Progress in Brain Research Vol 08 Biogenic Amines

PROGRESS I N BRAIN RESEARCH VOLUME 8 BIOGENIC AMINES PROGRESS I N BRAIN RESEARCH ADVISORY BOARD W. Bargmann E. De ...

Author: Harold E. & Williamina A. | editors Himwich

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PROGRESS I N BRAIN RESEARCH VOLUME 8

BIOGENIC AMINES

PROGRESS I N BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

E. De Robertis J. C. Eccles J. D. French

H. HydCn

J. Ariens Kappers S. A. Sarkisov

Kiel Buenos Aires Canberra Los Angeles

Goteborg Amsterdam Moscow

J. P. SchadC

Amsterdam

T. Tokizane

Tokyo

H. Waelsch

New York

N. Wiener

J. Z . Young

Cambridge (U.S.A.) London

PROGRESS I N BRAIN RESEARCH VOLUME 8

BIOGENIC A M I N E S EDITED BY

H A R O L D E. H I M W I C H AND

W I L L I A M I N A A. H I M W I C H Galesburg State Research Hospital, Galesbuug, Ill. (U.S.A.)

ELSEVIER P U B L I S H I N G C O M P A N Y AMSTERDAM

/

LONDON

1964

/

N E W YORK

ELSEVIER P U B L I S H I N G C O M P A N Y

335 JAN

V A N G A L E N S T R A A T , P.O. B O X

21 1 ,

AMSTERDAM

AMERICAN ELSEVIER P U B L I S H I N G COMPANY, I N C .

5 2 V A N D E R B I L T A V E N U E , N E W Y O R K , N.Y. 10017

ELSEVIER P U B L I S H I N G COMPANY LIMITED 1 2 B , R I P P L E S I D E COMMERCIAL ESTATE R I P P L E R O A D , B A R K I N G , ESSEX

L I B R A R Y OF C O N G R E S S C A T A L O G C A R D N U M B E R

WITH

94

ILLUSTRATIONS A N D

42

64-15284

TABLES

ALL RIGHTS RESERVED T H I S B O O K O R A N Y P A R T T H E R E O F M A Y N O T BE R E P R O D U C E D I N A N Y F O R M , I N C L U D I N G P H O T O S T A T I C O R M I C R O F I L M FORM, WITHOUT WRITTEN PERMISSION FROM T H E PUBLISHERS

V

List of Contributors

J. AXELROD, Laboratory of Clinical Sciences, National Institute of Mental Health, Bethesda, Md. (U.S.A.).

H. H. BERLET,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.). H. BLASCHKO, Department of Pharmacology, Oxford University, Oxford (Great Britain). B. B. BRODIE,Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Md. (U.S.A.). C. BULL,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.). A. CARLSSON, Department of Pharmacology, University of Goteborg, Goteborg (Sweden).

E. COSTA,Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Md. (U.S.A.). E. DE ROBERTIS, Instituto de Anatomia General y Embriologia, Facultad de Ciencias Midicas, Buenos Aires (Argentina). V. DI CARLO,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

B. FALCK,Department of Histology, University of Lund, Lund (Sweden). D. X. FREEDMAN, Department of Pharmacology and Psychopharmacology Laboratory, Department of Psychiatry, Yale University School of Medicine, New Haven, Conn. (U.S.A.). E. M. GAL,Neurochemical Research Division, Department of Psychiatry, College of Medicine, State University of Iowa, Iowa City, Iowa (U.S.A.).

K. F. GEY,Medical Research Department, Hoffmann-La Roche & Co., Ltd., Base1 (Switzerland). N. J. GIARMAN, Department of Pharmacology and Psychopharmacology Laboratory, Department of Psychiatry, Yale University School of Medicine, New Haven, Conn. (U.S.A.).

H. GREEN,Research and Development Division, Smith Kline and French Laboratories, Philadelphia, Pa. (U.S.A.).

VI

LIST OF CONTRIBUTORS

J. A. HARVEY,Departments of Pharmacology, Psychology and Anatomy, The

University of Chicago, Chicago, 111. (U.S.A.). A. HELLER,Departments of Pharmacology, Psychology and Anatomy, The University of Chicago, Chicago, Ill. (U.S.A.). H. E. HIMWICH,Thudichum Psychiatric Research Laboratory, Galesburg State Hospital, Galesburg, Ill. (U.S.A.).

