  may 
be  a  function  of  the  developmental  potential  of  the  normal  neuroblast  pre- 
cursor of  the  mouse  neuroblastoma. 

The  formation  of  synapses  between  the  various  neuroblastoma  clones  and 
dissociated  cells  from  cardiac  and  skeletal  muscle  was  investigated.   The 
rate  of  contraction  of  single  cardiac  cells  or  of  colonies  in  vitro  was 
determined  by  recording  pulsations  on  video-tape  and  displaying  the  output 
on  recorder  paper,  using  a  video-analogue  output.   Neuroblastoma  cells  were 
found  to  form  a  strong  physical  connection  with  muscle  cells.   Stimulation 
of  neuroblastoma  cells  altered  the  rate  of  contraction  of  muscle  cells. 


3/i 


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Serial  No.    NHLI-124 


These  and  additional  results  suggest  that  neuroblastoma  cells  are  capable 
of  synapsing  in  vitro;  however,  further  work  is  needed  co  substantiate  this 
possibility. 


Honors  and  Awards:   None. 
Publications: 


Seeds,  N.  W.,  Oilman,  A.  G.,  Amano,  T.  and  Nirenberg,  M.  W.:   Regulation 
of  axon  formation  by  clonal  lines  of  a  neural  tumor.   Proc.  Natl   Acad 
Sci.  66:  160-167,  1970.  '—^ * 


i 


r^ 


3/jf^ 


Serial  No.    NHLI-125 


1.  Biochemical  Genetics 

2.  Medical  Genetics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Mammalian  Peptide  Chain  Termination 

Previous  Serial  Number:   None 

Principal  Investigator:   Arthur  Beaudet 

Other  Investigators:   C.  T.  Caskey  and  J.  L.  Goldstein 

Cooperating  Units:   None. 

Project  Description: 


Mammalian  peptide  chain  termination  has  been  studied  in  vitro  using  a 
modification  of  the  formyl  methionine  (fMet)  release  assay  developed  for 
bacterial  studies.   Release  of  fMet  from  rabbit  reticulocyte  fMet  tRNA^^'ribo- 
some  intermediates  requires  a  protein  release  factor  (R) ,  terminator  codon 
template,  and  GTP.   Release  factor  has  been  purified  to  >95%  homogeneity 
prepared  from  rabbit  reticulocytes,  and  partially  purified  from  guinea  pig 
liver  and  Chinese  hamster  liver.   The  purified  reticulocyte  R  factor  has  a 
native  molecular  weight  by  G200  Sephadex  chromatography  of  -   255,000,  is 
composed  of  subunits,  has  a  ribosomal-dependent  GTPase  activity,  and  is 
active  in  the  fMet  release  assay  using  the  templates  UAAA,  UAGA,  or  UGAA. 
Multiple  release  factors  of  differing  codon  specificity  have  not  been  foimd. 
The  release  reaction  is  inhibited  by  GDPCP  which  is  consistent  with  the 
demonstrated  GTPase  activity  being  essential  for  peptide  chain  termination. 
The  release  reaction  is  also  inhibited  by  sparsomycin,  anisomycin,  and 
gougerotin,  inhibitors  of  the  mammalian  peptidyl  transferase.   This  extends 
the  correlation  found  in  bacterial  systems  implying  peptidyl  transferase 
participation  in  peptide  chain  termination.   These  antibiotics  do  not  in- 
hibit R  factor  ribosomal  dependent  GTPase  activity.   More  recently  we  have 
studied  the  formation  of  R* radioactive  terminator  codon -ribosome  inter- 
mediates.  GDPCP  and  ethanol  can  be  used  to  stabilize  such  intermediates  for 
quantitation  by  Millipore  filter  retention.   These  studies  suggest  that 
recognition  sites  for  all  three  terminator  codon  are  part  of  a  single  large 
R  factor. 

Honors  and  Awards:   None. 

Publications : 

Goldstein,  J.,  Beaudet,  A.,  and  Caskey,  C.T.:   Peptide  chain  termination 
with  mammalian  release  factor.   Proc.  Natl.  Acad.  Sci.  U.S.  67:  99-106, 
1970. 


3/r 


Serial  No.  NIiLI-125 


Beaudet,  A.,  and  Caskey,  C.  T.:  Mammalian  peptide  chain  termination  II. 
Codon  specificity  and  GTPase  activity  of  release  factor.   Proc.  Natl. 
Acad.  Sci.  U.S.  68:  619-624,  1971. 


nA 


3/^ 


4 


Serial  No.    NHLI-126 

1.  Biochemical  Genetics 

2.  Macromolecules 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Biochemistry  of  Rare  Constituents  of  tRNA 

Previous  Serial  Number:   NHLI-338 

Principal  Investigators:   Maurille  Fournier,  Marcia  Litwack,  Jane  Marmor, 

Donald  Powers 

Other  Investigator:   Alan  Peterkofsky 

Cooperating  Units:   Dr.  Herbert  Dickerman,  Johns  Hopkins  University; 

B.  P.  Doctor,  Walter  Reed  Army  Institute  of  Research 


Project  Description: 

A.   Biological  Studies  of  Isopentenyl  Adenosine  in  Transfer  RNA  (Marcia 
Litwack  and  Alan  Peterkofsky) . 

Isopentenyl  adenosine  (iPA)  occurs  only  in  those  species  of  tRNA  re- 
sponding to  codon  whose  first  letters  begin  with  U.   Our  previous  studies 
showed  that,  in  Lactobacillus  acidophilus,  mevalonic  acid  was  the  precursor 
of  iPA.   iPA  content  of  tRNA  can  be  varied  dependent  on  the  concentration  of 
mevalonic  acid  in  the  culture  fluids.   Our  studies  have  shown  that,  while 
the  gram-negative  organism  Escherichia  coli  contains  the  thiomethylated 
derivative  of  iPA  in  its  tRNA,  the  tRNA  of  the  gram-positive  Lactobacillus 
contains  unmodified  iPA,  as  does  mammalian  liver  tRNA. 

We  have  now  completed  our  studies  on  a  comparison  of  the  biological 
properties  of  iPA-rich  and  iPA-poor  tRNA.   All  attempts  to  show  a  require- 
ment for  iPA  in  tRNA  for  maximtnn  rate  or  extent  of  aminoacylation  or  peptide- 
bond  synthesis  have  been  negative.   We  conclude,  in  conflict  with  some  other 
data  in  the  literature,  that  iPA  is  not  necessary  for  the  acceptance  or 
transfer  functions  of  tRNA  in  Lactobacillus . 

B.   The  Importance  of  Methylated  Bases  for  the  Biological  Activity  of  the 
Methionine  tRNA  (Jane  Marmor  and  Alan  Peterkofsky) . 

This  project,  initiated  last  year,  has  been  brought  to  a  conclusion. 
While  the  properties  of  methyl-deficient  tRNA  for  several  amino  acids  have 
been  previously  examined,  extending  the  study  to  include  the  tRNA  for 
methionine  was  felt  to  be  warranted.   Methionine  tRNA  is  unique  in  several 
respects:   It  is  a  substrate  for  the  enzyme  that  forms  N-formyl-methionyl- 
tRNA,  it  specifically  recognizes  initiation  factors,  and  it  is  the  only  tRNA 
that  does  not  form  a  complex  with  elongation  factors.   We  have  compared  the 


w 


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3/y 


Serial  No,  NHLI-126 


properties  of  normal  and  methyl-poor  methionyl-tRNA  in  a  variety  of  test 
systems  and  found  the  following:   (a)  of  the  two  normally  occurring  iso- 
acceptors  of  met-tRl-JA,  the  non-formylatable  species  is  acylated  more  quickly 
than  the  fcrmylatable  species  and  both  of  these  are  aminoacylated  by  purified 
methionyl-tRNA  synthetase  more  rapidly  than  is  methyl-poor  met-tRNA;  (b)  on 
reverse-phase  chromatography,  methyl-poor  met-tRNA.,  met-tRNAj^  and  met-tRNA-p 
can  all  be  separated  from  each  other;  (c)  methyl-poor  met-tRNA  serves  as  a 
substrate  for  the  transformylating  enzyme,  is  recognized  by  initiation 
factors  and  is  excluded  from  recognition  by  elongation  factors  in  a  manner 
indistinguishable  from  normally  methylated  met-tRNA. 

C.  The  Involvement  of  Leucine  in  tRNA  Modification  (Maurille  Fournier  and 
Alan  Peterkofsky) . 

It  has  previously  been  shown  that  when  "relaxed-control"  E.    coli  is 
deprived  of  methionine,  new  species  of  tRNA  (detected  by  column  chromato- 
graphy) are  formed.   Since  methionine  is  the  source  of  methyl-groups  of  tRNA, 
these  new  species  are  methyl-deficient.   We  have  now  found  that  new  species 
of  tRNA  also  accumulate  when  "relaxed  control"  E.    coli  are  starved  of 
leucine.   New  RNA  synthesis  is  necessary  for  the  phenomenon  since  the 
appearance  of  new  species  is  prevented  by  leucine  starvation  of  stringent 
control  cells  or  uracil  starvation  of  "relaxed  control"  cells.   Leucine 
starvation  leads  to  formation  of  new  species  that  accept  the  amino  acids 
leucine,  arginine  and  histidine.   This  distribution  suggests  that  leucine 
is  involved  in  a  modification  reaction  specific  for  those  tRNA's  that  recog- 
nize codons  beginning  with  C.   This  is  in  keeping  with  previous  data  from 
this  and  other  laboratories  showing  that  tRNA's  recognizing  codons  beginning 
with  U  contain  isopentenyl  adenosine  and  those  recognizing  codons  beginning 
with  A  contain  N-(purin-6-ylcarbamoyl)-threonine.   Further  studies  on  these 
new  species  of  tRNA  will  continue. 

D.  The  Biochemistry  of  N- (purin-6-ylcarbamoyl) -threonine  in  tRNA  (Donald 
Powers  and  Alan  Peterkofsky). 

The  most  recently  discovered  minor  constituent  in  tRNA  is  N-(purin-6- 
ylcarbamoyl)-threonine  (PCT) .   Since  it  has  been  found  in  the  tRNA  for  the 
amino  acids  isoleucine,  methionine,  serine  and  lysine,  it  has  been  suggested 
that  its  occurrence  will  be  restricted  to  those  tRNA's  whose  codons  begin 
with  A.   We  have  initiated  a  program  to  study  the  biosynthesis  and  distri- 
bution of  PCT  in  tRNA.   By  exposure  of  a  "relaxed  control"  threonine  auxo- 
troph  of  E,    coli  to  threonine  under  conditions  where  RNA  synthesis  is  allowed 
and  protein  synthesis  is  blocked,  we  have  been  able  to  specifically  incor- 
porate label  from  radioactive  threonine  into  PCT.   This  is  evidence  that 
threonine  is  at  least  a  partial  precursor  of  PCT.   Our  plan  for  the  immediate 
future  is  to  utilize  specifically  labeled  tRNA  to  deduce  which  species  of 
tRNA  contain  PCT.   This  should  allow  us  to  test  the  distribution  hypothesis 
mentioned  above. 


3/4 


Serial  No.   NHLI-126 


E.   In  vitro  Transfer  RNA  Synthesis  and  Modification  (Maurille  Foumier, 
B.  P.  Doctor  and  Alan  Peterkofsky) 

We  have  recently  initiated  a  program  to  purify  those  regions  of  E.    coli 
DNA  carrying  the  information  for  tRNA  synthesis  (tRNA  cistrons).   Our 
interest  in  this  area  is  threefold:   (a)   We  are  interested  in  studying  the 
factors  controlling  the  synthesis  of  tRNA  at  the  transcriptional  level; 

(b)  we  would  like  to  know  the  nature  of  precursor  tRNA's  if  such  exist; 

(c)  we  wish  to  study  those  in  vitro  modification  reactions  that,  thus  far, 
have  not  been  amenable  to  examination.   By  a  DNA-RNA  hybridization  procedure, 
we  have  been  able  to  prepare  enriched  tRNA  cistrons.   These  tRNA  cistrons 
were  used  as  a  template  with  RNA  polymerase.   Further  studies  in  this  area 
are  in  progress. 

Honors  and  Awards:   None. 

Publications ; 

Foumier,  M.  ,  Doctor,  B.  P.,  and  Peterkofsky,  A.:   Unique  transfer  RNA 
subspecies  formed  by  leucine  statn^ation  of  relaxed  control  E^.  coli. 
Fed.  Proc.  29:  468,  1969. 


Foumier,  M.  ,  Brenner,  D.  J.,  Peterkofsky,  A.,  and  Doctor,  B.  P.:   In 
vitro  RNA  synthesis  using  purified  E.  coli  tRNA  cistrons.   British 
Biophysical  Society,  Jan.  1971. 

Gonano,  F.  ,  Stem,  R.  ,  Littauer,  U.  ,  Fleissner,  E,  .  and  Peterkofsky,  A.: 
tRNA  meti-deficienti  nella  sintesi  proteica.   Italian  Molecular  Biology 
Society,  Rome,  1970. 

Litwack,  M.  and  Peterkofsky,  A.:  Transfer  RNA  deficient  in  N"- (A  -iso- 
pentenyl)  adenosine  due  to  mevalonic  acid  limitation.  Biochemistry  10: 
994-1006,  1971. 

Marmor,  J.  B.,  Dickerman,  H.  W.  and  Peterkofsky,  A.:   Studies  in  methyl- 
deficient  methionine  transfer  ribonucleic  acid  from  Escherichia  coli . 
J.  Biol.  Chem.  246:  3464-3473,  1971. 


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Serial  No.     NHLI-127 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Genes  for  Neuronal  Properties  Expressed  in  Neuroblastoma  x  L 
Cell  Hybrids. 

Previous  Serial  Number:   None. 

Principal  Investigator:   John  D.  Minna. 

Other  Investigators:   Marshall  Nlrenberg,  Takehlko  Amano,  and  Devera  Glazer 

Cooperating  Units:   Phillip  G.  Nelson  and  John  H.  Peacock,  Behavioral  Biology 
Branch,  National  Institute  of  Child  Health  and  Human 
Development . 

Project  Description: 

Mutant  clones  of  mouse  neuroblastoma  were  selected  by  mutagenesis  and 
exposure  to  6-thioguanine.   The  electrically  excitable  neuroblastoma  cells 
were  fused  with  electrically  passive  L  cells  having  a  hitherto  undescrlbed 
electrical  marker.   Hybrid  clones,  examined  10-40  generations  after  fusion, 
were  found  to  be  electrically  excitable.   The  results  show  that  at  least 
part  of  the  genetic  information  from  neuron  differentiation  can  be  function- 
ally expressed  in  neuroblastoma  x  L  cell  hybrids.   No  evidence  for  a  pleio- 
troplc  repressor  terminating  neuron  differentiation  was  found.   In  fact, 
most  N  X  L  cell  hybrids  were  more  active  electrically  than  the  parental 
neuroblastoma  line.   Somatic  cell  hybrldlzatlcn  applied  to  normal  neuro- 
blasts should  provide  a  relatively  simple  means  of  establishing  clonal  lines 
of  cells  derived  from  different  types  of  neurons.   The  levels  of  acetyl- 
cholinesterase of  N  X  L  cell  hybrid  clones  were  determined  30-50  cell  gener- 
ations after  cells  were  fused.   The  neuroblastoma  parent  and  all  N  x  L  cell 
hybrids  contained  acetylcholinesterase;  appreciable  amounts  of  enzyme  were 
not  detected  in  the  L  cell  parent  or  in  L  x  L  hybrids.   The  results  show 
that  neuroblastoma  genes  for  acetylcholinesterase  continue  to  be  expressed 
after  fusion. 

Results  obtained  with  neuroblastoma  clearly  show  that  rapidly  dividing 
cells  still  retain  the  ability  to  express  neuronal  functions  and  that  some 
neuronal  genes  remain  active  in  somatic  cell  hybrids.   The  possibility  that 
neurons  may  be  capable  of  initiating  a  program  of  neuron  differentiation  in 
recipient  cells  also  deserves  consideration. 


Honors  and  Awards 


None. 


j:te) 


Serial  No.  NHLI-127 


Publications ; 


Minna,  John,  Nelson,  Phillip,  Peacock,  John,  Glazer,  Devera  and 
Nirenberg,  Marshall:   Genes  for  neuronal  properties  expressed  in  neuro- 
blastoma X  L  cell  hybrids,   Proc.  Natl.  Acad.  Sci.  68:  234-239,  1971. 

Nelson,  P.  G. ,  Peacock,  H.  H. ,  Amano,  T.  and  Minna,  J.:   Electrogenesis 
in  mouse  neuroblastoma  cells  in  vitro.   J.  Cell.  Physiol.,  in  press. 


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Nelson,  P.  G.,  Peacock,  H.  H. ,  and  Amano,  T. :   Responses  of  neuro- 
blastoma cells  to  iontophoretically  applied  acetylcholine.   J.  Cell. 
Physiol. ,  in  press. 


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Serial  No.    NHLI-128 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Acetylcholinesterase  in  Neuroblastoma  Cells. 

Previous  Serial  Number:   None 

Principal  Investigator:   Arthur  J.  Blume 

Other  Investigator:   Marshall  Nirenberg 

Cooperating  Units:   None 

Project  Description: 

We  have  previously  shown  that  neuroblastoma  cells  contain  acetyl- 
cholinesterase and  both  excitatory  and  inhibitory  acetylcholine  receptors 
and  that  these  factors  respond  to  regulatory  mechanisms  in  vitro.   Thus, 
purification  and  characterization  of  the  factors  were  initiated  to  define 
the  relationship  between  acetylcholinesterase  and  acetylcholine  receptors 
and  the  nature  of  the  regulatory  mechanisms.   Acetylcholinesterase  was  found 
to  be  associated  with  membranes;  however,  the  enzyme  now  has  been  purified 
extensively  and  is  now  being  characterized. 

Honors  and  Awards:   None. 

Publications : 

Blume,  A.,  Gilbert,  F. ,  Wilson,  S.,  Farber,  J.,  Rosenberg,  R.  and 
Nirenberg,  M. :   Regulation  of  acetylcholinesterase  in  neuroblastoma 
cells.   Proc.  Natl.  Acad.  Sci.  U.S.A.  67:  786-792,  1970. 


