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ANMJAL REPORT

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

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

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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325

335

340
342
343

344
348

352
354

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359

361

365
366

369

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380

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386

388
391
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395

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

801
805

807

810

812

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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-

time -dependent
ischemia . Pre-
ffer considerably

is remarkably
respiratory
siveness to hor-
ies also are in

changes in myo-
tional aberrations,
for impaired myo-
s whereby ischem-
ion, can be

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

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.

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

mm^msmms^'

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.

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.

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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.

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.

3.
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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

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.

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.

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^

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.

■a
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-1-

plT

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

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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-1

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.

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

0-J^

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^"

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

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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lU:

CO)

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.

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.

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

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

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

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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 '

•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 <<^

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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WBI"

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

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

tmamns^sai

■ysaxaspnuf-^-'.

Serial No, NHLI-24(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 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.

-1-

rj-

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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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

♦

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.

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

[IT

Serial No. NHTJ-32

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: 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.

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.

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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

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

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.

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

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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.

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

w-^

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.

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

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

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

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

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.

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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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

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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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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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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-

■?f

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.

/CI.

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

/**^

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.

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

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.

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.

Previous Serial No.: None

Principal Investigators:

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

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.

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.

/ff

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.

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

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

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.

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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.

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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

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

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.

/Sc

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"

*»«„

/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>

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.

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.

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

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

■

■a o

B 3

6 o

7^ 1

3
(1) 1

< :

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

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

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

/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:

//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:

Biochemical

studies of histaminase.

The

data

showed

that

histaminase

activity

was associated with diamine oxidase activity

in all

the

tissues stud

ied.

e deamination of 6-^H-histamine and

6-%-

-putrescine

i.7as

inhibited to

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

^»

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

Lung

Bone marrow

Spleen

Liver, kidney, prostate

Heart

Plasma

2.8-4. 6+

Submaxillary gland, skin

testicles, brain

*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^

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.

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

100

Ammonium sulfate

100

1st DEAE Sephadex

122

Sephadex G 100

200

2nd DEAE Sephadex

274

Isoelectric Focusing

343

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/

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

«"

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

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.

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?

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

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

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.

^37

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

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.

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

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.

/?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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/<yf

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

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.

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.

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.

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

-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

^*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

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.

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.

JUS"

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

«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

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

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.

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

6.4 X 10"^

2.8
3.8

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

2 X 10~3 M

4 X 10"-^ M
8 X 10-3 M

44
58

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 .

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.

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^

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.

^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.

^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

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

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¥^

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^

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.

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:

3t¥3

ATPase

120

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.

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*^

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-

-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

^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.

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

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.

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

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%

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

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)

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

«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)

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.

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

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

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.

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

[

\^.

sf^.

]

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

I „

' o

■D

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,

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

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) .

3oi

■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.

1 2o7

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

^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.

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.

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

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

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. '—^ *

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.

3/^

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

ipfl

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.

■>

—1

3/f

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.

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.

—J*

-s

Nelson, P. G., Peacock, H. H. , and Amano, T. : Responses of neuro-
blastoma cells to iontophoretically applied acetylcholine. J. Cell.
Physiol. , in press.

\a D

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t (T>

5 3

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T Q)

3 -■

'-•

3x/

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.

Jix

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.

JXl

'■o o \

RJ CD

|£! o

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-

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

■'n

Ja7

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

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/

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

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

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

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

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.

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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.

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

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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^/

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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

■V

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39f

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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-/

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

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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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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

■^

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

^■

— ».

■)

—J

-•

3srf

ump^

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/

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 .

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

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.

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

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