T. KAKEFUDA,City of Hope Medical Center, Duarte, Calif. (U.S.A.). A. R. KELLY,Henry Ford Hospital, Detroit, Mich. (U.S.A.). H. KOENIG, Neurology Service, V. A. Research Hospital; and Department of Neurology and Psychiatry, Northwestern University Medical School, Chicago, 111. (U. S.A.). E. KUNZ,Medical Research Department, Hoffmann-La Roche & Co., Ltd., Basel (Switzerland). J. S. LUKASZEWSKI, Henry Ford Hospital, Detroit, Mich. (U.S.A.).

F. D. MARSHALL, JR., Neurochemical Research Division, Department of Psychiatry, College o f Medicine, State University of Iowa, Iowa City, Iowa (U.S.A.). R. Y. MOORE,Departments of Pharmacology, Psychology and Anatomy, The University o f Chicago, Chicago, Ill. (U.S.A.). A. PLETSCHER, Medical Research Department, Hoffmann-La Roche & Co., Ltd., Basel (Switzerland). W. B. QUAY,University o f California, Berkeley, Calif. (U.S.A.). E. ROBERTS, City of Hope Medical Center, Duarte, Calif. (U.S.A.). J. L. SAWYER, Research and Development Division, Smith Kline and French Laboratories, Philadelphia, Pa. (U.S.A.).

S. M. SCHANBERG, Department of Pharmacology and Psychopharmacology Labora-

r

f tory, Department of Psychiatry, Yale University School of Medicine, New Haven, Conn. (U.S.A.).

R. R. SCHOPBACH, Henry Ford Hospital, Detroit, Mich. (U.S.A.). TH. L. SOURKES, Allan Memorial Institute of Psychiatry, McGill University, Montreal, Quebec (Canada). S. VARON,City of Hope Medical Center, Duarte, Calif. (U.S.A.).

H. WEINSTEIN, City of Hope Medical Center, Duarte, Calif. (U.S.A.).

A. M. WELCH,Department of Pharmacology, Medical College o f Virginia, Richmond, Va. (U.S.A.). B. L. WELCH,Laboratory of Population Ecology, Department of Biology, College of William and Mary, Williamsburg, Va. (U.S.A.). V. P. WHITTAKER, Biochemistry Department, Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge (Great Britain).

Other volumes in this series:

Volume 1: Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener and J. P. Schadb

Volume 3 : The Rhinencephalon and Related Structures Edited by W. Bargmann and J. P. Schade

Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schade

Volume 5 : Lecticres on the Diencephalon Edited by W. Bargmann and J. P. Schade

Volume 6 : Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade

Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova

Volume 9 : The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich

Volume 10: Structure and Function of the Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. Schadt

Volume 11 : Organization of the spinal Cord Edited by J. C. Eccles and J. P. Schade

Volume 12: Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. Schade

Vdume 13 : Mechanisms of Neural Regeneration Edited by M. Singer and J. P. Schade

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schade

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IX

Contents

.................................

V

......................................

XI

List of contributors Preface

Historical introduction: Specific interactions between catecholamines and tissues H. Blaschko (Oxford, Great Britain) . . . . . . . . . . . . . . . . . . .

.....

1

.......

9

. . . . . . . . . . . . . . . . . . . . . . . . .

28

Functional significance of druz-induced changes in brain monoainine levels A. Carlsson (Goteborg, Sweden) . . . . . . . . . . . . . . . . . . . Cellular localization of monoamines B. Falck (Lund, Sweden) . . . .

Accumulation of exogenous monoamines in brain in vivo and its alteration by drugs A. Pletscher, K. F. Gey and E. Kunz (Basel, Switzerland) . . . . . . . . . . The effect of central nervous system lesions in the rat on brain serotonin A. Heller, J. A. Harvey and R. Y . Moore (Chicago, Ill.) . . . . . . The hydroxylation of tryptophan by pigeon brain in vivo E. M. Gal and F. D. Marshall, Jr. (Iowa City, Iowa)

. . . . .

45

. . . . . . . . . . 53

. . . . . . . . . . . . . . . . .

Circadian and estrous rhythms in pineal and brain serotonin W. B. Quay (Berkeley, Calif.) . . . . . . . . . . . . .

..............

56 61

Action of serotonin, allied compounds and monoamine oxidase inhibitors on peripheral nerve fibers 64 V. Di Carlo (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . General discussion

.................................

67

Drug-induced changes in the subcellular distribution of serotonin in rat brain with special reference to the action of reserpine N. J. Giarman, D. X. Freedman and S. M. Schanberg (New Haven, Conn.) . . . . . . . 72 The uptake and release of catecholamines and the effect of drugs J. Axelrod (Bethesda, Md.) . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

81

Investigations on the storage sites of biogenic amines in the central nervous system V. P. Whittaker (Cambridge, Great Britain) . . . . . . . . . . . . . . . . .

....