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Serial  No.        NHLI-129 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Cyclic  AMP  Metabolism  in  Neuronal  and  Glial  Clonal  Cell  Lines 

Previous  Serial  Number:   None 

Principal  Investigator:   Alfred  G.  Gilman 

Other  Investigator:   Marshall  Nirenberg 

Cooperating  Units :   None 

Project  Description: 

A  simple,  rapid  assay  for  adenosine  3' 5 '-cyclic  monophosphate  was 
developed  that  is  based  upon  competition  between  labeled  and  unlabeled  cAMP 
for  a  protein,  presumably  a  cAMP-dependent  protein  kinase.   The  nucleotide- 
protein  complex  is  then  adsorbed  and  washed  on  a  cellulose  ester  filter. 
The  assay  is  sensitive  to  0.05-0.10  pmoles  of  cAMP,  and  is  being  used  to 
explore  cAMP  metabolism  in  neuroblastoma  and  other  cell  types  that  are  grown 
in  vitro. 


Honors  and  Awards: 
Publications : 


None 


Gilman,  A.G.:   A  protein  binding  assay  for  adenosine  3*5 '-monophosphate, 
Proc.  Natl.  Acad.  Sci.  67:  305-312,  1970. 

Rail,  T.  W.  and  Gilman,  A.G.:   The  role  of  cyclic  AMP  in  the  nervous 
system.   Neurosciences  Research  Program  Bulletin  8:  221-323,  1970. 


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ANNUAL  REPORT  OF  THE 
LABORATORY  OF  BIOCHEMISTRY 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1970  through   June  30,  1971 

SECTION  ON  ENZYMES 

Research  in  the  Section  on  Enzymes  has  been  concerned  with  the 
cellular  regulation  of  nitrogen  metabolism,  amino  acid  dissimilation, 
the  mechanism  of  action  of  vitamine  Bi2  coenzymes,  the  anaerobic  dis- 
similation of  nicotinic  acid  and  the  regulation  of  homocysteine  bio- 
synthesis. 

Regulation  of  Glutamine  Metabolism,  (a)  Glutamine  synthetase.   Previous 
studies  demonstrated  that  the  activity  of  glutamine  synthetase  (GS)  in 
E.    coli  is  regulated  by  adenylylation  and  deadenylylation  of  a  single 
tyrosyl  hydroxyl  group  on  each  one  of  the  enzyme's  12  subunits  (Reactions 
1  and  2). 


GS  +  12  ATP  ?^  GS  (AMP) 12  +  12  PPi 
GS  (AMP)i2  +  12  Pi  ?=^  12  ADP 


(1) 
(2) 


Adenylylation  is  accompanied  by  changes  in  catalytic  potential,  in  divalent 
cation  requirement  (from  Mg++  to  Mn++) ,  in  pH  optimum,  and  in  sensitivity 
to  inhibition  by  end  products  of  glutamine  metabolism.   The  average  state 
of  adenylylation  is  determined  by  the  relative  rates  of  reactions  1  and  2 
which  are  modulated  in  reciprocal  fashion  by  concentrations  of  glutamine, 
a-ketoglutarate,  UTP  and  ATP.   It  has  now  been  established  that  deadenylyl- 
ation involves  phosphorolysis  (rather  than  hydrolysis  as  previously  assumed) 
of  the  phos|ihodiester  linkage  binding  AMP  to  enzyme,  yielding  ADP  as  the 
reaction  product  (reaction  2) .   The  energy  of  the  adenylyltyrosine  bond  is 
thus  conserved  in  the  generation  of  a  pyrophosphate  bond.   It  was  further 
established  that  a  single  protein  complex  (Pj)  containing  one  adenylyltrans- 
f erase  subunit  (ATase  subunit ,  MW  =  70,000  MW)  and  a  subunit  required  for 
deadenylylation  (DA  subunit,  MW  =  60,000)  is  involved  in  catalyzing  both 
reactions  1  and  2.   Either  Pj  alone  or  its  ATase  subunit  by  themselves  can 
catalyze  reaction  1;  however,  intact  Pi  is  required  for  reaction  2,  and  its 
catalytic  potential  and  sensitivity  to  metabolite  control  is  dependent  upon 
a  second  protein  component  (Pn)  of  50,000  MW.   With  different  enzyme  pre- 
parations (Pj  plus  Pxi)  ,  the  sensitivity  of  reaction  2  to  activation  by 
a-ketoglutarate,  ATP  and  UTP  and  its  inhibitability  by  glutamine  are  variable. 
The  possibility  that  the  activity  of  Pn  is  also  modulated  by  covalent 
attachment  of  a  nucleotide  is  indicated  by  its  inactivation  upon  exposure 
to  snake  venom  phosphodiesterase. 

It  has  been  established  that  E^.  coli  contains  multiple  molecular 
forms  (hybrid  molecules)  of  glutamine  synthetase  that  differ  from  one 
another  in  the  number  (0  to  12)  and  distribution  of  adenylylated  subunits. 
Heterotropic  interaction  between  adenylylated  and  unadenylylated  subunits 
in  these  hybrid  enzyme  molecules  lead  to  marked  changes  in  catalytic 
potential,  in  affinities  for  substrates  and  in  susceptibility  to  urea 
denaturation.   Hybrids  produced  by  subunit  dissociation  and  reassociation 


iar- 


I 


of  mixtures  of  adenylylated  and  unadenylylated  enzymes  are  indistinguishable 
from  naturally  occurring  hybrid  forms  produced  by  partial  adenylylation 
or  deadenylylative  reactions. 

[  I 
From  the  relationship  between  specific  activity  of  Co   activated 
enzyme  and  the  average  state  of  adenylylation  it  is  deduced  that  adenylyl- 
ation of  a  subunit  leads  to  its  inactivation  and  (because  of  heterotropic 
interactions)  to  nearly  complete  inactivation  of  unadenylylated  subunits 
that  are  in  direct  contact  with  the  adenylylated  subunit.   It  is  a  con- 
sequence of  such  heterologous  interactions  that  the  greatest  effect  on 
catalytic  activity  is  achieved  by  the  adenylylation  of  only  1  to  3  of  the 
enzyme's  12  subunits  and  almost  complete  inactivation  is  obtained  when 
only  9  subunits  are  adenylylated.   This  non-linear  relationship  between 
catalytic  potential  and  state  of  adenylylation  could  be  of  physiological 
significance  since  the  affinity  of  the  adenylyltransf erase  for  glutamine 
synthetase  decreases  rapidly  with  state  of  adenylylation,  making  complete 
adenylylation  difficult. 

Differential  effects  of  various  divalent  cations  (i.e.,  Co"*"*",  Mg   , 
Mn++,  Ca++  and  Zn++)  on  pH  optimum,  affinity  for  substrates,  catalytic 
potential,  inhibitability  by  end  products  of  gl-utamine  metabolism  and  U.V. 
spectrum  of  glutamine  synthetase  indicate  that  each  cation  provokes  a  unique 
conformation  of  the  enzyme. 

Calorimetric  studies  of  the  interaction  between  divalent  cations  and 
enzyme  (carried  out  in  collaboration  with  Dr.  P.D.  Ross  of  NIAMD)  show  that 
2  protons  are  displaced  from  the  enzyme  during  the  binding  of  each  of  the 
first  12  equivalents  of  either  Mn++  or  Mg"++  to  the  enzyme.   One  proton  is 
released  instantaneously  and  the  second  in  a  slow  process  (half  time  =  0.5  - 
0.9  min)  probably  involving  a  conformational  change  in  the  protein.   The 
kinetics  of  this  slow  reaction  are  similar  to  those  of  the  spectral  shifts 
that  accompany  binding  of  Mn++  or  Mg++.   Despite  these  rather  large  per- 
turbations in  the  microenvironment  of  certain  aromatic  amino  acids  that 
accompany  binding  of  divalent  cations  to  glutamine  synthetase,  these  cations 
have  no  demonstrable  effect  on  the  secondary  or  quaternary  structure  as 
determined  by  circular  dichroism  (CD)  and  optical  rotatory  dispersion 
measurements  (310-218  nm) .   From  CD  measurements,  it  was  estimated  that  the 
ot-helical  content  of  native  glutamine  synthetase  is  37%. 

In  an  effort  to  distinguish  between  unique  configurations  of  un- 
adenylylated enzyme  (E's)  ,  adenylylated  enzyme  (E]^)  and  their  dissociated 
subunits,  antibodies  were  prepared  to  each  species  of  enzyme  and  subunits. 
Eo  and  Ej^   could  not  be  distinguished  from  one  another  on  the  basis  of  com- 
plement fixation  using  antiserum  to  either  form  of  enzyme.   The  results 
reinforce  conclusions  based  on  other  data  that  adenylylation  does  not 
provoke  a  substantial  change  in  enzyme  structure.   However,  differences 
in  behavior  of  antibodies  prepared  to  subunits  indicate  that  adenylylation 
introduces  a  distinct  antigenic  determinant  on  the  enzyme.   A  cross  reaction 
between  antibodies  of  E.    coli  glutamine  synthetase  and  the  syn^^h^tases  from 
Salmonella  typhimurium,  Klebsiella  pneumoniae,  Pseudomonas  putida  and 
Azotobacter  vinelandii  indicate  a  significant  degree  of  homology  bewteen 
these  enzymes.   No  cross  reaction  was  obtained  with  glutamine  synthetases 


ja.6 


from  various  species  of  Bacilli,  Clostridia ,  Streptomyces ,  Neurospora, 
Methanosarcina  or  Acanthomoeba.   (b)  Glutamate  Synthetase.   Tempest,  et  al. 
(Biochem.  J.  117,  405  (1970))   recently  reported  the  discovery  of  a  new 
enzyme,  glutamate  synthetase,  that  catalyzes  the  TPNH  dependent  conversion 
of   a-ketoglutarate  and  glutamine  to  two  moles  of  glutamate.  (reaction  1). 

Glutamine  +  a-ketoglutarate  +  TPNH  -y  2  glutamate  +  TPN  (1) 

Glutamate  +  NH4+  +  ATP  — >  Glutamine  +  ADP  +  Pi  (2) 

a-Ketoglutarate  +  NH4+  +  ATP  +  TPNH  -v-  glutamate  +  ADP+TPN+Pi        (3) 

When  coupled  with  glutamine  synthesis  (reaction  2)  this  new  reaction  leads 
to  the  overall  reaction  3  which  represents  a  new  highly  exergonic  pathway 
for  the  synthesis  of  glutamate.   The  new  glutamate  synthetase  has  now  been 
purified  to  near  homogeneity  from  extracts  of  E^.  coli :   It  has  a  molecular 
weight  of  about  750,000  as  determined  by  ultracentrifugation  and  gel 
filtration.   The  S20w  -   22S;  pH  optimum  =  7.8.   The  protein  contained  1  mole 
of  bound  flavin  (FAD  +  FMN)  per  200  gms  which  was  reduced  by  TPNH  but  not  by 
DPNH.   The  apparent  dissociation  constants  (Kms)  for  glutamine  and   a-keto- 
glutarate are  170  and  30  uM  respectively.   These  Kms  and  the  low  Kms  for  the 
substrates  of  glutamine  synthetase  permit  rapid  synthesis  of  glutamate  by 
reaction  3  at  concentrations  of  NH4"^  that  preclude  significant  glutamate 
formation  by  the  classical  glutamate  dehydrogenase.   Therefore  the  new 
pathway  of  glutamate  synthesis  becomes  important  when  the  intracellular 
concentration  of  NH^"*"  is  low.   Its  role  in  glutamate  synthesis  is  further 
indicated  by  the  fact  that  its  formation  is  repressed  by  high  concentration 
of  glutamate.   It  is  noteworthy  that  in  this  new  pathway  glutamine  is  a 
precursor  of  glutamate  synthesis,  and  therefore  of  all  amino  acids  derived 
from  glutamate  either  directly  or  via  transamination  reactions.   The  pre- 
viously unexplained  inhibitions  of  glutamine  synthetase  by  alanine,  glycine 
or  serine  are  now  recognized  as  simple  cases  of  feedback  inhibition  of  the 
first  enzyme  in  the  biosynthetic  pathway  by  ultimate  end  products. 
(c)   Glutaminase.   It  is  known  that  E.  coli  contains  two  glutaminases  with 
different  pH  optima  (pH  5  and  pH  7) .   As  part  of  the  study  on  regulation 
of  nitrogen  metabolism  in  E.    coli,  the  properties  of  these  glutaminases  were 
investigated.   Growth   studies  with  E.    coli  B  showed  that  the  pH  5  enzjone 
increases  markedly  during  stationary  phase  whereas  the  pH  7  enzyme  is 
highest  during  the  log  phase  and  early  stationary  phase.   Preliminary 
experiments  indicate  that  highest  activity  of  the  pH  7  enzyme  is  obtained  in 
the  presence  of  excess  nitrogen  and  growth  is  limited  by  carbohydrate 
supply.   The  pH  7  enzyme  has  been  purified  over  1700  fold  from  crude  extracts. 
It  is  a  cold-labile  enzyme  and  is  inactivated  at  0°  and  is  partially 
reactivated  by  either  glutamine  or  glutamate.   The  enzyme  is  stabilized  by 
glutamate  and  borate.   It  exhibits  complex  kinetics  suggestive  of  cooperative 
substrate  interactions  and  has  almost  no  activity  at  pH  5. 

Amino  Acid  Metabolism.   The  B12  coenzyme  dependent  lysine  mutases  of 
Clostridium  sticklandii  and  Clostridium  M-F  have  been  studied  in  detail 
as  regards  reaction  mechanism  and  characterization  of  the  proteins.   Both 
a-D-lysine  mutase  and  L-3-lysine  mutase  consist  of  two  dissimilar  protein 
moieties,  and  B12  coenzyme  serves  as  carrier  of  the  migrating  hydrogen  in 
each  reaction.   The  cobalamide  protein  moiety  of  a-D-lysine  mutase  contains 


m 


■'n 


Ja7 


c 


pyridoxal 
substrate 
mutase  re 
carbonyl 
much  more 
are  other 
propertie 
pathways- 


phosphate  and  this  binds  the  w-amino  group  of  the  amino  acid 
s  and  probably  serves  as  intermediate  amino  group  carrier  in  the 
action.   L-a-lysine  mutase  appears  to  contain  pyruvate  as  its 
compound  rather  than  pyridoxal  phosphate.   Both  of  the  mutases  are 
complicated  in  their  requirements,  such  as  activation  by  ATP,  than 
Bi2  coeazyme  linked  enzymes  previously  studied.   Their  complex 
s  are  probably  involved  in  regulation  of  the  two  parallel  metabolic 
fermentation  of  D-a-lysine  and  L-a-lyf.ine  in  which  they  participate. 


The  product  of  L-g-lvsin-^  mutase,  3,5-diaminohexanoic  acid,  contains 
two  asymmetric  centers  (C-3  and  C-5)  giving  rise  to  two  pairs  of  enantiomers 
that  differ  in  their  melting  points.   Since  a  knowledge  of  the  steriochem- 
istry  of  the  enzymic  product  would  aid  in  understanding  the  mechanism  of  the 
mutase  reaction,  these  isomers  were  obtained  by  chemical  synthesis.   It  has 
been  established  that  only  the  low  melting  isomer  is  enzymatically  active. 
From  NMR  spectral  studies  of  benzoylated  amino  lactam  derivatives  of  the 
isomeric  amino  acid  it  was  deduced  that  the  enzymatically  active  isomer  has 
the  erythro  configuration.   Since  the   6-lysine  substrate  has  the  S-con- 
figuration  the  new  asymmetric  center,  C-5,  in  3,5-diaminohexanoic  acid  must 
therefore  have  an  S-conf iguration. 

The  peptide  growth  factor  requirement  of  Clostridium  sticklandii 
is  satisfied  by  a  fraction  isolated  from  a  casein  digest  that  contains 
only  two  major  acidic  peptide  components.   The  amino  acid  composition  of 
this  peptide  fraction  is  glutamic  acid,  valine,  leucine,  isoleucine,  alanine 
and  some  serine  and  threonine.   Based  on  molecular  weight  estimations,  the 
active  peptide (s)  should  consist  of  5  to  7  amino  acid  residues.   Experiments 
are  currently  in  progress  to  achieve  final  isolation  and  determination 
of  structure  of  the  required  peptide. 

The  ATP-forming  glycine  reductase  system  of  C^.  sticklandii,  after 
fractionation  into  three  soluble  highly  purified  protein  components  still 
shows  an  absolute  requirement  for  orthophosphate  in  order  to  reduce  glycine 
to  acetate  and  ammonia  with  dithiothreitol  as  electron  donor.   Marked 
sensitivity  to  hydroxylamine  suggests  a  carbonyl  compound  cof actor  is  involved. 

Methane  biosynthesis.    Since  the  early  reduction  steps  in  methane  formation 
from  carbon  dioxide  and  formate  are  completely  unknown,  I'^C  formate  reduction 
to  methane  by  Methanococcus  vannielii  extracts  is  being  investigated.   A 
nucleotide  fraction  in  the  extracts,  which  becomes  highly  radioactive 
during  the  reaction,  has  been  purified  and  shown  to  serve  as  a  precursor  of  ■; 
labeled  methane.   Chemical  characterization  of  this  radioactive  material, 
now  in  progress,  should  determine  whether  it  has  an  intermediary  role  in 
the  process.   Marked  stimulation  of  methane  biosynthesis  from  formate  by 
several  one-carbon  derivatives  of  uracil  suggests  that  the  presumed  inter- 
mediate may  be  a  modified  uridine  nucleotide. 

Anaerobic  dissimilation  of  nicotinic  acid.   In  the  nicotinic  acid  fermentation 
pathway  the  obligatory  roles  of  two  inducible  enzjTnes  which  together  re- 
arrange the  carbon  skeleton  of  the  original  substrate  to  a  symmetrical  inter- 
mediate account  for  the  results  of  earlier  isotope  studies.   One  of  these 
enzymes,  the  B]^2  coenzyme-dependent   a-methyleneglutarate  mutase,  catalyzes 


:J3.0 


a  carbon-carbon  bond  cleavage  reaction  and  this,  followed  by  a  second  iso- 
merization  catalyzed  by  methylitaconate  isomerase,  forms  the  symmetrical 
dicarboxylic  acid,  dimethylmaleic  acid.   The  mechanisms  of  the  hydrogen 
transfers  catalyzed  by  both  enzymes  have  been  studied. 