90

Electron microscope and chemical study of binding sites of brain biogenic amines E. De Robertis (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . .

. . . .

118

Acidic glycolipoprotein granules (lysosomes) as probable binding sites of biogenic amines H. Koenig (Chicago, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137

General discussion

142

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Biochemical-pharmacological studies with 5-hydroxytryptophan, precursor of serotonin H . Green and J . L. Sawyer (Philadelphia, Pa.) . . . . . . . . . . . . . . . . . .

..

150

Concept of the neurochemical transducer as an organized molecular unit a t sympathetic nerve endings E. Costa and B. B. Brodie (Bethesda, Md.) . . . . . . . . . . . . . . . . . . . . . . 168 Cerebral and other diseases with disturbance of amine metabolism Th. L. Sourkes (Montreal, Quebec) . . . . . . . . . . . . .

............

186

X

CONTENTS

An effect of aggregation upon the metabolism of d ~ p a m i n e - l - ~ H B. L. Welch and A. M. Welch (Richmond, Va.) . . . . . . .

. . . . . . . . . . . . 201

Effects of marplan on catecholamine and serotonin metabolism in the human R. R. Schopbach, A. R. Kelly and J . S. Lukaszewski (Detroit, Mich.) . . . . . . . . . . 207 Effects of isocarboxazid on spontaneous and drug-induced extrapyramidal alterations C. Bull and H. H. Berlet (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . .

. .

21 I

y-Aminobutyric acid binding and content in density gradient subfractions of mouse brain H. Weinstein, S. Varon, E. Roberts and T. Kakefuda (Duarte, Calif.) . . . . . . . . . . 21 5 General discussion

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Summary H. E. Himwich (Galesburg, Ill.)

219

. . . . . . . . . . . . . . . . . . . . . . . . . .

226

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246

Author index.

XI

Preface

The purpose of the symposium on Binding Sites of Brain Biogenic Amines and therefore that of this monograph is to present the latest word on a subject which is of interest not only for theoretical scientific values but also because it represents the sharp advancing edge of progress in the fields of neuropharmacology and psychopharmacology. The most recent advances are well-known by the workers active in these fields, an ‘invisible college’ in which knowledge of the latest discoveries is shared and discussed, and it is these latest results that are now made available to all interested individuals. In order to accomplish this end we invited papers from some of the leading investigators in the various fields involved in the elucidation of the roles played by brain biogenic amines, both from America, Canada and Argentina in the Western hemisphere and from Sweden, Great Britain, The Netherlands and West Germany from across the Atlantic Ocean. The program was so structured that the first of a series of three chairmen presented a historical introduction and that role was ably fulfilled by Dr. H. Blaschko of Oxford. The other two chairmen were Dr. lrvine H. Page of Cleveland and Professor Peter Holtz of Frankfurt, and their remarks are found in the unstructured discussion closing each of the three sessions. In these discussions not only the chairmen and lecturers but also some of the members of the audience, of whom there were more than 400, took part. In the historical introduction, mentioned above, Dr. Blaschko stresses the development of our ideas of specificity of enzyme action. Among the other high points are data concerning central noradrenergic, dopaminergic and serotonergic synapses of Arvid Carlsson (Goteborg) and Bengt Falck (Lund), the latter using his new method of fluorescent microscopy. The observations of Eduardo De Robertis (Buenos Aires) and V. P. Whittaker (Cambridge) reveal cholinergic and non-cholinergic vesicles in the synapses of the brain. The former probably contain the binding sites of acetylcholine but the fine localizations of serotonin, noradrenaline and dopamine within the nerve endings proper are still undetermined. In support of ideas of noradrenergic and dopaminergic neurotransmission is the biochemical evidence of the aromatic amino acid decarboxylase in the formation of these neurohumors and the interrelationships of monoamine oxidase (MAO) and catechol-0-methyltransferase (COMT) in the termination of neurotransmission as shown by Julius Axelrod (Bethesda), Arvid Carlsson (Goteborg), Erminio Costa and Bernard B. Brodie (Bethesda), and Alfred Pletscher, K. F. Gey and E. Kunz (Basel). The gap in our knowledge of serotonin metabolism is strikingly demonstrated by the experiments of Harry Green and John L. Sawyer (Philadelphia) who find that exogenous brain serotonin can be catab-