Regulation  of  methionine  biosynthesis.   Previous  work  in  this  laboratory  has 
shown  that  the  synthesis  of  homocysteine,  the  immediate  precursor  of 
methionine,  is  accomplished  in  3  sequential  steps,  which  differ  slightly 
in  different  microorganisms,  and  are  catalyzed  by:  a)  homoserine  transacylase, 
utilizing  succinyl  CoA  in  Salmonella  and  acetyl  CoA  in  Neurospora;  b)  cysta- 
thionine  Y~synthase;  and  c)   g-cystathionase.   In  Neurospora  the  cysta- 
thionine synthase,  which  had  been  shown  to  be  subject  to  end  product 
inhibition  by  S-adenosylmethionine,  has  now  been  found  to  be  completely 
inactive  in  the  absence  of  polyglutamate  derivatives  of  N^-methyltetra- 
hydrofolate.   This  allosteric  activation  serves  to  prevent  over-production 
of  homocysteine  in  the  absence  of  the  methylfolate  needed  for  its  conversion 
to  methionine.   It  is  noteworthy  that  in  Neurospora  it  is  the  second  enzyme 
of  the  homocysteine  synthetic  pathway  that  is  subject  to  regulation.   The 
intermediary  role  of  cystathionine  in  methionine  synthesis  in  yeast  is  still 
uncertain.   Now  significant  cystathionine  synthase  activity  can  be  detected 
in  extracts  of  yeast  protoplasts,  but  the  reaction  is  not  affected  by 
addition  of  methylfolates  and  is  slower  than  the  direct  formation  of  homo- 
cysteine from  acetylhomoserine  and  sulfide.   In  Salmonella  also,  where  the 
first  enzyme  of  the  pathway  is  subject  to  end  product  inhibition,  a  re- 
gulatory role  for  methylfolates  could  not  be  demonstrated.   The  mechanism 
regulating  the  synthesis  of  the  transsuccinylase  in  Salmonella  appears  to  be 
different  from  that  regulating  the  second  and  third  enzjrmes.   When  the 
growth  rate  of  a  mutant  which  responds  to   either  methionine  or  vitamin  B12 
is  slowed  in  a  chemostat  by  limiting  the  concentration  of  methionine  the 
second  and  third  enzymes  are  derepressed  in  a  normal  fashion,  but  it  is 
virtually  impossible  to  derepress  the  trans-succinylase.   This  result  is  not 
observed  with  vitamin  62^2  limitation,  suggesting  that  exogenous  methionine 
is  a  preferential  source  of  a  component  involved  in  controlling  the 
synthesis  of  the  transsuccinylase. 


Jlf 


i 


i 


Annual  Report  of  the 

Section  on  Cellular  Physiology 

Laboratory  of  Biochemistry 

National  Heart  and  Lung  Institute 

July  1,  197  0  through  June  30,  1971 

The  research  program  of  the  Section  on  Cellular  Physiology  continues  to  be 

directed  toward  two  broad  areas  of  biochemistry  and  cellular  physiology; 

structure  and  structure-function  relationships  of  proteins  on  the  one 

hand  and  the  role  of  cell  membranes  in  synthetic  activities  of  cells  on 

the  other,  encompassing  programs  in  the  areas  of  (1)  the  structure  and 

biochemical  activities  of  the  proteins  of  the  contractile  system  of  muscle; 

(2)  the  structure  of  fibrinogen;  (3)  radiation  damage  in  proteins;  and 

(4)  the  mechanism  of  protein  synthesis  and  its  relationship  to  cell  structure. 

The  following  is  a  resume  of  some  major  developments  in  the  research  of 

the  section. 

Proteins  of  the  contractile  system  of  muscle. 
Myosin-actin  interactions: 

It  is  now  generally  recognized  that  contraction  of  muscle  involves  the 
interaction  of  the  two  proteins,  actin  and  myosin,  with  ATP.   Clearly,  it 
is  of  importance  to  determine  the  strength  of  binding  and  the  binding  ratio 
of  actin  to  myosin  both  in  the  presence  and  absence  of  ATP.   While  there  is 
a  voluminous  literature  on  the  characteristics  of  actin-myosin  complexes, 
no  stoichiometry  can  be  deduced  for  this  system  under  conditions  of 
physiological  ionic  strength  where  both  proteins  exist  as  aggregates.   How- 
ever, heavy  meromyosin  (HMM)  a  prodvict  of  brief  tryptic  digestion  of  myosin 
constituting  about  7  5%  of  the  mass  of  the  myosin  molecule  is  soluble  and 
monomeric  at  low  ionic  strength  and  its  binding  to  actin  can  be  studied  in 
the  analytical  ultracentrifuge. 


Actin  under  conditions  of  physiological  ionic  strength,  or  higher,  exists 
as  a  double  stranded  linear  polymer  and  rapidly  sediments  in  the  ultra- 
centrifuge.   In  binding  experiments  with  HMM  any  unbound  HMM  is  left  in  the 
supernatant  and  can  be  quantitated  with  the  photoelectric  scanning  system. 
Such  studies  under  a  variety  of  conditions  of  ionic  strength,  temperature 
and  initial  actin  concentrations,  have  demonstrated  that  the  maximum  molar 
binding  ratio  of  HMM: actin  monomer  in  the  absence  to  ATP  is  1:2.   Further- 
more, under  all  these  conditions,  the  binding  constant  was  too  high  to 
measure  —  greater  than  2X10^M. 


This  binding  is  many  fold  stronger  than  that  in  the  presence  of  ATP  as 
deduced  from  kinetic  measurements.   The  binding  ratio  of  1:2  is  in  agreement 
with  the  fact  that  myosin  and  HMM  are  2-headed  molecules  so  that  apparently 
each  head  binds  to  a  separate  actin  monomer. 

In  the  presence  of  ATP  actin  strongly  activates  the  HMM  ATPase  and  therefore 
the  extent  of  actin-HMM  binding  can  be  estimated  from  kinetic  measurements. 
These  measurements  suggest  that  in  the  presence  of  ATP  the  binding  between 
actin  and  HMM  is  much  weaker  tte n  in  the  absence  of  ATP,   In  an  effort  to 
directly  examine  the  binding  in  the  presence  of  ATP,  the  analytical 


53o 


ultracentrifuge  was  used.   Unexpectedly  it  was  found  that  under  conditions 
where  kinetic  measurements  suggest  that  the  actin  and  HMM  are  completely 
complexed,  the  ultracentrifuge  showed  that  50%  of  the  HMM  was  in  fact 
dissociated  from  the  actin.   This  suggests  that  during  the  cycle  of  actin- 
HMM  interaction,  much  of  the  HMM  exists  in  a  "refractory"  form  in  which  it 
cannot  bind  to  actin.   Since  HMM  is  a  2-headed  molecule,  it  is  quite  likely 
that  only  one  head  of  the  molecule  emerges  from  its  refractory  period  at  a 
time,  so  that,  in  effect,  only  one  head  of  the  HMM  binds  to  actin  in  the 
presence  of  ATP.   This  would  of  course  contrast  with  the  situation  in  the 
absence  of  ATP  where  both  heads  bind  simultaneously  and  this  difference  in 
the  number  of  heads  binding  might  account  for  the  strong  binding  which  occurs 
in  the  absence  of  ATP  as  opposed  to  the  weak  binding  in  the  presence  of  ATP. 

The  observation  that  much  of  the  HMM  is  in  a  refractory  form  where  it 
cannot  bind  to  actin  in  the  presence  of  ATP  may  have  significant  implications 
for  the  situation  in  vivo.   The  flexability  of  contracting  muscle  as  opposed 
to  rigor  muscle  may  occur  because  in  the  rigor  state  all  of  the  myosin 
bridges  are  bound  to  the  actin  filament  whereas  in  contracting  muscle  only 
a  fraction  of  these  bridges  are  bound.   In  support  of  this  hypothesis 
X-ray  diffraction  studies  of  contracting  muscle  show  that  less  than  50% 
of  the  myosin  bridges  are  closely  associated  with  actin.   Therefore  the 
occurrence  of  a  refractory  period  during  which  the  myosin  molecule  cannot 
bind  to  actin  may  play  a  key  role  in  the  cyclic  interaction  of  actin  and 
myosin  which  causes  contraction  in  vivo. 

Actin: 


G-Actin,  the  major  protein  of  the  thin  filaments  in  the  contractile  appara- 
tus of  muscle  —  and,  apparently,  of  other  cells  as  well  —  has  a  molecular 
weight  of  45,000  and  consists  of  a  sequence  of  410  amino  acid  residues.   As 
an  initial  goal  in  elucidating  the  primary  structure  of  actin,  it  was 
decided  to  attempt  isolation  of  all  of  the  peptide  fragments  of  actin 
resulting  from  cyanogen  bromide  cleavage  of  the  molecule.   Cyanogen  bromide 
specifically  attacks  methionyl  residues  cleaving  the  adjacent  peptide  bond 
and  converting  the  methionine  to  carboxyl  terminal  homoserine  of  the 
resulting  peptide  fragments.   Siibsequent  isolation  of  all  of  the  methionine 
containing  peptides  resulting  from  hydrolysis  of  actin  by  trypsin  should 
result  in  a  set  of  overlapping  peptides.   Matching  up  of  these  two  sets  of 
peptides  should  then  result  in  the  ordering  of  the  cyanogen  bromide 
fragments  and  provide  a  base  for  further  sequence  analysis.   There  are 
sixteen  methionine  residues  in  actin  leading  to  seventeen  cyanogen  bromide  t> 

fragm.ents.   The  previous  report  dealt  with  the  isolation  of  all  seventeen 
of  these  and  the  results  have  been  published.   With  respect  to  the  sixteen 
overlaps  all  but  one  have  been  successfully  identified.   Although  the 
missing  overlap  can  be  inferred,  efforts  continue  to  positively  identify 
it.   With  the  placing  of  the  seventeen  cyanogen  bromide  peptides  in  sequence 
the  initial  goal  of  this  project  is  complete.   From  this  information  and 
sequence  studies  in  another  laboratory  we  now  have  about  70%  of  the  410 
amino  acid  residues  of  actin  in  sequence. 

Myosin: 

The  myosin  molecule  is  composed  to  two  identical  polypeptide  chains  each 

of  200,000  molecular  weight.   In  addition  about  15%  of  the  mass  of  all 

2  53/ 


i 


I 


native  myosin  preparations  consist   of  smaller  polypeptide  chains  of  about 
20,000  molecular  weight.   It  has  not  been  possible,  so  far,  to  separate 
the  latter  material  from  the  large  chains  except  under  denaturing  conditions. 
While  the  small  chains  are  widely  regarded  as  a  functional  part  of  the 
myosin  molecule,  this  has  not  been  proved.   No  stoichiometry  in  the  structure 
has  been  demonstrated,  a  problem  that  is  complicated  by  the  fact  that  this 
low  molecular  weight  fraction  is  composed  of  four  components  in  unequal 
amounts.   Conflicting  figures  in  the  literature  are  probably  confused  by  the 
fact  that  myosin  prepared  by  "standard"  laboratory  procedures  always  contains 
5-10%  of  low  molecular  weight  contaminants  readily  removable  by  any  one  of 
three  chromatographic  procedures.   Some  time  ago,  before  the  presence  of 
the  small  chains  in  myosin  was  recognized  it  was  observed  in  this  laboratory 
that  myosin  possessed  histidine  as  an  amino  terminal  group.   Subsequently, 
another  laboratory  reported  the  presence  of  a  blocked  N-terminal,  identified 
as  acetyl  serine.   It  has  now  been  determined  that  the  large  chains  of 
myosin  possess  the  amino  terminal  histidine,  while  the  small  chain  fraction 
possesses  one  component,  readily  separable  from  the  rest  by  chromatography, 
which  contains  a  blocked  amino  terminal,  tentatively  identified  as  acetyl 
serine.   In  addition,  the  small  chain  fraction  possesses  amino  terminal 
alanine,  about  six-tenths  of  a  mole  per  mole  of  small  chains,  and  a  minor 
amount,  about  0.1  mole,  of  either  aspartic  or  glutamic  acid.   Disc  gel 
electrophoresis  of  the  small  chains  shows  the  presence  of  two  major  and 
two  minor  components.   With  the  high  recovery  of  N-terminal  alanine  it 
must  be  assumed  that  two  of  the  four  components  have  amino  terminal  alanine 
and  the  other  two  recognized  N-terminal  structures  account  for  the  other 
two  components. 

Myosin  from  human  blood  platelets: 

Starting  with  human  platelet  concentrates,  two  myosin-like  proteins  have 
been  isolated.   Both  proteins,  like  myosin,  have  ATPase  activity  that  is 
stimulated  by  Ca""""*"  and  EDTA  and  inhibited  by  Mg+'''.   Both  proteins  bind 
to  rabbit  skeletal  actin  and  are  released  by  Mg-ATP.   In  the  details  of 
their  enzyme  activities  these  two  proteins  resemble  smooth  muscle  myosin. 
The  two  proteins  differ  in  molecular  weight.   On  disc  gel  electrophoresis 
in  the  presence  of  sodium  dodecylsulfate  the  heavier  of  the  two  proteins, 
coelectrophoreses  with  the  heavy  chains  (200,000)  of  rabbit  skeletal 
myosin.   The  lighter  of  the  two  proteins  shows  a  subunit  molecular  weight 
of  about  100,000  in  the  same  electrophoretic  system. 

Electron  microscopic  confirmation  of  structural  similarities  to  myosin  has 
also  been  obtained.   The  heavier  of  the  platelet  proteins  forms  aggregates 
at  low  salt  concentration  which  are  similar  to  the  "thick  filaments" 
formed  by  muscle  myosin.   Moreover,  the  lighter  species  forms  the 
"arrowhead"  structures  with  rabbit  skeletal  actin  characteristic  of  the 
interaction  of  skeletal  muscle  heavy  meromyosin  and  actin. 


33X 


Annual  Report  of  the 
Section  on  Cellular  Biochemistry  and  Ultrastructure 
Laboratory  of  Biochemistry 
National  Heart  and  Lung  Institute 
July  1,  1970  through  June  30,  1971 

Research  has  continued  in  the  three  related  areas  of:   1)  the  molecular  and 
ultrastructural  basis  of  movement  in  motile,  non-muscle  cells,  2)  the  bio- 
chemistry and  ultrastructure  of  the  plasma  membrane,  and  3)  phagocytosis  and 
pinocytosis .   In  general  the  soil  amoeba,  Acanthamoeba  castellanii ,  has  pro- 
vided the  experimental  material  but  none  of  the  phenomena  under  study  is 
unique  to  amoebae .   The  amoeba  does  provide  a  readily  cultured,  homogeneous 
population  of  cells  which  are  relatively  highly  motile,  which  are  nutrition- 
ally exclusively  dependent  on  phagocytosis  and  pinocytosis,  and  which  may 
have  a  simpler  plasma  membrane  than  mammalian  cells  . 

(1)  The  analogy  between  amoeba  actin  (obtained  as  a  pure  protein  last  year 
in  this  laboratory)  and  muscle  actin  has  been  developed  further  by  demonstra- 
ting major  similarities  in  the  amino  acid  composition  of  three  of  the  peptides 
produced  by  cleavage  of  each  protein  by  cyanogen  bromide.   One  of  these,  the 
peptide  that  contains  the  unusual  amino  acid  3-methylhistidine,  may  have 
identical  amino  acid  composition  in  the  amoeba  and  rabbit  actins  . 

We  have  now  purified  some  200-fold  an  amoeba  ATPase  with  enzymatic  properties 
essentially  identical  to  those  of  muscle  myosin;  i.e.,  a  K  ,  EDTA- stimulated 
ATPase  that  is  also  active  as  a  Ca   -ATPase  but  which  is   inhibited  by  Na   and 
Mg   .   The  amoeba  ATPase  is  activated  30-fold  by  the  addition  of  muscle  actin 
in  the  presence  of  Mg"^.  Although  myosin-like  ATPases  have  previously  been 
found  in  other  motile  cells  this  is  the  first  instance  in  which  it  has  been 
possible  to  show  activation  of  a  non-muscle  ATPase  by  actin.   A  very  interest- 
ing observation  has  been  that  the  amoeba  ATPase  seems  to  have  a  molecular 
weight  of  about  130,000  which  is  much  lower  than  the  molecular  weights  of  all 
other  myosin-like  ATPases  (about  450,000)  .   This  difference  may  explain  the 
inability  thus  far  to  demonstrate  the  presence  of  thick,  myosin-like  filaments 
in  the  amoebae.   Thin,  actin  filaments  are  present. 

The  biochemical  and  ultrastructural  studies  with  the  Acanthamoeba  have  pro- 
vided some  of  the  strongest  evidence  for  the  fundamental  similarities 
between  the  processes  of  cell  motility  and  muscle  contraction. 

(2)  Last  year  we  reported  the  procedure  for  the  isolation  of  highly  purified 
amoeba  plasma  membranes  which  contain  a  very  active  alkaline  phosphatase,  and 
described  the  ratio  of  lipid  to  protein  and  the  composition  of  the  phospho- 
lipids, sterols  and  glycerides .  We  have  now  found  that  the  amoeba  plasma 
membrane  contains  a  uniquely  high  concentration  of  non-lipid  phosphorus 

(1  |imole/mg  protein)  and  a  very  high  concentration  of  carbohydrate  (at  least 
1  nmole  of  "glucose"  equivalents /mg  protein)  .   The  delipidated  membrane  has 
been  fractionated  into  a  high  molecular  weight  fraction  which  contains  all  of 
the  carbohydrate  and  non-lipid  P  and  a  small  percentage  of  the  protein,  and 
a  low  molecular  weight  fraction  which  contains  only  protein.   The  protein 
fraction  seems  to  contain  relatively  few  components  (only  two  or  three  bands 

1  333 


i 


'^ 


are  seen  in  polyacrylamide  gel  electrophoresis  of  the  whole  membranes)  none 
of  which  has  a  molecular  weight  greater  than  15,000  and  half  of  which  have 
molecular  weights  less  than  5000  by  the  usual  criteria.   The  carbohydrate 
contains  glucose,  mannose,  xylose,  and  several  unidentified  sugars.  The 
polymer  that  contains  the  non- lipid  P  has  not  been  identified  but  the  P  is 
not  released  upon  hydrolysis  in  3N  HCl  at  100°  for  3  hours  . 

As  originally  speculated  from  functional  considerations,  it  now  seems  probable 
that  the  amoeba  plasma  membrane  is  indeed  much  simpler  in  protein  composition 
than  the  corresponding  membranes  of  mammalian  and  bacterial  cells  and  thus 
may  provide  a  very  useful  system  for  establishing  the  molecular  organi7.ation 
of  a  biological  membrane. 