XI1

PREFACE

olized rapidly at a time when M A 0 is completely inhibited. In contrast, Nicholas J. Giarman, D. X. Freedman and S. M. Schanberg resolve a contradiction in the literature on the mechanism of action of reserpine in depleting brain amines, and Theodore L. Sourkes (Montreal) completes the picture with a paper on disturbances of amine metabolism in disease. The papers of this symposium are summarized after the third session by Harold E. Himwich (Galesburg). The discerning reader will readily see that much new material is presented for the first time at this symposium. Today we use many disciplines to unravel the scientific problems stimulated by the imagination of the investigator. The disciplines are sometimes used singly, but mostly in a multidiscipline fashion, and this modern attitude to research is reflected in the various disciplines used in the papers of this monograph. They consist of a list of morphological sciences : neuroanatomy, neurohistology and neuropathology, and another of the functional sciences : neurophysiology, neurochemistry and neuropsychopharmacology. But the separate employment of morphological and functional sciences today is becoming more apparent than real as indicated by the terms histochemistry and its most recent development, fluorescent microscopy, and by the combined use of ultracentrifugation and electron microscopy, and this joint employment of the anatomic and physiologic disciplines is well-exemplified in the present monograph. Such a book as this, though it may be stimulating to the student at the medical college, is beyond his full understanding because the various papers are presented by experts who naturally assume a vast fund of information on the part of their audience. On the other hand, preclinical scientists : the neurohistologist, neuropathologist, neurophysiologist, neurochemist, neuropsychopharmacologist, and the clinical scientists : the neurologist and psychiatrist, will find this monograph useful. To the uninitiated, the subject of brain biogenic amines may seem to represent a narrow field, but it is really a vast one with many facets and requires the converging of many disciplines for its elucidation. For those investigators desiring to enter this complex field which is rapidly expanding, these papers represent an open sesame. Such a volume, too, is indispensable for workers in laboratories where the problems of neuropharmacology and psychopharmacology are advanced and also where investigators seek basic parameters for correlation with behavior, normal and abnormal. We take pleasure in thanking Dr. Thomas T. Tourlentes, the Superintendent of our hospital, for his aid and encouragement in the various details concerned with the preparations for this symposium which actually began two years before it took place. And during the course of the symposium he undertook the administration of so many of the details which were necessary for the success of the symposium and this monograph. It was in cooperation with Dr. Tourlentes that the social gatherings, during the course of the symposium, took place, gatherings which afforded the necessary circumstances for the interpersonal communication which means so much to the lecturers and guests as well. We also wish to thank Miss Florence 0. Johnson, the Assistant Superintendent in Charge of Non-Medical Affairs, for her behind-the-scenes aid which she gave throughout. It would be difficult to acknowledge the cooperation of the large number of individuals of the laboratory and hospital who so willingly

PREFACE

XI11

helped during the actual progress of the symposium, but our photographer, Mr. Lloyd Tenneson and his staff, our secretaries, Mrs. Ned Wilmot and her secretarial staff, and Mr. Salter and his group who took care of so many essential details in the management of the symposium, are among the many who contributed. Finally, we gratefully acknowledge the financial assistance afforded by the foundations and drug companies who far-sightedly furnished the financial aid to make the symposium possible, and a list of these organizations follows : National Science Foundation; Manfred Sake1 Foundation ; Abbott Laboratories; Burroughs Wellcome and Company; Ciba Pharmaceutical Company ; Eli Lilly and Company ; Geigy Chemical Corporation ; Hoffniann-La Roche & Co., Ltd.; Merck, Sharp and Dohme Postgraduate Program; Pitman-Moore Company; Sandoz, Inc. ; Schering Corporation; Searle and Company; Smith Kline and French Laboratories, Inc.; E. R. Squibb and Sons; SterlingWinthrop Research Institute; Strasenburg Laboratories; Wallace Laboratories, and Wyeth Laboratories. H. E. HIMWICH

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1

Historical Introduction: Specific Interactions between Catecholamines and Tissues H . BLASCHKO Department of Pharmacology, Oxford University, Oxford (Great Britain)