(3)   Both  the  plasma  membrane  and  the  motile  process  are  involved  in  endo- 
cytosis  .   Recent  biochemical  and  ultrastructural  studies  have  confirmed  our 
previous  impression  that  solute  molecules  enter  the  amoeba  only  by  pinocytosis 
The  magnitude  of  this  continuous  process  is  such  that  we  estimate  from  bio- 
chemical and  electron  microscopic  data  that  the  amoeba  surface  membrane  turns 
over  approximately  once  a  minute.   Scanning  electron  microscopy  has  yielded 
striking  three  dimensional  images  of  the  amoebae,  illuminated  the  mechanisms 
by  which  they  capture  particles  during  phagocytosis,  and  shown  the  relation- 
ship of  the  amoeba  to  the  substrate  during  motion. 


33^ 


Project  Title: 


Serial  No.  NHLI-  130 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Enzymes 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Regulation  of  Bacterial  Purine  and  Pyrimidine  Base  and 
Nucleoside  Utilization 


Previous  Serial  Number:   NHLI -200 
Principal  Investigator:   Joy  Hochstadt-Ozer 
Other  Investigators:   None 


Cooperating  Units: 


Cashel,  NINDS 
Levinthal,  NIAMD 


Project  Description: 

Objectives:   Previous  studies  from  this  laboratory  indicated  that  phospho- 
ribosyltransferases  (PRT'ases)  in  Bacillus  subtilis  might  be  involved  in 
purine  uptake.   Isolation,  characterization,  and  study  of  regulation  of 
adenine  PRT'ase  in  E.  coli  showed  that  this  enzyme  is  membrane-associated, 
can  be  released  from  the  pericytoplasmic  space  upon  osmotic  shock  of 
viable  cells,  is  responsible  for  adenine  transport  by  a  group  translocation 
mechanism  and  that  both  PRT'ase  activity  and  uptake  are  regulated  by  5' 
nucleotides.   The  present  project  is  concerned  with  extending  the  studies 
so  far  concentrating  on  adenine  toward  the  elucidation  of  the  mechanism, 
regulation  and  coordination  of  all  nucleic  acid  precursors,  and  to  examine 
the  genetic  and  membrane  topographic  and/or  functional  interrelationship  of 
the  carrier  and/or  enzymes  responsible  for  the  control,  uptake  and  utili- 
zation of  nucleotide  precursors.   This  system  seems  furthermore  to  be  ideal 
as  a  model  for  study  of  nascent  membrane  functional  units  as  appropriate 
genetic  reagents  and  reconstitution  techniques  are  being  developed  in  it. 

Major  Findings: 

Adenine  Phosphoribosyl transferase:   Regulation  by  combinations  of 
nucleotides:   A  variety  of  combinations  of  two  '5  nucleotides  were  tested 
to  determ.ine  possible  cooperativity  between  effectors.   6-OH  nucleotides 
could  antagonize  the  effect  6-NH  nucleotides  to  a  small  but  significant 
extent.   The  mechanism  of  antagonism  appeared  to  be  by  competing  at  the 
same  inhibitory  site  (possibly  the  catalytic  site  since  '5  nucleotides  each 
inhibit  by  competing  with  the  substrate  PRPP) . 

Guanine  Hypoxanthine-Xanthine  Phosphoribosyltransferase(s) :   Further 
purification  of  a  several  hundred  fold-purified  enzyme  preparation  on 
Ecteola-Cellulose,  has  led  to  the  identification  of  at  least  three  protein 


33  S' 


B9(!CaK~wvTT':::'  J^i 


% 


I 


Serial  No.  NHLI-130 

peaks  each  exhibiting  all  three  phosphoribosyl  activities.   Since  these 
activities  were  not  separable  by  gel  filtration  chromatography  they  appear 
to  have  similar  molecular  weights.   Genetic  analysis  to  determine  whether 
these  represent  modified  forms  of  a  single  gene  product  or  separate  gene 
products  are  in  progress.   Each  peak  of  activity  is  being  analysed  for 
possible  differences  in  enzymatic  and  regulatory  behavior.   Major  differences 
have  not  been  observed  with  respect  to  hypoxanthine  as  substrate.   Studies 
to  determine  differences  with  respect  to  guanine,  xanthine,  8-azaguanine, 
and  6  Mercaptopurine  as  substrate  are  in  progress. 

Mutants  of  Purine  Utilization.   Though  analog  resistant  mutants  of 
purine  utilization  have  been  reported  for  E.  coli  these  have  been  found  to 
be  unstable  and  to  revert  to  PRT'ase  activity  unless  maintained  on  the 
selective  medium.   The  occurrence  of  the  several  (possibly  interconvertable) 
PRT'ase  forms  may  contribute  to  this  genetic  "instability"  in  E.  coli. 
Neither  drug  selection  alone  nor  purine  plus  penicillin  selection  produced 
stable  mutants  from  either  normal  or  mutagenized  stocks.   Combination  of 
drug  resistance,  purine-penicillin  selection  and  screening  by  replica  plating 
of  mutagenized  cells  in  sequence  is  currently  being  employed  as  a  means  of 
selecting  double  or  triple  mutants  should  such  multiple  mutations  be  requi- 
site to  loss  of  a  purine  PRT'ase  activity.   Alternatively  use  of  phage 
transduction  for  the  construction  of  an  appropriate  deletion  if  the  PRT'ase 
determinants  are  clustered  has  been  undertaken  in  collaboration  with 
Dr.  Mark  Levinthal,  Laboratory  of  Molecular  Biology,  NIAMD.   For  this,  a 
Salmonella  typhimurium  strain  carrying  a  deletion  in  the  Proline  A  B  region 
in  which  8-Azaguanine  resistance  was  fortuitously  found,  was  chosen  as 
starting  material.   Under  appropriate  conditions  hypoxanthine  uptake  levels 
and  PRT'ase  levels  in  this  strain  are  consistent  with  the  PRT'ase  function- 
ing in  utilization  in  a  manner  similar  to  that  found  for  E.  coli.   Charac- 
terization of  Salmonella  enzyme  and  its  interspecific  transfer  to  E.  coli 
may  provide  a  means  of  genetic  analysis  without  loss  of  PRT'ase  activity. 
The  possibility  that  purine  PRT'ases  represent  an  essential  function  in 
E,  coli  is  thus  being  considered,  and  in  addition  to  utilization  of 
Salmonella  reagents,  conditional  lethal  mutants  are  being  sought  in  E.  coli. 

Mechanism  of  Uptake  of  Adenosine  and  Inosine.   The  mechanism  of  uptake 
of  adenosine  (and  inosine)  was  studied  in  isolated  membranes  and  found  to 
have  two  steps:   1)  Cleavage  to  adenine  (and  hypoxanthine)  and  ribose-1-P 
by  nucleoside  phosphorylases  and  2)  Group  translocation  by  adenine  (hypo- 
xanthine) phosphoribosyltransferases.   Though  the  kinetic  and  regulatory 
parameters  of  the  adenine  and  hypoxanthine  systems  differ  markedly  from  one 
another,  the  response  to  any  effector  or  rate  limiting  condition  for  adenine 
(hypoxanthine)  uptake  was  identical  to  its  effect  on  adenosine  (inosine) 
uptake.   5'  Nucleotides  are  inhibitors  in  both  systems. 

Mechanism  of  Cytosine,  Cytidine,  Uracil,  and  Uridine  Uptake.   Uracil 
PRT'ase  seems  to  be  involved  with  uracil  uptake  by  a  mechanism  similar  to 
adenine  uptake.   Cytosine  PRT'ase  is  lacking  in  E.  coli  and  cytosine  is 
probably  taken  up  as  uracil.   Cytidine  and  uridine  uptake  do  not  follow 
cytosine  and  uracil  uptake  as  identically  as  adenosine  and  inosine  uptake 


53^ 


Serial  No.  NHLI- 130 

follow  adenine  and  hypoxanthine  uptake,  respectively.   Nucleoside  phospho- 
rylase  for  cytidine  was  found  to  be  lacking  in  our  strain  of  E.  coli. 
'5  Nucleotides  are  inhibitors  of  all  nucleoside  and  base  uptake  by  purified 
TT.embranes,  however. 

Effect  of  ppGpp  on  Uptake  and  Enzymes  of  Nucleic  Acid  Precursors 
Metabolism  and  Interconversion.   The  production  of  ppGpp  ("Magic  Spot")  by 
stringent  strains  of  E.  coli  and  its  effect,  inhibition  of  RNA  synthesis, 
has  been  well  established  by  Dr.  Cashel  in  the  Laboratory  of  Molecular 
Biology,  NINDS.   Dr.  Cashel,  in  collaboration  with  the  laboratory  of 
Dr.  Ira  Pastan,  has  recently  found  ppGpp  exerts  a  regulatory  effect  on 
protein  synthesis  in  vitro.   Possible  coordination  of  regulation  of  gene 
action  and  of  existing  enzymatic  activities  by  this  compound  in  effecting 
complete  control  of  nucleic  acid  synthesis  has  been  studied  by  determination 
of  its  effect  on  uptake  of  bases  and  nucleosides  and  enz3mies  of  nucleotide 
synthesis  and  interconversion.   PpGpp  is  a  potent  inhibitor  of  adenine, 
adenosine,  hypoxanthine,  inosine,  guanosine,  and  uridine  uptake  but  not 
uracil,  cytosine,  and  cytidine  uptake.   It  is  a  potent  inhibitor  of  adenine, 
guanine,  and  hypoxanthine  PRT'ase  activities  but  not  purine  nucleoside  phos- 
phorylases.   It  is  an  inhibitor  of  adenosine  deaminase  and  uridine  phosphory- 
lase  but  not  uracil  PRT'ase.   The  level  of  inhibition  on  guanine  utilization 
or  prevention  of  deamination  of  intracellular  adenine  derivatives  (otherwise 
convertable  to  guanine  nucleotides)  when  compared  with  intracellular  ppGpp 
levels  in  stringent  cells  deprived  of  an  essential  amino  acid  can  adequately 
account  for  immediate  cessation  of  nucleic  acid  synthesis  in  such  cells. 

Dual  Isotope  Experiments.   Commercial  availability  of  uniformly  labeled 
^^C  bases  and  nucleosides  with  specific  activities  greater  than  10  times 
that  commercially  available  at  the  beginning  of  this  project  (1968)  have 
permitted  the  following  experiments  to  be  performed.   Membrane  vesicles  were 
preloaded  with  either  ^h  or  ^'^C  adenine,  adenosine,  or  hypoxanthine.   Then 
membranes  were  washed  free  of  extravesicular  tracer  and  incubated  with  ^^C 
or  -^H  adenine,  adenosine,  or  hypoxanthine  and  PRPP.   The  results  indicated 
that  extravesicular  labeled  material  is  preferred  to  the  preloaded  material 
as  a  substrate  for  phosphorylation  ("5  nucleotide  synthesis). 

Significance  to  Bio-Medical  Research:   This  project  investigates  the  nature 
and  regulation  of  structure-function-relationships  of  cell  membranes  as 
involved  in  purine  and  pyrimidine  utilization.   Understanding  of  this 
organelle  possessed  by  all  living  cells  (and  known  to  be  altered  in  a  number 
of  pathologic  states),  is  essential  for  a  conceptual  framework  of  cell 
function,  both  normal  and  pathologic.   The  experimental  program  utilizes 
enzyme-transport  systems  capable  of  assay  both  in  situ  (transport  activity) 
and  as  homogeneous  proteins  in  aqueous  solutions  (catalytic  activity). 
Such  independent  assay,  in  the  complex  milieu  of  the  membrane  and  in  iso- 
lation has  the  advantage  of  allowing  comparison  of  the  properties  of  mem- 
brane components  in  the  biological  as  well  as  in  well  defined  environments 
while  experimentally  preserving  the  ability  to  monitor  retention  of  at 
least  one  biologic  function  in  either  environment.   The  enzyme -trans port 
systems  themselves  are  those  involved  in  utilization  of  nucleic  acid 

3  337 


mmmm 


i 


I 

I 


Serial  No.  NHLI-130 

precursors  and  deficiency  of  one  of  them  (hypoxanthine  system)  has  been 
identified  in  man  (Lesch-Nyhan  Syndrome).   The  multiple  clinical  anomalies 
associated  with  absence  of  the  hypoxanthine  phosphoribosyltransferase 
(previously  thought  to  act  in  a  "salvage"  capacity  only)  indicates  the 
necessity  of  a  further  research  into  the  role  recycling  of  nucleic  acid 
precursors  play  In  the  cell  economy. 

Proposed  Course  of  Project: 

A.  EnzjTnes,  Membranes,  and  Transport.  Characterization  of  the  multiple 
peaks  of  PRT'ase  activity  for  each  purine  will  be  continued  to  determine 
the  genetic  or  chemical  determinant(s)  conferring  such  differentiability. 

B.  Role  of  the  PRT'ases  in  purine  transport.   Conditions  will  be  determined 
to  control  the  amount  of  PRT'ase  associated  with  membrane  vesicles  during 
membrane  isolation.   The  amount  of  enzyme  and  its  state  of  association  with 
the  membrane  will  be  correlated  with  the  PRT' as e -membrane  interaction  neces- 
sary to  carry  out  transport. 

C.  Reconstitution  experiments.   The  ability  of  isolated  enzyme  to  stimulate 
transport  when  added  to  isolated  membrane  vesicles  and  intact  cells  will  be 
evaluated.   Attention  will  be  paid  to  the  conditions  necessary  for  such 
reconstitution.   These  experiments,  where  initial  attempts  to  remove  con- 
siderable PRT'ase  from  membranes,  will  precede  reconstitution,  should  com- 
plement experiments  mentioned  above  in  B.   Purified  enzyme  will  also  be 
reacted  with  membranes  prepared  from  mutants  lacking  PRT'ase  and  uptake 
activity  in  an  attempt  to  confer  these  functions  on  such  mutant  membrane 
preparations. 

D.  Comparative  enzymology  of  PRT'ase  derived  from  sonically  disrupted  cells 
and  isolated  from  membrane  vesicles.  Using  a  number  of  specific  probes  useful 
in  determining  protein  structure  and  reactive  groups  (e.g.,  -SH  reagents, 
tryosine  reagents,  lysine  reagents,  acetylation) ,  the  nature  of  PRT'ase- 
raembrane  association  will  be  investigated.   The  protection  to  various  rea- 
gents, conferred  by  membrane-enzyme  association  or  conversely  heightened 
reactivity  for  (a)  PRT'ase  activity,  and  (b)  transport,  will  be  investigated. 
The  effect  of  nucleotide  inhibitors  on  these  interactions  will  be  subse- 
quently studied.   Serological  techniques  will  also  be  employed  in  evaluation 
of  the  nature  of  the  membrane-PRT' ase  interaction.   Antibody  to  purified 
soluble  enzyme  will  be  prepared  and  cross-reaction  patterns  between  soluble 
enzyme  in  various  states  of  subunit  interaction,  and  association  with  the 
membrane  will  be  studied.   Conversely  antibody  to  membrane  vesicles  and 
partially  purified  enzyme  derived  from  the  membranes  will  be  prepared  and 
exhaustively  absorbed  with  soluble  enzyme  that  has  been  subjected  to  the 
entire  purification  procedure.   The  ability  of  the  preabsorbed  antisera  to 
alter  purine  transport  and/or  membrane  PRT'ase  activity  would  serve  as  indi- 
cation of  other  factors  involved,  in  addition  to  the  PRT'ase  itself  (as 
purified) . 


33fi 


Serial  No.  NHLI- 130 

E.  Genetic  relationships.   The  genetic  studies  in  progress  outlined  above 
will  be  continued  in  hopes  of  constructing  a  series  of  mutants  for  purine 

utilization  which  will  be  used  as  reagents  for  further  study  of  mechanism  . 

and  regulation  of  membrane  plus  enzymes  in  the  uptake  process.   The  genetic  n 

and  enzyme  analysis  of  such  mutants  with  respect  to  membrane  and  soluble  i 

enzymatic  activity,  the  ability  of  the  soluble  enzyme  to  associate  with 
wild-type  membranes  and  vice  versa  should  provide  insight  into  the  genetic 
control  of  membrane-enzyme  interaction,  transport,  and  the  variety  of  genetic 
events  by  which  such  control  may  be  achieved.   The  variety  of  genetic  mech- 
anisms will  be  ascertained  by  appropriate  mapping  experiments  using  trans- 
duction in  Salmonella  and  by  use  of  a  combination  of  transduction  and  con- 
jugation in  E.  coll.   The  PRT'ase  and  uptake  activities  of  a  variety  of 
appropriate  strains  carrying  suitable  genetic  markers  to  act  as  recipient 
and/or  donors  of  genetic  material  in  such  experiments  have  already  been 
characterized  in  preparation  for  this  line  of  experimentation. 

F.  Biosynthesis.  The  appearance  of  enzyme  as  soluble  versus  membrane  bound 
will  be  studied  utilizing  a  variety  of  techniques.  Pulse-labeling  of  cellu- 
lar protein  will  be  employed  as  one  approach.  Determination  of  the  specific 
activity  of  radioisotope  in  PRT'ase  purified  from  membrane  vesicles  and 

from  the  pericytoplasmic  space  after  subjecting  intact  cells  to  osmotic  shock 
treatment  should  indicate  whether  the  partitions  that  have  been  observed  to 
date  are  random  or  whether  they  have  a  biological  basis  related  to  "age"  or 
enzyme  molecules.   Another  approach  to  the  biogenesis  of  membrane  enzymes 
involves  enzyme  induction.   Since  purine  PRT'ases  have  been  found  to  be 
induced  significantly  upon  "forcing"  cellular  dependence  on  them,  the 
appearance  of  membrane-associated  PRT'ase  activity  under  these  conditions 
will  also  be  studied  by  pulse  labeling  techniques.   Attempts  can  then  be 
made  to  correlate  enzjmie  appearance  with  other  concomitant  biosynthetic  pro- 
cesses in  the  membranes  (e.g.,  phospholipid  composition  and  turnover, 

assuming  appropriate  controls  will  be  developed).   Mutants  requiring  an  ' 

unsaturated  fatty  acid  such  as  those  developed  by  R.  Vagelos  have  been  used 
to  correlate  membrane  phospholipid  synthesis  with  the  appearance  of  sugar 
transport  function  and  can  be  also  used  to  study  appearance  of  purine(pyri- 
midine)  transport  function.  t 


33? 


{ 


Serial  No.   NHLI-131 


Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 


I 


PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  the   3-Lysine  Mutase  Complex 

Previous  Serial  Number:   none 

Principal  Investigators:   John  J.  Baker 

Chris  van  der  Drift 

Other  Investigator:   T.C.  Stadtman 

Project  Description: 

Objectives:    6-Lysine  mutase  is  a  complex  enzyme  system  that  consists 
of  a  cobamide  protein  of  M.W.  160,000  and  a  smaller  protein  of  M.W.  60,000. 
Hie  reaction  catalyzed  by  this  complex  is  L-g-lysine  ?^  L-erythro-3,5  diamino- 
hexanoic  acid.   The  cof actors  required  for  activity  of  this  complex  are  a  B12 
coenzyme,  dithiothreitol ,  magnesium,  a  monovalent  cation,  ATP,  and  pyruvate. 
The  roles  of  ATP  and  pyruvate  in  the  action  of  this  enzyme  complex  are  not 
understood.   Consequently  the  major  objectives  are  to  elucidate  the  role  of 
these  cofactors  and  to  purify  the  protein  components  to  homogeneity. 

Major  Findings: 

B-Lysine  mutase  activity  was  measured  by  the  acid  ninhydrin  procedure 
of  Chinard.   Since  this  assay  is  long  and  cannot  readily  measure  initial 
rates,  a  spectrophotometric  assay  was  developed  by  coupling  the   3-lysine 
mutase  reaction  with  3,5-diaminohexanoic  acid  dehydrogenase  to  form  DPNH , 
3-keto-5  aminohexanoic  acid,  and  ammonia  from  3,5  diauninohexanoic  acid  and 
DPN.   The  dehydrogenase  was  partially  purified  from  the  C_^  sticklandii 
extracts  until  free  of  DPNH  oxidase  activity.   The  purification  steps  were 
ammonium  sulfate,  G-25,  DEAE  cellulose  and  hydroxylapatite.   In  addition 
the  components  of  the  3-lysine  mutase  complex  were  purified  from  the  same 
extract.   The  cobamide  protein  comes  off  the  DEAE  column  about  50%  pure. 
The  smaller  component  was  found  in  a  different  salt  fraction  and  has  been 
purified  by  DEAE  cellulose  chromatography.   This  smaller  component  catalyzes 
the  activation  of  the  cobamide  protein  by  ATP.   Some  cobamide  protein  fractions 
give  excellent  initial  rates  in  the  spectrophotometric  assay  without   ATP, 
but  rapidly  lose  their  ability  to  convert   3-lysine  to  3,5-diaminohexanoic 
acid  unless  ATP  and  the  smaller  protein  component  are  present. 

The  role  of  pyruvate  in  the  function  of  the  enzyme  complex  is  unknown. 
tiaBtii^    (10-*^  M)  ,  hydroxylamine  (10-5  M)  ,  and  phenylhydrazine  (10"^  M)  are 
potent  inhibitors  of   3-lysine  mutase.   Preliminary  experiments  in  which  the 
enzyme  was  reduced  with  tritiated  NaBH^  followed  by  acid  hydrolysis  have 


3*/o 


Serial  No.   NHLI-131 


shown  that  several  tritiated  products  were  obtained.   Unlike  several  other 
eniymes  that  contain  electrophilic  centers,  neither  lactate  or  alanine 
were  products  of  the  reduction. 

Proposed  Course  of  Action: 

Future  experiments  will  be  concerned  with  the  further  purification 
of  the  protein  components  involved  in  the   g-lysine  mutase  complex.   In 
addition,  the  electrophilic  center  will  be  labeled  with  tritium  and/or 
I'^C,  the  protein  will  be  hydrolyzed  by  both  acid  and  protolytic  enzjones, 
and  the  electrophilic  center  identified.   Experiments  with  l^C  and  32p 
labeled  ATP  may  determine  why  ATP  is  required  for  the  enzyme  to  turnover. 


3*4/ 


( 


Serial  No.  NHLI-i32 


Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 


PHS-FriLI 
Individual  Project  Report 
July  1,  1970  through  June  1,  1971 

Project  Title:   Genetics  of  E^.  coli  Glutamine  Synthetase 

Principal  Investigator:   Mary  Anne  Berberich 

Other  investigators:   none 

Project  Description: 

objectives:   Genetic  studies  on  the  glutamine  synthetase  enzyme 
system  in  E^.  coli  have  been  initiated  to  further  elucidate  the  relationships 
among  the  enzymes  involved  in  nitrogen  assimilation  in  this  organism.   Since 
the  end  products  produced  by  each  of  the  enzymes  involved,  as  well  as  the 
concentration  of  inorganic  nitrogen  in  the  growth  medium,  influence  the 
activity  and,  in  some  instances,  the  amount  of  enzyme  produced,  these 
studies  were  undertaken  with  a  view  toward  determining  whether  there 
exists  some  regulatory  system  for  this  group  of  enzjnnes  which  operates  at 
the  genetic  level. 

Major  Findings: 

A  strain  o  f  E^.  coli  K]^2  which  is  prototrophic,  streptomycin  resistant 
and  non-permissive  was  chosen  as  the  parent  strain.   By  a  combination 
of  mutagenesis  with  diethylsulfate  and  penicillin  selection  techniques, 
a  group  of  mutants  with  desirable  growth  requirements  have  been  isolated 
and  this  group  is  currently  being  characterized  as  to  their  enz^Tnic 
defect . 


Proposed  Course  of  Research: 

In  particular,  the  relative  positions  of  glutamine  synthetase, 
glutaminase,  glutamate  dehydrogenase  and  glutamate  synthetase  on  the  E.    coli 
chromosome  will  be  studied  by  transduction  and  mating  techniques.   The 
first  stage  of  this  research  project  is  currently  in  progress,  namely, 
the  selection  of  mutants  minus  or  defective  for  the  above  enzymes  to  be 
used  in  mapping  studies. 

Attempts  are  also  being  made  to  screen  for  suitable  specific-type 
inhibitors  of  glutamine  synthetase  which  would  make  it  possible  to  select 
for  constitutive  mutants.   These  types  would  be  useful  as  well  in  this 
laboratory's  biochemical  work  both  for  production  purposes  and  for  studies 
of  protein  structure. 

It  is  hoped  that  enough  mutants  will  be  obtained  for  glutamine 
synthetase  so  that  a  fine  structure  genetic  analysis  may  be  carried  out  and 
compared  with  the  biochemical  evidence  which  suggests  that  the  structural 
subunits  of  this  protein  are  identical. 

1  J^i 


Serial   No.     NHLI-133 


Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   A  Dithiol  Dehydrogenase  of  Clostridium  sticklandii. 

Principal  Investigator :   Lauren  M.  Cagen 

Other  Investigators:   T.C.  Stadtman 

Cooperating  Units:   none 

Project  Description: 

Objective:   A  dithiol  dehydrogenase  has  recently  been  discovered  in 
extracts  of  C.    sticklandii.   This  enzyme  differs  from  a  previously  reported 
mercaptan  dehydrogenase  in  this  organism  in  being  specific  for  dithiol 
compounds.   This  enzjone  will  be  purified  and  characterized. 

Maj  or  Findings : 

The  reduction  of  tetrazolium  dye  by  dialyzed  extracts  of  C^.  sticklandii 
is  greatly  stimulated  by  FAD  and  by  a  dialyzable  cofactor  present  in  boiled 
extracts.   FMN  does  not  substitute  for  FAD.   The  enzjone  will  reduce  added 
FAD  in  the  absence  of  tetrazolium  dye.   The  cofactor  in  boiled  extracts 
does  not  seem  to  be  a  metal,  but  hasnot  yet  been  identified.   KCN  also 
stimulates  the  reduction  of  tetrazolium  dye.   It  has  not  been  established 
whether  this  stimulation  is  due  to  chelation  of  an  enzyme  bound  metal, 
discharge  of  an  enzyme  per  sulfide  group,  or  to  some  other  activity  of  the 
cyenide.   Preliminary  purification  experiments  with  DEAE  cellulose,  poly- 
cry  lemide  gel,  and  ammonium  sulfate  have  been  carried  out. 

Proposed  Course  of  Action: 

The  enzyme  will  be  further  purified  and  characterization  of  the 
cofactor(s)  in  boiled  extracts  will  be  undertaken.   The  nature  of  the  cyanide 
stimulation  will  be  studied  as  well  substrate  specificity  and  ability  of 
the  enzyme  to  link  DPNH  oxidation  to  amino  acid  reduction  by  £.  sticklandii 
extracts.   It  will  be  seen  whether  the  dithiol  dehydrogenase  is  distinct 
from  the  DPNH  dehydrogenase  also  present  in  these  extracts. 


s 

U    fD 
5    3 

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1 

Part  B      No 


3¥3 


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i 


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Project  Ticie: 


Serial  No.  NHLI-134 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Enzymes 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  ],  1970  through  June  30,  1971 

1)  Methionine  Biosynthesis  and  its  Regulation  in  Fungi  and 

Bacteria, 

2)  Mechanisms  of  Pyridoxal  Phosphate  Enzyme  Catalyzed 

Elimination  and  Replacement  Reactions. 

3)  Microbial  Genetics  of  Microtubular  Proteins  and  their 

Assembly. 


Previous  Serial  Number:   NHLI-i-98 

Principal  Inv/estigators:   Martin  Flavin 

Michael  Savin 


Other  Investigators: 
Collaborating  Units: 

Project  Description: 


Clarence  Slaughter  (technician) 

J.  Selhub  and  W.  Sakami,  Dept.  of  Biochemistry, 
Western  Reserve  Medical  School;  T.  Fukasawa  and 
K.  Kurahashi,  Institute  for  Protein  Research, 
Osaka  University 


Objectives:   Many  amino  acids  have  additional  functions  besides  serving  as 
building  blocks  for  protein  but  methionine  is  conspicuous  in  this  respect. 
It  contributes  to  membrane  phospholipid,  in  the  form  of  the  methyl  groups  of 
choline,  as  much  as  to  protein,  and  in  addition  is  a  source  of  polyamines, 
a  general  methylating  agent,  via  adenosylmethionine,  and  the  initiator  of 
protein  synthesis.   It  would  then  not  be  surprising  if  the  regulation  of 
its  synthesis  were  to  reveal  some  unusual  features.   Previous  work  in  this 
laboratory  has  shown  that  the  synthesis  of  homocysteine,  the  immediate 
precursor  of  methionine,  is  accomplished  in  3  sequential  steps,  which  differ 
slightly  in  different  microorganisms,  and  are  catalyzed  by:   a)  homoserine 
transacylase,  utilizing  succinyl  CoA  in  Salmonella  and  acetyl  CoA  in 
Neurospora;  b)  cystathionine  /-synthase;  and  c)  p-cystathionase.   Our 
objective  is  to  complete  the  characterization  of  these  enzymes  and  deter- 
mine the  mechanisms  by  which  their  synthesis  and  activity  is  regulated. 
Cystathionine  7-synthase,  which  has  been  isolated  in  pure  form  from 
Salmonella,  is  a  pyridoxal-P  enzyme  which  catalyzes  a  unique  spectrum  of 
elimination  and  replacem.ent  reactions,  and  is  being  used  to  study  the  mech- 
anisms of  these  reactions.   A  new  objective  is  to  obtain  microbial  mutants 
with  alterations  in  the  primary  structure  of  their  microtubular  proteins, 
and  eventually  to  study  the  structure  and  assembly  of  this  organelle. 


3^^ 


Serial  No.  NHLI-134 


Major  Findings: 

Nine  genes  are  known  in  Neurospora,  mutation  in  which  results  in  a 
nutritional  requirement  for  methionine.   As  reported  last  year,  extracts 
of  mutants  corresponding  to  4  of  these  genes  lack  cystathionine  synthase 
activity.   Two  of  these  mutants  (me-3  and  me-7)  respond,  as  expected,  to 
cystathionine  as  well  as  methionine.   The  others  (me-1  and  me-6)  respond 
only  to  methionine  and  their  extracts  have  additional  deficiencies:   me-1 
lacks  methylene  tetrahydrofolate  reductase  activity,  and  me-6  lacks  the 
folate  polyglutamate  derivatives  needed  for  the  non-Bj^o  homocysteine 
transmethylase.   The  puzzling  absence  of  cystathionine  synthase  activity 
from  the  latter  2  mutants  has  now  been  explained  by  the  discovery  that 
this  enzyme  activity  is  completely  dependent  on  the  presence  of  polyglu- 
tamate derivatives  of  N^-methyltetrahydrofolate.   This  allosteric  acti- 
vation serves  to  prevent  over-production  of  homocysteine  in  the  absence 
of  the  methylfolate  needed  for  its  conversion  to  methionine;  methylfolate 
also  antagonizes  the  allosteric  inhibition  of  cystathionine  synthase  by 
S-adenosylmethionine.   It  is  noteworthy  that  in  Neurospora  it  is  the 
second  enzyme  of  the  homocysteine  synthetic  pathway  that  is  subject  to 
regulation. 


4 


Last  year  cystathionine  synthase  was  reported  to  be  absent  from 
extracts  of  some  species  of  Neurospora;  these  extracts  have  now  been  found 
to  have  activity  when  methylfolates  are  added.   The  intermediary  role  of 
cystathionine  in  methionine  synthesis  in  yeast  is  still  uncertain.   We 
have  now  found  significant  cystathionine  synthase  activity  in  extracts  of 
yeast  protoplasts,  but  the  reaction  is  not  affected  by  addition  of  methyl- 
folates and  is  much  slower  than  the  direct  formation  of  homocysteine  from 
acetylhomoserine  and  sulfide.   The  latter  2  reactions  are  catalyzed  by 
the  same  enzyme,  as  in  Salmonella  and  in  contrast  to  Neurospora. 

The  regulation  of  homocysteine  synthesis  in  Salmonella  has  been  rein- 
vestigated in  relation  to  the  questions:   do  methylfolates  play  any  role, 
and  can  any  clue  be  obtained  as  to  why  the  first  enzyme  of  the  pathway 
appears  to  be  uncontrolled  in  Neurospora.   The  results  have  not  so  far 
answered  these  questions.   In  Salmonella  the  first  enzyme,  homoserine 
trans-succinylase,  is  subject  to  synergistic  end  product  inhibition  by 
methionine  +  S-adenosylmethionine.   It  is  of  interest  that  the  synergism 
is  not  observed  in  the  absence  of  succinyl  CoA,  i.e.  when  the  activity  is 
assayed  by  an  alternate  reaction  involving  the  exchange  of  labeled  homo- 
serine into  succinylhomoserine.   New  procedures  allowing  precise  measure- 
ment of  the  trans-succinylase  in  crude  extracts  have  enabled  us  to  rein- 
vestigate the  regulation  of  the  synthesis  of  the  3  enzymes  of  the  pathway. 
A  mutant  lacking  S-adenosylmethionine  synthase,  scored  as  resistant  to 
ethionine,  was  first  identified  in  Neurospora  in  this  laboratory.   A 
similar  E.  coli  mutant  has  been  shown  by  others  to  be  constitutive  for  the 
second  and  third  enzymes,  and  we  have  shown  that  this  is  also  the  case  for 
the  first  enzyme.   The  question  of  whether  S-adenosylmethionine,  or  some- 
thing derived  from  it,  it  also  the  corepressor  for  all  3  enzymes  in 


i' 


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Biaiasfl*;';><c^i^:^i^«**»^^■«i^^^  .:**«•■■ 


L 


Serial  No.  NHLI-1,34 

Salmonella  is  unresolved  as  we  have  not  yet  identified  a  mutant  lacking 
S-adenosylmethionine  synthase  in  this  organism.   Other  results  suggest 
that  the  mechanism  regulating  the  synthesis  of  the  first  enzyme  differs 
from  that  for  the  second  and  third  in  Salmonella.   When  the  growth  rate  of 
a  mutant  which  responds  to  either  methionine  or  vitamine  B,^  is  slowed  in 
a  chemostat  by  limiting  the  concentration  of  methionine  the  second  and 
third  enzymes  are  derepressed  in  a  normal  fashion,  but  it  is  virtually 
impossible  to  derepress  the  trans-succinylase.   This  result  is  not  observed 
with  vitamin  B12  limitation,  suggesting  that  exogenous  methionine  is  a 
preferential  source  of  a  component  involved  in  controlling  the  synthesis  of 
the  trans-succinylase.   Two  possibly  related  observations  are  that  trans- 
succinylase  activity  is  much  reduced  in  stationary  phase  cells  of  wild  type 
grown  on  minimal  medium,  and  that  the  second  and  third  enzymes  are  dere- 
pressed in  mutants  lacking  the  first. 

In  collaboration  with  colleagues  at  the  University  of  Osaka,  where  the 
senior  investigator  spent  2  months  as  a  guest  of  the  Naito  foundation,  a 
study  was  begun  of  the  role  of  S-adenosylmethionine  in  the  processes  of 
host  DNA  modification  and  restriction,  utilizing  bacterial  mutants  which 
have  alterations  in  S-adenosylmethionine  synthase. 

The  feasability  of  using  fungi  for  genetic  and  chemical  studies  of 
microtubular  proteins  was  explored.   Colchicine  binding  protein  was  not 
detected  in  extracts  of  Neurospora  or  yeast.   Colchicine  and  colcemid 
(10~-^  M)  did  not  prevent  meiotic  division  in  Neurospora.   In  preliminary 
experiments,  the  vegetative  growth  of  Neurospora  (wild  type  and  a  mutant 
with  apparently  generally  increased  permeabilitj') >  Saccharomyces ,  and 
Schizosaccharomyces  was  not  inhibited  by:   colchicine,  colcemid,  vinblastin, 
podophyllotoxin,  or  griseofulvin.   In  view  of  these  results  we  have  under- 
taken to  aquaint  ourselves  vjith  the  genetics  of  Chlamydomonas .  a  micro- 
organism with  abundant  extranuclear  microtubules. 

Proposed  Course  of  Project: 

The  principle  problems  in  homocysteine  synthesis  in  Neurospora  are  to 
understand  why  it  is  the  second  enzyme  in  the  pathway  that  is  subject  to 
metabolic  control,  and  to  elucidate  the  chemical  nature  of  cystathionine 
synthase,  in  particular  to  determine  whether  the  latter  consists  of  a  very 
weakly  associated  aggregate  of  2  proteins  coded  by  the  me-3  and  me-7  genes, 
one  of  which  might  have  a  purely  regulatory  function,  or  whether  one  of 
the  proteins  is  cystathionine  synthase  and  the  other  an  enzyme  which  acti- 
vates it,  perhaps  by  methylation  (allosteric  inhibitor  and  activator  are 
both  reactive  methylating  reagents). 