The study of the catechol amines has a history that goes back over a period of more than a hundred years. In recent years the early history of the subject has been repeatedly reviewed (Blaschko, 1950a, 1957; Hagen and Welch, 1956). The specific chemical properties of the catechol amines were first described by Vulpian in 1857, and a few years later Henle described the reaction of the adrenal medulla with potassium bicarbonate, a reaction which gave rise to the name “chromaffin” (Kohn) or “pheochrome” (Poll) cell (for literature, see Boyd, 1960). The early work on the chemical nature of the adrenal hormones was supplemented by the studies of Balfour and others on the development of the adrenal medulla: these studies have recently been reviewed by Boyd (1960). Balfour traced the origin of the chromaffin tissue (and of the sympathetic ganglion cells) to the neural crest and thus laid the foundation for our ideas of the relationship between nerve cells and catechol-amine secreting cell, ideas that found independent support in the observations of Poll on chromaffin ganglion cells in Annelids (Poll and Sommer, 1903). It was here that the idea of a nerve cell as a cell endowed with chemical specificity was first discussed (Gaskell, 1916). Vulpian called the reaction of the adrenal medulla with ferric chloride its “signe particulier” ;in other words, he postulated a specific chemical ability for the chromaffin cells. The discovery of adrenaline and the elucidation of its chemical structure confirmed these ideas, and more recently noradrenaline and dopamine have been discovered in the chromaffin tissue and in nervous tissue. There is no reason to suppose that the number of the naturally occurring amines of this group is exhausted. A future historian may have to add an appendix to this list. With the development of our knowledge of the chemistry and distribution of the catechol amines, there arose also the question as to the metabolic pathway of their formation. It is impossible, in this brief review, to discuss all the early work on this subject, but I should like here to acknowledge my own indebtedness to the work of Otto Neubauer, who in 1928 published an article on protein metabolism in which the metabolic pathways of the amino acids were discussed. In Neubauer’s article the possible pathways of adrenaline formation were reviewed in general terms but these did not include the pathway now generally accepted; the References p . 718

2

H. BLASCHKO

present ideas were formulated (Blaschko, 1939) after the discovery by Holtz of the enzyme dopa decarboxylase. Enzymes are prototypes of substances that exhibit chemical specificity. It was in connection with enzyme specificity that Emil Fischer introduced the picture of the lock and the key. T h s picture does not give an idea of the forces active between substrate and enzyme; it stresses the importance of a fit between these two. The enzymes are not the only structures that exhibit specificity. In our field of interest, we have to distinguish at least these sites: (a) those involved in catecliol amine formation, (6) those responsible for catechol amine storage, (c) the receptors in the effector organs, and ( d ) those concerned in biological inactivation. In all these sites specificity is not absolute. In Emil Fischer’s terminology, we can say that the locks can be opened not only by the appropriate keys; they can also be picked. This picking of the locks is really what the drugmakers exploit: they study the active sites in order to make synthetic compounds that react with the specific sites. I should like to discuss the development of our knowledge of some of these sites and their specificity requirements. 1 shall at first discuss the enzyme dopa decarboxylase. In 1950, in a volume dedicated to Otto Meyerhof, the substrate specificity of dopa decarboxylase was first discussed (Blaschko, 1950b). It was then proposed that in the reaction between the enzyme and its substrate, the amino acid, three groups took a part: (1) The group -R. (2) The amino group. ( 3 ) The carboxyl group. R

I

H-C-NH2 COOH

(a) The amino group

It was the finding in 1939 that in contrast to dopa, N-methyl-dopa was not decarboxylated by dopa decarboxylase that was made the basis for the suggestion that a primary amine was the precursor of adrenaline (Blaschko, 1939, 1959). At that time pyridoxal phosphate had not yet been discovered, but it is now known that the inability to react with N-substituted amino acids is a property of many pyridoxal enzymes. In 1950 a brief reference was made to the work of Snell, who had suggested that in transamination reactions there occurred the formation of a Schiff’s base between the amino group of the substrate and the carbonyl group of the pyridoxal: H

I

-C-NHz

t

I O=C + -C-N-C ‘\ I /

R1

H

/ \

Ri

Such a reaction would account for the fact that the N-methylated compound was metabolically inert. A fuller formulation of the enzyme-substrate reaction for pyridoxal enzymes was