It  may  not  be  possible  to  pursue  Project  2  during  the  coming  year. 
There  are  2  problems  of  particular  interest  at  this  stage  (see  last  year's 
report  by  B.  Posner) .   The  capacity  of  cystathionine  7-synthase  of 
Salmonella  to  catalyze  the  stereospecif ic  or  stereoselective  exchange  of 
OL   and  p  hydrogens  in  many  amino  acids,  particularly  diastereoisomeric 
amino  acids,  provides  an  opportunity  to  define  the  geometry  of  the  active 


3^^ 


Serial  No.  NHLI-134 

site  and  the  steric  course  of  pyridoxal-P  catalyzed  reactions  in  general. 
The  second  relates  to  the  observation  that  in  the  reaction  succinylhomo- 
serine  -•  a-ketobutyrate  there  is  partial  direct  transfer  of  both  a  and  p 
hydrogen  to  carbon  4.   A  study  of  the  conditions  under  which  either  the  a 
or  the  p  transfer  will  prevail  over  the  other  should  illuminate  the  nature 
of  the  general  base  group(s)  which  attack  these  protons  and  the  route  by 
which  the  latter  reach  carbon  4. 

We  plan  to  search  for  Chlaymydomonas  mutants  in  which  either  cell 
division  or  flagellar  regeneration  is  resistant  to  inhibition  by,  or 
dependent  on,  colchicine  and  related  substances.   These  mutants  should 
have  either  altered  apparatus  for  assembling  microtubules  or,  hopefully, 
alterations  in  the  structure  of  the  colchicine  binding  protein.   We  hope 
also  to  devise  improved  methods  for  the  assay  and  isolation  of  microtubular 
proteins  through  affinity  labeling  and  chromatography. 

Publications : 

1.  Kerr,  D.  S.:   0-Acetylhomoserine  and  sulfhydrylase  from  Neurospora: 
Purification  and  consideration  of  its  function  in  homocysteine  and 
methionine  synthesis.   J.  Biol.  Chem.  246:   95-102,  1971. 

2.  Selhub,  J.,  Savin,  M.  A.,  Sakami,  W. ,  and  Flavin,  M. :   Synchronization 
of  converging  metabolic  pathways:  Activation  of  the  cystathionine  /-synthase 
of  Neurospora  crassa  by  methyltetrahydrofolate.   Proc.  Nat.  Acad.  Sci.  68: 
312-314,  1971. 


3.   Guggenheim,  S.  and  Flavin,  M. :   Cystathionine  7-synthase  from 
Salmonella:  Spectral  changes  in  the  presence  of  substrates.   J.  Biol, 
(in  press,  June  1971). 


Chem. 


4.   Flavin,  M. :   Alternate  pathways  of  methionine  biosynthesis  and  their 
regulation.   Tanabe  sjrmposium  on  amino  acid  metabolism  (in  press,  1971). 

Abstracts: 

1.   M.  A.  Savin  and  M.  Flavin:   Regulation  of  homocysteine  biosynthesis  in 
Salmonella.   Proc.  Amer.  Soc.  Biol.  Chem.  (in  press.  May  1971). 


39^/ 


ws^sssmfn^t.  ^i  ■ 


i 


I 


Serial  No.  NHLI-135 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Enzymes 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   Enzyme  Structure  and  Mechanisms  of  Action  and  Control 

Previous  Serial  Number:   NHLI-199 

Principal  Investigator:   Ann  Ginsburg 

Other  Investigators:   S.  Barbara  Hennig 
Carlos  E.  Caban 
John  B.  Hunt 
Mary  Anne  Berberich 
Joseph  E.  Ciardi 

Cooperating  Units:   P.  D.  Ross,  D.  M.  Segal,  and  D.  R.  Davies,  NIAMD 

Project  Description: 

Objectives:   1)  To  study  the  physical  and  chemical  properties  of  glutamine 
synthetase  from  Escherichia  coli,  particularly  with  respect  to  the  corre- 
lation of  the  structure  and  catalytic  function  of  this  enzyme,   2)  To  study 
conformation  and  stabilization  changes  of  a  protein  macromolecule  effected 
through  the  specific  binding  of  small  molecules,  and  the  relationship  of 
such  effects  to  enzyme  catalysis  and  regulation.   3)  To  purify  and  study 
the  ATP-glutamine  synthetase  adenylyl transferase  from  E.  coli,  with  emphasis 
on  the  mechanism  of  action  and  the  physical  structure  of  this  enzyme. 

Major  Findings: 

1.   Studies  on  the  ATP:  glutamine  synthetase  adenylyltransferase  (ATase) 
from  E.  coli  (Principal  Investigators:   S.  B.  Hennig,  C.  E.  Caban,  and 
J.  E.  Ciardi).   Two  active  molecular  forms  of  this  enzyme  were  found:   A 
relatively  small  protein  of  /«-'70,000  mol.  wt.  appears  to  be  derived  fron  a 
protein  of  '^  130,000  mol.  wt.  during  purification  and  storage  at  4°.   In 
collaborative  studies  with  Drs.  Wayne  B.  Anderson  and  E.  R.  Stadtman,  it 
was  shown  by  co-purification,  polyacrylamide  gel  electrophoresis  and  heat- 
inactivation  studies  that  the  larger  adenylyltransferase  also  possesses  the 
deadenylylating  activity  (DA)  attributable  to  the  P^-protein  component  of 
the  DA  two  protein  (Pj  and  P^,)  system.   In  addition,  the  Pj-ATase,  but  not 
the  smaller  ATase,  has  appreciable  L-glutamate  (or  L-glutamine)  stimulated 
ATP-PPj^  exchange  activity.   (The  latter  activity  is  presumably  expressed 
through  an  ability  of  the  ATase  to  catalyze  a  partial  as  well  as  a  complete 
reversal  reaction  in  the  presence  of  Mg^^,  ATP,  pyrophosphate,  and  allos- 
teric  activator.)   In  preliminary  dissociation  studies,  the  P-r-protein 
appeared  to  be  composed  of  two  subunits  of  unequal  size.   The  inability  of 
the  small  active  ATase  to  catalyze  either  Pj-DA  or  ATP-PP^^  exchange 

1  3¥B 


■i 


Serial  No,  NHLI-135 

activities  has  suggested  that  a  subunit  other  than  the  small  ATase  in  the 
P-,.-protein  complex  is  necessary  for  the  expression  of  these  activities. 
Whether  or  not  the  same  catalytic  site  of  the  ATase  when  it  is  a  part  of  the 
Pr-protein  complex  is  involved  in  both  the  attachment  and  removal  of  AMP 
from  glutamine  synthetase  (in  adenylylation  and  deadenylylation  reactions, 
respectively)  is  unknown.   (It  is  known  that  specific  metabolities  have 
reciprocal  effects  on  the  adenylylation  and  deadenylylation  reactions.)   A 
physical  characterization  of  the  two  active  molecular  forms  of  ATase  is  in 
progress . 

2.   On  the  binding  of  effectors  to  glutamine  synthetase  of  E.  coli. 
(2a)  The  binding  of  feedback  inhibitors  to  glutamine  synthetase  was  further 
investigated  by  Dr.  M.  A.  Berberich.   The  potential  use  of  Sepharose-bound 
glutamine  synthetase  (catalytically  active  dodecameric  aggregates)  or  of 
Sepharose-bound  subunits  (catalytically  inactive)  to  study  the  interaction 
of  effectors  with  this  proteins  was  explored  more  thoroughly.   Due  to  non- 
specific binding  of  all  low-molecular  weight  compounds  tested,  it  was  con- 
cluded finally  that  columns  of  Sepharose-bound  protein  could  not  be  used  to 
study  the  specific  binding  of  small  molecules  by  the  protein.   Since 
Sepharose-4B  itself  does  not  bind  effectors,  the  non-specific  binding  ob- 
served is  a  function  of  the  Sepharose-protein  matrix. 

14 
The  binding  characteristics  of  L-   C-tryptophan  in  the  presence  of  a 

high  level  of  AMP  (0.01  M)  and,  conversely,  of  ^C-AMP  in  the  presence  of 
0.01  M  L-tryptophan  to  native  glutamine  synthetase  was  determined.   The 
results  show  that  there  is  an  interaction  between  these  allosteric  inhibitor 
sites.   Tryptophan  at  10  mM  concentration  causes  a  7-fold  decrease  in  the 
affinity  of  glutamine  synthetase  for  AMP,  which  binds  to  twelve  apparently 
independent  (non-interacting)  sites.   AMP  at  10  mM,  (but  not  at  1  mM)  con- 
centration causes  a  3-fold  decrease  in  affinity  of  the  enzyme  for 
L-tryptophan.   Although  the  binding  of  L-tryptophan  by  glutamine  synthetase 
remains  cooperative  with  10  mM  AMP  present,  the  number  of  L-tryptophan 
binding  sites  appears  to  increase  to  >  12/mole  enzyme.   The  changes  in  the 
binding  of  L-tryptophan  appear  to  be  induced  when  glutamine  synthetase  is 
>  707o  saturated,  with  AMP;  only  87o  saturation  of  the  enzyme  with  L-tryptDptian, 
however,  significantly  lowers  the  enzyme  affinity  for  AMP. 

(2b)  A  calorimetric  study  of  the  interaction  of  Mn   with  glutamine  synthe- 
tase was  performed  by  Dr.  J.  B.  Hunt  in  cooperation  with  Dr.  P.  D.  Ross 
(NIAMD) .   These  results  show  that  2  protons  are  displaced  from  the  enzyme 
during  the  binding  of  each  of  the  first  twelve  equivalents  of  Mn2+.   One 
proton  is  released  instantaneously  and  the  second  proton  is  released  in  a 
slow  process  that  is  first  order  with  respect  to  time  and  has  a  half  time 
at  37°  of  0.5-0.9  min  or  at  25°  of  3-4  min.   The  slow  thermal  process  may 
be  attributed  to  a  conformational  change  in  the  protein  that  is  associated 
with  an  endothermic  ^H  of  /^+  3  kcal  per  mole  subunit.   The  binding  of 
Mn   to  glutamine  synthetase  is  largely  entropy  driven  with  AS  0^    +35 
cal/degree/mole  subunit.   Approximately  two  protons  also  are  released  from 
the  enzyme  for  each  equivalent  of  Mg^"*"  bound.   The  kinetics  of  the  slow 
thermal  process  during  the  binding  of  Mn2+  (or  Mg2+)  were  similar  to  those 


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Serial  No.  NHLI-135 

obtained  by  difference  spectra  measurements  at  290.3  nm  at  either  37°  or  25°. 
The  first-order  rate  constants  for  the  protein  conformational  change  at  25° 
and  37°  indicate  that  the  heat  of  activation  for  this  process  is  large 
( '^  +  25  kcal  per  mole  subunit) .   Despite  the  rather  large  perturbations  in 
the  tyrosyl  and  tryptophanyl  spectral  regions  induced  by  the  binding  of  Mn"^"*" 
(or  Mg^"^)  to  glutamine  synthetase,  these  cations,  (as  also  either  Co   or 
Zn^"*") ,  have  no  detectable  effect  on  the  secondary  or  quaternary  structure  as 
determined  by  circular  dichroism  (CD)  and  optical  rotatory  dispersion  (ORD) 
measurements  (310-218  nm) .   From  CD  and  ORD  measurements,  it  was  estimated 
that  the  a-helical  content  of  native  glutamine  synthetase  is  r^  377„;  6  M 
guanidine-HCl  converts  the  enzyme  structure  to  1007o  random  coil. 

3.   X-Ray  crystallography  of  glutamine  synthetase  from  E.  coli  (D.  M.  Segal 
and  D.  R.  Davies,  NIAMD) .   Glutamine  synthetase  preparations  in  extreme 
adenylylation  states  (i.e.  glutamine  synthetase  with  one  or  twelve  equiva- 
lents of  3'-adenylyl  groups  per  mole  enzyme)  have  been  provided.   The 
eventual  growth  of  satisfactory  crystals  for  X-ray  analysis  is  anticipated. 

Significance  to  Bio-Medical  Research:   The  regulation  and  control  of 
enzymic  activities  jji  vivo  is  of  fundamental  importance  in  cellular  meta- 
bolism.  Through  studies  ^  vitro  these  processes  can  be  understood  more 
fully.   The  study  of  structural  changes  that  can  be  induced  in  a  protein 
macromolecule  are  important  in  understanding  cellular  processes  on  a  mole- 
cular basis. 

Proposed  Course  of  Project: 

1.  Studies  of  the  binding  of  substrates  and  other  effectors  to  glutamine 
synthetase  of  E.  coli  will  be  continued.   In  particular^  allosteric  linkage 
between  different  effectors  of  the  enzyme  will  be  investigated.   Direct 
binding  methods,  measurement  of  proton  release  from  the  enzyme  during  metal 
binding  by  micro-pH  methods,  micro-calorimetric  experiments  (in  collabora- 
tion with  P.  D.  Ross,  NIAMD),  and  possibly  also  NMR  and  epr  studies  will  be 
employed  in  these  studies. 

2.  Differential  sedimentation  techniques  will  be  standardized  using  the 
Beckman  Model  E.  ultracentrifuge  in  order  to  measure  gross  conformational 
changes  induced  in  macromolecules  by  various  effectors.   Simultaneously 
induced  micro-structural  perturbations  will  be  monitored  by  spectral, 
optical  rotatory  dispersion,  and  circular  dichroic  measurements. 

3.  Physical-chemical  properties  of  the  active  molecular  forms  of  ATP: 
glutamine  synthetase  adenylyl transferase  will  be  determined  by  sedimentation- 
equilibrium,  isoelectric  focusing,  electrophoresis,  standardized  Sephadex- 
gel  filtration,  and  fluorescent  techniques.   Selective  chemical  modification 
of  the  sulfhydryl  groups  (or  other  amino  acid  residues)  of  this  protein  will 
be  attempted  in  order  to  study  structure-function  relationships.   Techniques 
to  dissociate  the  Pj -adenylyl transferase  will  be  explored  for  the  purpose 

of  studying  the  subunit  structure  and  subunit  reassociation. 

4.  Attempts  will  be  made  to  isolate  and  characterize  a  3' -5'  cyclic 
AMP-dependent  protein  kinase  from  E.  coli. 


3SZ> 


Serial  No.  NHLI-135 

5.   The  X-ray  crystallographic  studies  on  glutamine  synthetase  by 
Drs.  D.  M.  Segal  and  D.  R.  Davies  (NIAMD)  will  continue. 

Publications: 

1.  Stadtman,  E.  R. ,  Ginsburg,  A,,  Ciardi,  J.  E. ,  Yeh,  J.,  Hennig,  S.  B. , 
and  Shapiro,  B.  M. :   Multiple  molecular  forms  of  glutamine  synthetase  pro- 
duced by  enzyme  catalyzed  adenylylation  and  deadenylylation  reactions. 
Advan.  Enz.  Regulation  8:   99-118,  1970. 

2.  Ginsburg,  A.:   The  preparation  of  5'-   C-adenylyl  glutamine  synthetase 
of  Escherichia  coli,  H.  Tabor  and  C.  W.  Tabor  (eds.),  Methods  in  Enzymology, 
Vol.  XVII  Part  A,  Academic  Press,  Inc.,  New  York  and  London,  1970,  p.  927- 
936. 

3.  Ginsburg,  A.  and  Stadtman,  E.  R. :   Multienzyme  Systems.   Ann.  Review  of 
Biochemistry  39:   429-472,  1970. 

4.  Deuel,  T.  F. ,  Ginsburg,  A.,  Yeh,  J.,  Shelton,  E. ,  and  Stadtman,  E.  R. : 
Bacillus  subtilis  glutamine  synthetase:  Purification  and  physical  charac- 
terization.  J.  Biol.  Chem.  245^:   5195-5205,  1970. 

5.  Anderson,  W.  B. ,  Hennig,  S.  B. ,  Ginsburg,  A.,  and  Stadtman,  E.  R. : 
Association  of  ATP:  Glutamine  synthetase  adenylyltransf erase  activity  with 
the  P-r  component  of  the  glutamine  synthetase  deadenylylation  system.   Proc. 
Natl.  Acad.  Sci.  (U.S.)  67:   1417-1424,  1970. 

6.  Hennig,  S.  B. ,  Anderson,  W.  B. ,  and  Ginsburg,  A.:   Adenosine  triphos- 
phate: Glutamine  synthetase  adenylyltransferase  of  Escherichia  coli:  Two 
active  molecular  Forms.   Proc  Natl.  Acad.  Sci.  (U.S.)  67:   1761-1768,  1970. 

7.  Hennig,  S.  B.  and  Ginsburg,  A.:  ATP:  Glutamine  synthetase  adenylyl- 
transferase from  Escherichia  coli:  Purification  and  properties  of  a  low- 
molecular  weight  enzyme  form.   Arch.  Biochem.  and  Biophys. ,  1971  (in  press). 


^s-/ 


i 


I 


Serial  No.  NHLI-136 


Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1970  through  June  1,  1971 

Project  Title:   Zinc  Induced  Paracrystalline  Aggregation  of  Glutamine 
Synthetase 

Principal  Investigator:   Richard  Miller 

Other  Investigators:   E.R.  Stadtman 

F.Z.  Smyrniotis 

Project  Description: 

Objectives:   1)   The  effects  of  zinc  upon  native  glutamine 
synthetase  were  studied  using  a  kinetic  spectrophotometric  and  spectro- 
fluoromatic  approach. 

2)   Purification  and  properties  of  glutamine-2-oxo- 
glutarate  aminotransferase,  oxido  reductase  (glutamate  synthetase). 

Zinc  induced  spectral  changes  and  zinc  induced  changes  in  TNS- 
enzyme  fluorescence  provides  evidence  for  a  marked  zinc  induced  change  in 
enzyme  conformation. 

Zinc  may  act  either  as  an  inhibitor  or  as  an  activator  of  glutamine 
synthetase  biosynthetic  activity  depending  upon  pH,  ATP  concentration 
and  Mg"^  concentration.   Furthermore,  it  has  recently  been  shown  that 
Zn++  can  support  y  -glutamyl  transfer  activity  of  unadenylylated  glutamine 
synthetase. 