HISTORICAL INTRODUCTION

3

subsequently given by Metzler, lkawa and Snell (l954), again with the formation of a Schiff’s base as the first product of the reaction between substrate and enzyme; this bond was believed to be reinforced by a chelating metal. The picture of the enzyme-substrate interaction, as originally developed by Snell and his colleagues, would account for the observations on the biosynthesis of catechol amines. However, since this picture was first proposed, several studies have appeared that appear to conflict with it. Connard and Nguyen-Chi (1958, 1959) reported that under certain conditions the dopa decarboxylase of guinea-pig liver was activated, and not inhibited, in the presence of small amounts of isoniazid. These studies have been extended to kynureninase and to the glutamic-aspartic transaminase of rat liver. In each instance the coenzyme, pyridoxal-5-phosphate, was replaced by its hydrazone, formed by pyridoxal phosphate and isoniazid or another hydrazide. Each time the apoenzyme was found to be completed by the corresponding hydrazone of pyridoxal phosphate, to give a catalytically fully active system. Gonnard considers these observations as incompatible with the scheme formulated by Snell (Gonnard and Nguyen-Philippon, 1961 ; Connard and BoignC, 1961). Similar observations have been reported from Dr. P. Holtz’s laboratory by Palm (1958), who used a preparation of dopa apodecarboxylase that would be activated by pyridoxal phosphate. Palm found that the hydrazone formed from isoniazid and pyridoxal phosphate was also able to complete the apoenzyme and, in fact, enzymic activity was maintained better by adding the hydrazone than by adding the free coenzyme. Palm (1958) and Holtz (1959), in discussing these results, favour an interpretation that differs from that put forward by Gonnard. They do not see in them a contradiction to Snell’s scheme; they believe that the hydrazone releases small amounts of free, catalytically active, pyridoxal phosphate. The latter is known to form, irreversibly, catalytically inactive products both with dopa and with dopamine, and the authors suggest that this irreversible inactivation is prevented while the coenzyme is present as a hydrazone. An entirely different line of inquiry stems from the discovery by Cori and his colleagues of pyridoxal phosphate in the enzyme phosphorylase. This finding suggests that a reaction with an amino group of a substrate cannot be the only way in which pyridoxal phosphate intervenes in an enzymic reaction. This work on phosphorylase has been continued by Krebs and Fischer, who have catalytically reduced the pure phosphorylase with sodium borohydride. The reduced enzyme, which was still catalytically active, was subjected to tryptic digestion. A peptide was isolated in which a pyridoxyl group is attached to the w-amino group of a lysine moiety in the peptide. The phosphorylase thus reduced was catalytically still active (Fischer et a/., 1958). Phosphorylase, one might argue, is an enzyme so different from dopa decarboxylase that a different mode of attachment of the pyridoxal group could be envisaged. However, this work has recently been extended to the glutamic-aspartic transaminase of liver (Hughes, Jenkins and Fischer, 1962) and here also by the use of sodium borohydride evidence has been obtained for the attachment of the carbonyl group of pyridoxal to the w-amino group of lysine. The transaminase is sufficiently closely References p. 718

4

H. B L A S C H K O

related to the decarboxylases to make one suspect that a similar mode of attachment might be present. At first sight, one would conclude that all these observations are incompatible with the idea that the amino acid as substrate reacts with the carbonyl group in the enzyme. However, one is reluctant to abandon the scheme proposed by Snell. The carbonyl group in pyridoxal is a very reactive, and therefore also a very vulnerable, one. It has been reported (Holtz, 1959) that this group can be protected not only by isoniazid but also by other amino compounds; of all these, lysine was found to confer the maximal protective effect. From these observations the proposal emerges that in the pyridoxal enzymes that act on amino acids the carbonyl group is protected by reaction with a n amino group, such as the w-amino group of lysine. When substrate is added, the amino group of the substrate displaces the protecting group from the carbonyl carbon which is thus uncovered. This is a picture that appears unorthodox to the enzyme chemist, but it seems to account for the observations made in recent years. This protecting action would be a rather unusual function for the enzyme protein. This is similar to the view taken by Snell in a recent article (Snell, 1961). At any rate, such a picture would retain what seemed useful in the earlier concepts.

(6) The group -R In 1950, the role of the phenolic hydroxyl groups in dopa decarboxylase and in the bacterial tyrosine decarboxylase were compared. Experimental evidence was given for the view that these two enzymes differed in this respect: in the bacterial enzyme the hydroxyl group in position para was important for rapid decarboxylation, in the mammalian enzyme this group was not essential; but it was the group in position meta to the side chain that was involved. The importance of the orientation of the link between the hydroxyl group and enzyme protein was illustrated by the fact that not only meta-tyrosine but also ortho-tyrosine were decarboxylated by dopa decarboxylase, whereas the bacterial enzyme decarboxylated tyrosine and meta-tyrosine, but not ortho-tyrosine. This picture was given support when the analogues of 3 :4-dopa became available: (a) 2:3-dopa was found to be decarboxylated by both the mammalian and the bacterial decarboxylase (Blaschko and Langemann, 1951); (6) 2 :6-dopa, the analogue with two o-hydroxyl groups, was decarboxylated by the mammalian, but not by the bacterial enzyme (Sourkes, 1955). These findings were in agreement with expectation. In the more recent work on the a-methylated amino acids, to be discussed below, the importance of the hydroxyl group in position meta has again become clear. Recent observations have added one important new factor still unknown in 1950: the probable identity of the 5-hydroxytryptophan decarboxylase with dopa decarboxylase. It is of interest, therefore, that it has now been shown by Erspamer and his colleagues (1961) that in the tryptophan series there is a close analogy to the findings with the hydroxyphenylalanines: in addition to 5-hydroxytryptophan,4-hydroxytryptophan is attacked, but other hydroxytryptophans are not decarboxylated. The