Major  Findings: 

A  previously  unknown  pathv/ay  for  glutamate  synthesis  has  been 
identified  in  Aerobacter  aerogenes  (tempest  et^  al . ,  Biochem.  J.  (19V0) 
117,  405). 

glutamine  +  a-ketoglutarate  +  TPNH  -  TPN  +  2,  glutamate 

The  enzyme  catalyzing  this  reaction  has  been  purified  to  near  homogeneity 
from  crude  extracts  of  E.    coli  W.   (NH^)2S04  acetone,  heat  and  gel 
filtration  steps  were  employed  in  the  purification.   The  protein  gave 
a  single  symmetrical  boundary  in  the  analytical  ultracentrifuge  (s20,w22S). 
The  molecular  weight  (750,000)  was  determined  by  ultracentrifugation'and 
gel  filtration.   As  isolated  the  enzyme  was  associated  with  approximately 
one  mole  of  flavin  per  200,000  gms  of  protein.   The  bound  flavin  which 
consisted  of  both  FMN  and  FAD  was  reduced  by  TPNH  but  not  by  DPNH. 


SS-x. 


Serial  No.  NHLI-136 


The  enzyme  pH  optimum  was  7.8.   The  Km  for   a-ketoglutarate  was  30 

yM  and  for  glutamine  it  was  170  ]M.  Preliminary  gorwth  experiments 

indicate  that  in  batch  cultures  of  E.  coli  w  there  is  no  relationship 
between  the  level  of  glutamate  synthetase  and  that  of  glutamine  synthetase, 

glutamate  dehydrogenase  or  free  NH^.  Preliminary  electron  micrographs 

indicate  that  glutamate  synthetase  is  a  large,  multisubunit ,  spherical 
molecule. 

Coupled  with  glutamine  synthetase,  (Km  for  NH3  1.0  mM) ,  glutamate 
synthetase  has  the  capacity  to  play  an  important  role  in  NH3  fixation  at 
levels  of  NH3  far  below  those  necessary  for  the  fxinction  of  glutamate 
dehydrogenase.   Furthermore,  the  lack  of  correlation  between  levels  of 
glutamate  synthetase  and  those  of  glutamine  synthetase,  glutamate 
dehydrogenase  and  free  NH3  in  batch  cultures  and  its  low  Km's  for 
substrates   suggest  that  glutamate  synthetase  may  be  of  major  importance 
in  NH3  metabolism  under  all  conditions. 

Proposed  Course  of  Action: 


Studies  are  planned  to  elucidate  further  the  kinetic  and 
physical  properties  of  glutamate  synthetase. 


i      i 


3^3 


■BsaBjjhggffWBiarBsrTiuJiaeB-ifiBBi^ 


c 


b 


Serial  No.   NFTT.T-I.S? 

Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  1971 

Project  Title:   Studies  on  L-asparaginase  in  anaerobic  bacteria 

Previous  Serial  Number  226 

Principal  Investigator:   J.M.  Poston 

Other  Investigators:   none 

Project  Description: 

Objectives:   Certain  neoplastic  tissues  have  been  shown  to  be 
adversely  affected  by  invlvo  administration  of  L-asparaginase.   This 
enzyme  lowers  the  concentration  of  circulating  L-asparagine  causing  the 
neoplastic  tissues  to  involute  and  die  while  normal  tissues  are  unaffected. 
The  susceptible  neoplastic  tissues,  unlike  normal  and  unsusceptible 
neoplastic  tissues,  do  not  possess  an  adequate  L-asparagine  synthetase  system. 
It  was  the  objective  of  this  study  to  isolate  an  organism  which  could  serve 
as  an  alternative  source  of  an  L-asparaginase,  especially  one  that  was 
specific  for  L-asparagine  and  had  no  glutaminase  activity.   This  would 
permit  the  study  of  the  effect  of  lowering  circulating  L-asparagine  without 
the  complication  of  lowering  the  circulating  glutamine  as  well. 

Major  Findings: 

As  was  previously  reported,  an  organism  was  isolated  from  soil  by 
enrichment  with  L-asparagine  and  glycerol.   It  possesses  L-asparaginase 
activity  with  an  apparent  K^  of  5  x  10~5  M  and  seems  to  have  negligible 
glutaminase  activity.   This  organism,   strain  7C1,  grows  well  in  a  medium 
containing  1%  L-asparagine,  1%  glycerol,  0.06  M  potassium  phosphate  (pH  7.0), 
trace  metals,  and  made  anaerobic  by  the  addition  of  0.03%  Na2S.   It  also 
grows  well  on  sterile  potato  plugs  either  anaerobically  or  aerobically. 
Dr.  Charles  Zierdt  of  the  Clinical  Pathology  Department  of  the  Clinical 
Center,  NIH,  has  tentatively  identified  7C1  as  a  strain  of  Klebsiella 
pneumoniae.   Although  this  is  a  facultative  organism,  7C1  grows  best  under 
anaerobic  conditions. 

Extracts  of  the  organism  made  by  several  methods  were  strongly  active 
and  retained  their  activity  upon  storage  at  -20°  C  for  long  periods.   The 
activity  is  not  affected  by  sulfhydryl  reagents  but  there  is  marked  inhibition 
by  high  concentrations  (0.01  M)  of  magnesium,  potassium  EDTA. 


3S^ 


Serial  No.  NHLI-137 


The  activity  has  been  refractory  to  purification.   This  seems  to  be 
due,  at  least  in  part,  to  increased  lability  upon  purification.   The 
activity  is  precipitated  between  30  and  40  percent  saturation  with 
ammonium  sulfate.   It  migrates  in  Sephadex  G-150  columns  as  would  be 
expected  for  a  protein  of  about  90,000  daltons.   Ion  exchange  has  not 
shown  any  effect  other  than  inactivation  of  the  enzyme  preparations. 

Affinity  chromatography  effected  striking  purification  but  the 
lability  of  the  activity  was  high  and,  as  yet,  no  satisfactory  method  of 
stabilization  has  been  found.   Heicamethylene  diamine  was  coupled  to 
Sepharose  4B  following  cyanogen  bromide  treatment  of  the  gel.   d (-)-3 -chloro- 
succinamic  acid  (prepared  from  L-asparagine  by  treatment  with  nitrous  acid 
in  the  presence  of  sodium  chloride)  was  coupled  to  the  hexamethylene  diamine 
side  chain  yielding,  thereby,   L-asparagine  attached  to  the  gel  through  a 
six-carbon  spacer  chain.   Passage  of  crude  extracts  or  partially  purified 
fractions  with  from  6  to  60  fold  purification.   Specific  activities  of 
these  preparations  soon  dropped  to  the  original  levels  or  lower. 

Proposed  Course  of  Action; 

Pending  availability  of  new  methods  of  purification  and  stabilization, 
further  work  on  this  project  has  been  terminated. 


Part  B      No 


SSS" 


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c 


Serial  No.   NHLI-138 

Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NHLI 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   1)   Radioisotopic  Assays  for  Glutamine  Synthetase  and 
Glutaminase. 
2)   Regulation  of  Glutaminase  Activity  in  E.  coli 

Previous  Serial  Number:   none 

Principal  Investigators:  Stanley  Prusiner 

Other  Investigators:   E.R.  Stadtman 
L.  Milner 

Project  Description: 

Objectives:    1.   To  develop  a  sensitive  and  rapid  assay  for  measure- 
ment of  gluuamine  synthetase  and  glutaminase  activities. 


2.   To  study  the  regulation  of  glutaminase  activity  in 


E.  coli. 


Major  Findings: 

1.  A  very  sensitive  and  rapid  assay  for  glutamine  synthetase  and 
glutaminase  has  been  developed  by  separating  glutamate  from  glutamine  on 
small  columns  containing  dowex  1-Cl.   The  procedure  has  been  shortened 
from  several  hours  to  a  few  minutes  by  the  design  of  a  vacuum  manifold 
system  which  rapidly  filters  the  sample  over  the  ion  exchange  resin.   The 
assay  has  also  been  extended  for  the  measurement  of  asparagine  synthetase 
and  asparaginase  activity. 

2.  Two  glutaminases  from  JE.  coli  have  been  reported  in  the  literature 
previously  with  pH  optima  at  5  and  7.   The  relative  amounts  of  these  two 
enzymes  vary  markedly  in  the  four  strains  of  E.  coli  examined:   ML,  W,  K-12, 
and  B.   Growth  studies  with  E_^    coli  B  have  shown  that  the  pH  5  enzyme  in- 
creases markedly  during  stationary  phase  while  the  pH  7  is  highest  during 
log  and  early  stationary  phase.   These  studies  were  complicated  by  the  fact 
that  the  pH  7  enzyme  is  cold  labile  and  both  glutamine  and  glutamate  have  a 
profound  activating  effect  on  the  catalytic  activity  of  the  pH  7  enzymes. 
Preliminary  experiments  suggest  the  pH  7  enzyme  gives  highest  activity  in 
crude  extracts  when  the  cells  are  CHO  limited  and  have  excess  nitrogen 
available.   The  enzyme  has  been  purified  more  than  1700  fold.   Glutaminase 
requires  glutamate  and  borate  for  stability  at  this  stage  of  purification. 
The  pH  optima  is  7  with  almost  no  activity  at  pH  5.   Substrate  saturation 
studies  3how   complex  kinetics  associated  with  cooperative  phenomena. 


3SZ 


Serial  No.  NHLI-138 
Proposed  Course  of  Project: 

1.  Continue  purification  of  glutaminase  to  homogeneity. 

2.  Physical  and  kinetic  characterization  of  the  purified  enzjone. 

3.  Correlation  of  enzymic  activity  in  crude  extracts  with  NH3  and 
glutamate  levels  of  cells  which  can  be  varied  depending  on  the  growth 
conditions  and  stage  of  harvest. 

4.  Determine  whether  adenylylation  system  affects  the  activity  of 
glutaminase. 

5.  Comparison  properties  pH  5  and  7  glutarainases. 

6.  Attempt  to  elucidate  function  of  glutaminases  by  studying  the 
properties  of  the  enzyme  in  the  isolated  and  crude  forms  and  by  genetic 
selection  for  mutations  in  the  metabolism  of  glutamine. 


Part  a    Yes 


I 


■^ 


3S-7 


Serial  No.  NHT,T-13P^ 


Publications 

1.  Prusiner,  S.  and  Milner,  L. ,  A  rapid  radioactive  assay  for  glutamlne 
synthetase,  glutaminase,  asparagine  synthetase,  and  asparaginase. 

Anal.  Biochem.  37.-'  ^29-438,  1970. 

2.  Prusiner,  S. ,  Milner,  L. ,  Long,  C.W. ,  and  Myers,  M. ,  Vacuum  manifold 
for  rapid  assay  of  enzymes  using  radioactive  tracers  and  ion  exchange 
chromatography.   Review  of  Scientific  Methods,  1971  (in  press). 


35S 


Serial  No.  NHLI-139 


Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NHLI 

Individual  Project  Report 

July  1,  1970  through  June  1,  1971 

Project  Title:   Studies  on  Lysine  Fermentation  in  Clostridia 
The  D-a-Lysine  Pathway 

Principal  Investigator:   J.  Wai-Kuo  Shih 

Other  Investigators:   T.C.  Stadtman 

Project  Description: 

Objectives:   It  has  been  shown  by  this  laboratory  that  Clostridium 
sticklandii   converts  DL-ct-lysine  to  a  mole  each  of  acetate  and  butyrate 
and  two  moles  of  ammonia.   The  cell  suspensions  utilize  both  isomers  of 
lysine  as  shown  in  Scheme  I. 


CH  -CH2-CH2-CH2-C-COOH 


NH, 


NH. 


CH  -CH  -CH  -COOH  +  CH  -COOH 


CH  -COOH  +  CH  -CH2-CH2-COOH 


Scheme  I 

However,  soluble  enzyme  preparations  fail  to  produce  acetate  from  carbons  5 
and  6  of  lysine  (D-cleavage  of  Scheme  I) . 

This  investigator  intends  to  study  the  metabolism  of  D-a-lysine  in 
this  organism  and  particularly: 

(1)  The  conditions  required  for  effective  fermentation  of  D-a-lysine 
by  cell  free  enzyme  preparations. 

(2)  The  systems  involved  in  the  utilization  of  2,5-diamino  hexanoate 
which  is  the  product  of  D-a-lysine  mutase.   This  reaction  is  thought  to  be 
the  first  step  in  the  D-cleavage  pathway. 


Major  Findings: 


,14 


(1)  For  the  differentiation  of  the  L-  and  D-  cleavages  6-C  -DL-  -lysine 
is  used  as  substrate.   Either  D-  or  L-  lysine  can  be  used;  presumably  this 
organism  contains  lysine  racimase  in  excess.   The  ratio  of  Cl^-acetate  to 
Cl4-butyrate  is  a  measure  of  the  preference  for  the  D-pathway.   Wild-type 
and  a  few  small-colony  mutants  of  Clostridium  sticklandii  were  examined. 


•a 

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3srf 


ump^ 


i 


Serial  No.    NHLI-139 


Cell  suspensions  of  small-colony  mutant  S3  and  wild-type  cultures 
grovm  on  an  arginine-lysine  medium  seemed  capable  of  fermenting  lysine  by 
the  D-cleavage  more  readily  than  regular  cultures. 

(2)   For  simplifying  the  tedious  assay  of  C-^^  acetate  and  C-'-'^  butyrate 
as  their  hydroxmates,  a  revised  method  was  employed.   An  acetokinase, 
specific  for  acetic  acid  was  used  to  convert  only  acetate  to  the  nonvolatile 
hydro>nnate.   The  ratio  of  residual  radioactivity  after  reaction  with  aceto- 
kinase and  exhaustive  evaporation  to  the  total  radioactivity  in  the  original 
sample  is  a  measure  of  the  acetic  acid  content  of  incubation  mixtures.   Thus, 
C^^-acetate  production  from  6-cl^  lysine  is  an  assay  of  the  D-lysine  pathway. 

Proposed  Course  of  Research: 

1.  Conditions  and  cof actor  requirements  favorable  for  D-cleavage 
by  the  cell  suspension  system  will  be  studied  in  more  detail.   Hopefully, 
the  information  gained  can  be  applied  to  soluble  enzyme  preparations.   It 
may  be  necessary  to  develop  a  revised  technique  of  preparing  cell  free 
systems. 

2.  Both  1-cl^  and  6-C-'-^-2 ,5-diamino  hexanoate  were  prepared. 
These  labeled  materials  will  be  applied  to  the  study  of  the  further 
metabolism  of  this  compound  and  may  help  elucidate  the  defect  in  the 
metabolism  of  D-lysine  by  cell-free  system. 


Part  B      No 


34o 


Serial  No.  NHLI-140 

Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NHLI 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:  1.  Anaerobic  metabolism  of  certain  amino  acids  and  other 
nitrogen  compounds  with  especial  reference  to  the  role 
of  Bi2  coenzyme  and  to  electron  transfer  and 
phosphorylation  reactions  involved. 

2.  Methane  biosynthesis  and  one-carbon  compound 
metabolism. 

Principal  Investigator:  T.C.Stadtman 

Other  Investigators:  Colin  G.D.Morley  (Visiting  Scientist,  terminated 

Sept.  30,  1970)  B-a-lysine  mutase  studies, 
David  Turner  (NIH  Postdoctoral  Fellow,  terminated 

Feb.  3,  1971)  Glycine  reductase  studies. 
H.Fu  Rung  (Visiting  Scientist,  terminated,  Oct.  16, 

1970).  Nicotinic  acid  metabolism 
James  Shih  (Visiting  Scientist,  see  individual 

report) 
Chris  van  der  Drift  (Visiting  Scientist,  see 

individual  report) 
John  Baker  (Postdocotral  Fellow,  see  individual 

report) 
Laren  Cagen  (Postodoctoral  Fellow,  see  individual 

report) 
Joseph  N.  Davis  (Technical  assistant  and  anaerobic 

Laboratory  operator) 
Jay  Jones  (Technical  assistant). 

Cooperating  units:  Dr.  Lin  Tsai  (Lab. Biochem.  , Section  on  Enzymes,  NHLI)  go 

Dr.  J.  Retey  (Zurich,  Switzerland) 


Project  Description: 

Objectives:  1.   Determination  of  mechanism  of  B]^2  coenzyme  dependent 
amino  group  migration  reactions  of  the  lysine  fermentation.   (a) 
Purification  and  characterization  of  D-a-lysine  mutase  were  carried  out 
jointly  with  Dr.  Colin  Morley.  Continuation  of  studies  on  further  metabo- 
lism of  D-a-lysine  and  2 ,5-diaminohexanoate  carried  out  by  Dr.  James  Shih 
in  cooperation  with  Dr.  Lin  Tsai. 

(b)  Purification  and  characterization  of   g-lysine  mutase  were  con- 
tinued by  Dr.  John  Baker  and  Dr.  Chris  van  der  Drift.   Investigation  of 
roles  of  ATP  and  pyruvate  are  in  progress. 

2.   Metabolism  of  nicotinic  acid.  .  characterization  of  the  Bi^2 
coenzyme  dependent  step  of  the  fermentation,  and  a  subsequent  isomer- 
ization  of  methylitaconate  to  dimethylmaleate  was  carried  out  by 
Dr.  Hsiang-fu  Kung  in  cooperation  with  Dr.  Lin  Tsai. 

1  36/ 


3 


Serial  No.  NHT.T-140 


3.  Nature  of  electron  transport  and  phosphorylation  process  linked 
to  glycine  reduction  in  Clostridium  sticklandii.  Purification  of  components 
of  the  multienzyme  system  by  Dr.  David  Turner.  Purification  of  flavoprotein 
electron  transport  protein  by  Dr.  Laren  Cagen. 

4.  Characterization  of  the  menadione-dependent  p-nitrophenyJ- 
phosphatase  of  Cj_  sticklandii.   Experimental  work  carried  out  by  Joe  N.  Davis. 

5.  Isolation  and  identification  of  acidic  peptide  growth  factor 
required  by  wild  type  C^.  sticklandii  for  growth.   Experimental  work  carried 
out  by  Jay  Jones. 

6.  Mechanism  of  methane  biosynthesis  from  formate  by  Me thanococcus 
vannielii  and  from  acetate  by  Methanosarcina  barker!. 