HISTORICAL INTRODUCTION

5

4-hydroxy compound is of interest because the amine formed has central actions. It might be added here that the very much slower decarboxylation of tryptophan itself gives a demonstration of the fact, already mentioned, that specificity is never absolute; for the bacterial tyrosine decarboxylase it has been long known that it will also act, but slowly, on phenylalanine. (c) StereospeciJcity

It was postulated in 1950 that stereospecificity arose as a consequence of the existence of three “anchorages” on the enzyme (Blaschko, 1950~).The amino acid decarboxylases were compared with the also stereospecific amino acid oxidases. R

I

H-Ca-NH2

I

COOH

It was pointed out that for the oxidases the involvement of the group -R was less obvious: many amino acids were acted upon by one and the same oxidase. If stereospecificity rested upon the presence of three anchorages, one would have to postulate that in the oxidases the a-hydrogen atom was involved, and that is in fact true: a-methylalanine has long been known not to be oxidized by the D-amino acid oxidase of mammalian tissues (Keilin and Hartree, 1936). For the decarboxylases, an involvement of the a-hydrogen atom was not obvious. It was puzzling therefore when subsequently it was shown by Sourkes, Heneague and Trano (1952) that a-methylated amino acids, e.g. a-methyldopa and a-methylmeta-tyrosine were not decarboxylated, but that they acted as inhibitors of dopa decarboxylase. In the past two years new work has been published which brings the observations on a-methyl-amino acids into line with the ideas on stereospecificity as originally proposed. It has been found that a-methyldopa and a-methyl-meta-tyrosine both are not only inhibitors of the decarboxylase but that they are in fact substrates (Udenfriend, Lovenberg and Weissbach, 1960; Smith, 1960; Brodie and Costa, 1962; Carlsson, 1962). Moreover, it is believed that the longlasting lowering of the catechol amine content of the brain is due not to the amino acid acting as an enzyme inhibitor, but to the properties of the amine formed from it by decarboxylation. Although the presence of the a-methyl group is important, it does not seem to interfere with the attachment of the substrate (see also Hagen and Cohen). It might be added that it has recently been found by Wilson and Snell (1962) that in another pyridoxal enzyme acting on serine, a-methylserine is able to serve as substrate; again it is shown that the a-hydrogen atom is not involved in the enzymesubstrate interaction. The considerations here discussed are naturally of theoretical interest. They make us believe that the substrate specificity of an enzyme follows rules that we can hope to understand more fully one day. But they are also of practical use for those interested to make therapeutically useful compounds. We have learned in recent years that References p . 718

6

H. B L A S C H K O

amino acid precursors can penetrate into the central nervous system and we believe that they there exert pharmacological actior s, became they are converted at or near the sites of action to the amines. The amines themselves cannot be used, because they do not reach these sites. This action was first demonstrated for a naturally occurring amino acid, L-dopa (Carlsson, Lindqvist and Magnusson, 1957) but, as was shown some time ago (Blaschko and Chrusciel, I960), synthetic amino acids will also cause pharmacological actions. Storage sites and efectors The term “sympathomimetic” amine was introduced in 1910 in a paper by Barger and Dale. The authors there discuss the possibility that some drugs of this group might act by releasing what we would call today the mediator. This idea, which they ascribe to Dixon and Hamill (1909), is not accepted, with these words: ‘‘If, ho*ever we concede to amino-ethanol-catechol (this is what we now call noradrenaline, H.B.) the power of acting directly, we cannot reasonably invoke the liberation of adrenine to explain the action of bases one stage further removed from it in structure, and, continuing the argument, we are ultimately bound to admit even the primary fatty amines to the list of substances acting directly, like the hormone adrenine”. Let us also quote Dixon and Hamill (1909): “. . . there is reason in the suggestion that excitation of a nerve induces the local liberation of a hormone which causes specific activity by combination with some constituent of the end organ, muscle or gland. If this be true of electrical stimulation it may be true also of drugs that they act by causing a liberation of the specific hormone, or in the case of paralytic agents preventing such liberation, , . .”. It is well known that the first idea here discussed by Dixon and Hamill has found its modern expression in the theory of chemical transmission of nervous impulses, but it may come as a surprise to many readers that the idea of drug-induced release of transmitter is also already discussed in their paper. This idea has been most strongly supported by the recent work of Burn and Rand (1958). According to their findings we can distinguish direct actions, on the receptors in the effector cell, and indirect actions, on the storage site. The apparent continuity that leads from the natural mediator substance to the simple aliphatic amines is accounted for by the fact that many substances act directly as well as indirectly. The direct actions of a drug can still be elicited, and are often potentiated, in the reserpinized or denervated organ; the indirect actions depend on the presence of a store of catechol amines. The isolation and study of the storage particles is a relatively recent achievement and its history covers only the last decade. However, the demonstration that storage organelles for catechol amines can be obtained in vitro (Blaschko, Hagen and Hagen, 1957), has provided a useful tool for the study of drug actions in vitro. There is the recent demonstration by Schiimann and Philippu (1962), in which it was shown that tyramine and other indirectly acting sympathomimetic amines displace adrenaline from the chromaffin granules in vitro. These findings have an important bearingalso upon the study of receptor specificity. If it is true that compounds like tyramine do not act at the receptor site in the effector