Major  Findings: 

1.  Lysine  fermentation  reactions: 

In  addition  to  its  participation  in  the  overall  amino  group 
migration  reaction  catalyzed  by  D-a-lysine  tnutase,  the  cobamide  protein 
moiety  of  the  enzyme  complex  catalyzes  a  slow  pyridoxal  phosphate  and 
Mg"*^  dependent  exchange  of  hydrogen  between  water  and  the  C-6-methylene 
group  of  D-lysine.   Several  lines  of  evidence  suggest  that  this  exchange 
reaction  and  the  nigration   of  the  amino  group  to  the  adjacent  carbon 
atom  are  related  processes  and  that  both  require  Schiffs   base  formation 
between  the  terminal  amino  group  of  lysine  and  pyridoxal  phosphate. 
Inhibition  of  the  cobamide  protein  moiety  by  treatment  with  tetranitro- 
methane  and  by  N-acetylimidazole,  reagents  known  to  modify  tyrosine  groups 
of  proteins,  suggests  that  as  in  certain  other  pyridoxal  phosphate  dependent 
enzymes,  a  tyrosine  may  be  important  for  activity  of  the  cobamide  protein 
moiety  of  D-a-lysine  mutase. 

Although  the  intact  D-a-lysine  mutase  complex  appears  to  require  ATP 
only  as  an  allosteric  activator  and  is  equally  stimulated  by  the  phosphonlc 
acid  analogues  of  ATP,  certain  preparations  of  the  separated  protein 
components  are  active  only  with  ATP.   Separated  components  of   3-lysine 
mutase  behave  in  the  same  manner  and  it  is  tentatively  concluded  that 
ATP  is  additionally  required  in  reaction (s)  involving  chemical  modification 
of  both  of  these  mutases. 

2.  Studies  of  Dr.  Kung  established  the  obligatory  intermediary  roles 
of  ct-methyleneglutarate  mutase  (B]^2  coenzyme  dependent)  and  methylitaconate 
isomerase  in  the  overall  fermentation  of  nicotinic  acid  to  acetic  and 
propionic  acids  and  ammonia  by  Clostridium  barker i.   The  expected  hydrogen 
migration  reactions  could  be  demonstrated  by  tritium  and  deuterium  studies. 
Both  enzymes  were  obtained  in  highly  purified  form  and  were  considerably 
characterized . 


4 


3C^ 


Serial  No.  NHLI-140 


3.   Dr.  Turner  further  purified  to  homogeniety  the  small  acidic 
sulfhydryl  protein  moiety  of  the  glycine  reductase  system  of  Clostridium 
sticklandii  and  also  succeeded  in  further  separation  of  the  enzyme  system 
into  two  highly  purified  somewhat  larger  protein  fractions.   The  three 
recombined  proteins  still  exhibited  absolute  requirement  for  phosphate  in 
order  to  reduce  glycine  to  acetate  and  ammonia.   Thus  extensive  purification 
seems  not  to  have  caused  loss  of  the  concommitant  phosphorylation  reaction. 
Marked  sensitivity  to  low  levels  of  hydroxylamine  suggests  a  carbonyl 
cof actor  requirement  for  the  reaction. 

4.  The  pure  menadione-dependent  and  SH~  dependent  alkaline  phosphatase 
of  C^.  sticklandii  which  utilizes  p-nitrophenyl  phosphate  as  its  only  known 
substrate,  failed  to  be  labeled  with  P32  labeled  substrate  under  a  variety 
of  experimental  conditions.   Current  attempts  to  determin  e  the  nature  of 
the  phosphorylated  intermediate  that  finally  is  hydrolyzed  to  orthophosphate 
involve  synthesis  of  the  monophosphate  ester  of  menadiol  (reduced  menadione). 
Utilization  of  this  quinol  monophosphate  would  further  suggest  a  role  of  the 
phosphatase  in  an  electron  transport  phosphorylation  process. 

5.  The  acidic  peptide  growth  factor  required  by  C.  sticklandii  has 
been  highly  purified  from  tryptic  digests  of  cesein.   Several  active 
peptide  fractions  which  contain  5  to  7  amino  acid  residues  per  peptide 

(as  judged  by  gel  filtration  studies)  have  been  isolated.   The  major  amino 
acids  in  the  active  peptide  fractions  so  far  analyzed  are:  glutamic  acid, 
valine,  leucine,  isoleucine,  alanine  and  some  glycine,  serine  and  threonine. 
All  other  amino  acids  present  in  casein  thus  seem  to  be  excluded  as 
necessary  components  of  the  required  peptide  growth  factor.   The  minimum 
composition  of  the  active  peptide (s)  is  not  yet  known. 

6.  Methane  biosynthesis  from  formate  by  extracts  of  Me thano coccus 
vannielii   depends  on  ATP  and  hydrogen  and  is  stimulated  by  various  one- 
carbon  derivatives  of  uracil  and  also  by  phosphoribosyl  pyrophosphate. 
When  l^C-formate  is  used  as  substrate,  a  highly  radioactive  nucleotide 
fraction  can  be  isolated  from  the  bacterial  extracts.   This  labeled  material 
which  is  anionic,  also  serves  as  substrate  for  l^C-methane  synthesis. 
Experiments  are  in  progress  to  characterize  this  material  chemically  and  to 
determine  whether  it  is  an  actual  intermediate  in  the  overall  reduction 
pathway. 

Proposed  Course  of  Research: 

Since  all  of  the  problems  mentioned  above  except  the  study  of 
nicotinate  metabolism   (2)  are  currently  in  progress,  the  general  direction 
of  the  research  outlined  in  each  instance  is  that  to  be  further  pursued  in 
the  months  to  come. 


3^3 


■aii—i«j  imuaLUuvutwrnwitLUij 


Serial   No.      NHLI-140 

Part   B 

Pub] ications 

1.  T.C.  Stadtman,  "Vitamin  B^^"  '  Science  l^l  859  (1971) 

2.  C.G.D.  Morley  and  T.C.  Stadtman,  Studies  on  the  Fermentation  of 
D-a-lysine.   Purification  and  properties  of  an  adenosinetriphosphate 
regulated  B12  coenzyme  dependent  D-a-lysine  mutase  complex  from 
Clostridium  sticklandii.  Biochemistry  9^  4890  (1970). 

3.  C.G.D.  Morley  and  T.C.  Stadtman,  STudies  on  the  Fermentation  of 
D-a-lysine.   On  the  Hydrogen  Shift  Catalyzed  by  the  Bx2  coenzyme 
Dependent  D-a-lysine  Mutase,  Biochemistry  (in  press) 

4.  C.G.D.  Morley  and  T.C.  Stadtman,  Studies  on  the  Fermentation  of  D-a-lysine 
On  the  role  of  pyridoxal  phosphate  in  the  B12  coenzyme  dependent  D-a-lysine 
mutase  reaction,  (in  preparation). 

5.  H.F.  Kung  and  T.C.  Stadtman,  Nicotinic  Acid  Metabolism.  VI.  Purification 
and  Properties  of   a-methyleneglutarate  Mutase  (B2^2~^^P^'^'^^'^'-)  ^^'^ 
Methylitaconate  Isomerase,  J.  Biol.  Chem.  246:3746  (1971). 

6.  H.F.  Kung,  L.  Tsai  and  T.C.  Stadtman,  Nicotinic  Acid  Metabolism.  VIII. 
Tracer  Studies  on  the  Intermediary  Roles  of   a-methyleneglutarate, 
Methylitaconate,  Dime  thy Imaleate  and  Pyruvate,  (in  preparation). 

7.  T.C.  Stadtman,  "B12  coenzyme  dependent  amino  group  migrations" 
The  Enzymes,  Vol  VI  Ed.  by  P.D.Boyer,  Academic  Press  (in  press). 

8.  A  Schwartz  and  T.C.  Stadtman,  Small  Colonies  of  Clostridium  sticklandii 
Resulting  from  Nitrosoguaindine  Treatment  and  Exhibiting  Defects  in 
Catabolic  Enzymes,  J.  Bact.  104,  1242  (1970). 


3iiC 


Serial   No.      NHTJ-141 

LaBoratory  of   Biochemistry 
Section   on   Enzymes 
Bethesda,   Maryland 

PHS-NHLI 

Individual  Project  Report 

July  1,  1970  through  June  30,  1971 

Project  Title:   1.   Effects  of  Divalent  Cations  and  Energy  Charge  Upon  the 
Activity  of  Glutainine  Synthetase 

Principal  Investigator:   Amiel  Segal 

Other  Investigator:   E.R.  Stadtman 

Project  Description: 

Objective:   1.   The  differential  effects  of  Cq-^,   Mn"*^,  Mg++,  Ca"'"'" 
and  Cd"'"'"  upon  the  pH  optimum.  Km  for  substrates,  catalytic  activity  and  UV 
spectrum  of  glutamine  synthetase  indicate  that  each  provokes  a  unique  "» 

conformation  of  the  enzyme.   Cadmium ^calcium  and  manganese  salts  inhibit  the 
Mg++  dependent  activity  of  unadenylylated  glutamine  synthetase,  whereas 
Co"'"^  is  an  activator  of  the  enzyme. 

Like  Mg++,  Co"^  supports  activity  only  of  unadenylylated  subunits,  but 
the  pH  optimujj  feedback  inhibitability,  and  dependence  upon  the  metal/ATP 
ratio  are  similar  to  those  of  Mn++  dependent  activity  of  adenylylated  enzyme. 
With  Mg++  or  Mn"^  the  enz5rme  exhibits  hyperbolic  saturation  functions  for 
glutamate  and  NH^  but  Co"*"!"  dependent  activity  exhibits  heterotTopic  and 
homotropic  interactions  with  these  substrates.   The  non  linear  relationship 
between  specific  activity  and  state  of  adenylylation  supports   the  conclusion 
that  heterologous  interactions  occur  between  adenylylated  and  unadenylylated 
subunits  in  hybrid  molecules.   The  data  suggest  that  adenylylation  of  a 
subunit  abolishes  its   activity  and  decreases  activity  of  adjacent  un- 
adenylylated  subunits.   These  heterologous  interactions  render  the  co 
enzyme  exquisitely  sensitive  to  regulation  by  adenylylation  and  deadenylylation. 

2.   Mg-*"*"  dependent  activity  of  unadenylylated  glutamine  synthetase  is 
a  function  of  the  energy  charge  of  the  reaction  mixture,  whereas  Mn"*"*" 
dependent  activity  of  adenylylated  enzyme  is  considerably  less  dependent  upon 
the  energy  charge.   The  response  of  the  enzyme  to  energy  charge  is  a  function 
of  the  Mn++  concentration,  with  increasing  sensitivity  to  energy  charge  at 
higher  Mn"*"*"  concentrations.   The  inhibitability  of  glutamine  synthetase  by 
glycine  and  alanine  increases  markedly  with  increasing  energy  charge.   These 
observations  are  consistent  with  similar  observations  made  on  other  bio- 
synthetic  enzymes. 

Proposed  Course  of  Research:   Temperarily  discontinued. 


36r 


Serial  No.     KTTT.T-142 


I 


Laboratory  of  Biochemistry 
Section  on  Enzjines 
Bethesda,  Maryland 

PHS-NHLI 
Individual  Project  Report 
July  1,  1970  through  June  30,  19  71 

Project  Title:   Antibodies  to  E^.  coli  Glutamine  Synthetase 

Previous  Serial  No.  NHLI-208 

Principal  Investigator:   Steven  R.  Tronick 

Other  Investigators:   E.R.  Stadtman 
J.E.  Ciardi 

Project  Description; 

Objectives:   1)   To  characterize  rabbit  antibodies  to  adenylylated 
and  unadenylylated  E^  coli  glutamine  synthetase. 

2)  To  determine  if  these  antibodies  react  with  crude 
glutamine  synthetase  preparations  from  various  microorganisms. 

3)  To  determine  if  cross-reacting  enzymes  are  modified 
by  adenylylation. 

Major  Findings: 

1)   Complement  fixation-  The  sensitive  micro-complement  fixation 
technique  of  Van  Vanukis  and  Levine  (Methods  in  Enzymol.  11,  928)  was  used 
in  order  to  detect  differences  in  the  conformation  of  adenylylated  (E12) 
and  unadenylylated  (Eg)  enzymes.   This  method  was  also  useful  in  detecting 
classes  of  antibodies  directed  to  different  antigenic  determinants   on  the 
enzymes.   No  differences  were  found  between  Eq  and  £12  using  antisera  to 
either  form  of  the  enzyme.   This  result  indicates  that  Eq  and  E]^2  have  very 
similar,  conformations.   The  peak  of  complement  fixation  (varying  enzyme 
against  constant  antiserum)  was  broad-indicating  many  antigenic  determinants 
on  the  enzymes.   0.007  ug/ml  of  enzyme  was  readily  detected  with  a  1:20,000 
dilution  of  antiserum.   The  following  results  suggest   that  adenylylation 
introduces  a  distinct  antigenic  determinant  on  the  enzjmie  and  its  subunits. 
Antiserum  to  £3^2  fixed  complement  with  £j^2~subunits  but  not  with  Eg-subunits. 
An  antiserum  to  E]^2  subunits  fixed  complement  with  adenylylated,  but  not  un- 
adenylylated subunits.   This  antiserum  fixed  complement  with  E12  (1/5  the 
amount  fixed  with  E]^2  subunits)  but  not  with  Eg.    Another  antiserum  to 
adenylylated  subunits  also  fixed  complement  with  E]^2~su'^'J"^ts  but  not  with 
Eg-subunits.  (However,  this  antiserum  fixed  complement  with  Eg  and  E] 2 : 
The  titer  for  Eg  and  E12  decreased  during  the  course  of  immunization,  while 
the  titer  for  Ei2~subunits  greatly  increased).  The  antisera  to  E]^2  ^'^'^  E12- 
subunits  did  not  bind  AMP  as  measured  by  equilibrium  dialysis,  ammonium 
sulfate  precipitation  of  Ab-Ag  complexes,  and  pr acipitation  of  Ab-Ag  complexes 
with  anti-rabbit  gamma  globulin.   In  order  to  further  characterize  antibody 

1  3U 


Serial  No. 


NHLI~142 


classes  in  each  antiserum,  competition  experiments  were  performed.   Although 
difficulties  were  encountered  in  obtaining  reproducible  data,  the  results 
suggest  that  subunits  bind  to  E12  and  Eg  antisera.   Also,  EO~subunits 
appeared  to  bind  to  Ei2~sutiunit  antiserum.   (Thus,  separation  of  Eg-  and 
Ei2~subunits  may  be  very  difficult) . 

Complement  fixation  was  also  used  to  determine  whether  pure  glutamine 
synthetases  from  other  organisms  are  antigenically  related  to  E^.  coli. 
Pseudomonas  putida  glutamine  synthetase,  obtained  from  Dr.  A.  Segal, 
partially  cross-reacted  (0.1   Hg  of  enzyme  fixed  65%  less  complement  than 
0.1   yg  of  ^.  coli  enzyme).   Bacillus  subtillis  glutamine  synthetase,  pre- 
pared by  Dr.  T.Deuel,  did  not  cross-react.   These  results  are  in  accord 
with  those  previously  reported  (No.  208,  1970)  using  the  method  of 
Ouchterlony. 

2)  Inactivation  of  enzyme  activity-  Gamma  globulin  purified  from 
antisera  to  Eq  and  E12  inhibited  the  y-glutamyl  transferase  activity 

of  Eq  and  Ei2'   %2  ^^^  inhibited,  by  either  antibody,  to  a  greater  extent 
than  was  Eg.   Both  Eq  and  E12  were  activated  (130%)  by  an  antiserum  to  E12- 
subunits.   Preliminary  experiments  indicated  that  the  state  of  adenylylation 
of  Eq  or  E12  was  not  changed  by  the  interaction  with  antibody. 

3)  Reaction  with  other  glutamine  synthetases-  Crude  extracts  from 
fourteen  microorganisms  were  examined  for  glutamine  synthetase  activity 
(y-glutamyl  transfer) ,  for  precipitin  line  formation  with  Eq  and  E]^2 
antisera  in  the  Ouchterlony  assay,  and  for  inhibition  of  transferase 
activity  by  anti-Eg  and  £^2  gamma  globulin.   Hethanosarcina  barkerii, 
Clostridium  sticklandii,  Nearospora  crassa.  Bacillus  sxibtilis,  and  Bacillus 
licheniformis  did  not  exhibit  transferase  activity  nor  did  they  form  pre- 
cipitin lines  in  the  Ouchterlony  assay.   Acanthamoeba  castellanii, 
Streptomyces  rutgersensis ,  and  Streptomyces  diastachromogenes  extracts 
displayed  transferase  activity  but  were  not  inhibited  by  high  levels  of 
antibody.   They  did  not  form  precipitin  bands.   Salmonella  typhimurium, 
Klebsiella  pneumoniae,  and  Proteus  mirabilis  crude  extracts  catalyzed  the 
Y-glutamyl  transfer  reaction  and  formed  precipitin  bands  which  fused  with 
the  precipitin  bands  of  pure  and  crude  E.    coli  glutamine  synthetase 
(indicating  antigenic  homology).   The  transferase  activity  in  these  extracts 
was  inhibited  by  antibody,  under  standard  conditions,  to  about  the  same 
extend  (25%)  as  the  E^.  coli  enzjnne.   The  transferase  activity  in  Pseudomonas 
putida  crude  extracts  from  cells  grown  on  4  mM  NH^"*"  (unadenylylated  enzyme) 
was  not  inhibited  by  antibody;  however,  the  activity  in  crude  extracts  of 
cells  grown  on  40  mM  NH4+  (adenylylated  enzyme)  was  inhibited  by  50%.   Both 
types  of  extracts  formed  precipitin  lines  which  spurred  with  the  E.    coli, 

S^.  typhimurium,  and  K.  pneumoniae  bands  (indicating  partial  antigenic 
homology) . 

Surprisingly,  crude  Azotobacter  vinelandii  extracts  formed  precipitin 
lines  with  antibody.   Transferase  activity  supported  by  Mg++  was  inhibited 
by  70%  with  a  standard  concentration  of  JE.  coli  antibody.   The  Mn++  supported 
activity  was  inhibited  by  only  30%  as  was  the  activity  without  added  metal 
ion.   NOrmal  gamma  globulin  had  no  effect  on  any  of  these  activities. 


U7 


I 


I 


Serial  No._jaaU:=i4_2 

Proposed  Course  of  Action : 

The  various  crude  glutamine  synthetases  that  cross-react  with 
antibody  to  the  E_.  coll  enz>Tne  have  all  been  derived  from  gram-negative 
organisms.   Several  more  gram-negative  and  gram-positive  orga'^isms  will 
be  assayed  in  order  to  determine  if,  in  general,  the  glutamine  synthetases 
from  gram-negative   organisms  are  antigenically  related  (to  E^.  coli)  . 
Attempts  will  be  made  to  determine  if  the  cross-reacting  e