HISTORICAL INTRODUCTION

7

cell, the specificity requirements of this site will have to be re-formulated. We must believe that the number of amines truly adrenaline- or noradrenaline-like in action is much smaller than the number of substances that Barger and Dale called sympathomimetic in 1910. SUMMARY

A brief review is given of the development of our ideas on specificity. One of the specific sites of importance in the formation of the catechol amines is the enzyme L-dopa decarboxylase. The significance of the phenolic hydroxyl groups, of the *-hydrogen atom and of the amino group is discussed. These ideas allow us to understand the importance of the rneta-hydroxy group and also the fact thpt the a-methylamino acids are decarboxylated. It is shown that the new ideas on the structure of pyridoxal enzymes are compatible with earlier pictures of the enzyme-substrate interaction. ACKNOWLEDGEMENT

Thanks are due to the European Office, U.S .Air Force Office of Aerospace Research, for their support (Grant USAF-EOAR 62-80). REFERENCES G., AND DALE,H. H., (1910); Chemical structure and sympathomimetic action of amines. BARGER, J . Physiol. (Lond.), 41, 19-59. BLASCHKO, H., (1939); The specific action of L-dopa decarboxylase. J. Physiol. (Lond.), 96, 5OP-51P. BLASCHKO, H., (l950a); Chemical control of nervous activity. B. Adrenaline and Sympathin. The Hormones. ZZ. G. Pincus and K. Thimann, Editors. New York, Academic Press (p. 602-631). BLASCHKO, H., (1950b); Substrate specificity of amino-acid decarboxylases. Biochim. biophys. Acta (Amst.), 4, 130-1 37. BLASCHKO, H., (1 950c) ; Action of local ho1 mones : remarks on chemical specificity.Proc. roy. SOC.B, 137, 307-3 I 1. BLASCHKO, H., (1957); Metabolism and storage of biogenic amines. Experientia (Busel), 13, 9-12. BLASCHKO, H., ( I 959); The development of current concepts of catecholamine formation. Phuumucol. Rev., 11, 307-316. BLASCHKO, H., AND C H R U ~ I ET.L L., , (1960); The decarboxylation of amino acids related to tyrosine and their awakening action in reserpine-treated mice. J. Physiol. (Lond.), 151, 272-284. BLASCHKO, H., HAGEN,J. M., AND HAGEN,P., (1957); Mitochondria1 enzymes and chromafin granules. J. Physiol. (Lond.), 139, 316-322. BLASCHKO, H., AND LANGEMANN, H., (1951); Enzymatic decarboxylation of 2:3-dihydroxyphenylalanine. Biochem. J., 48, VII. BOYD,J. D., (1960); Origin, development and distribution of chromaffin cells. Adrenergic Mechanisms. J. R. Vane, G. E. W. Wolstenholme and M. O’Connor, Editors. Ciba Foundation Symposium (p. 63-82). BRODIE, B. B., AND COSTA,E., (1962); Some current views on brain monoamines. Monoamines el Syst%meNerveux Central. Symposium. Geneva, Georg & Cie (p. 13-49). BURN,J . H., AND RAND,M. J., (1958); The action of sympathomimetic amines in animals treated with reserpine. J . Physiol. (Lond.), 141, 314-336. CARLSSON, A,, (1962); Pharmacological analysis of central nervous action. Proc. First Intern. Pharmacol. Meeting, Oxford, Pergamon Press, 8 (p. 71-74). CARLSSON, A., LINDQVIST, M., AND MAGNUSSON, T., (1957); 3,4-Dihydroxyphenylalanine and 5hydroxytryptophan as reserpine antagonists. Nature (Lond.j, 180, 1200.

8

H. B L A S C H K O

DIXON,W. E., AND HAMILL, P., (1909);The mode of action of specific substances with special reference to secretin. J . Physiol. (Lond.), 38, 314-336. ERSPAMER, V., GLASSER,A., PASINI,C., AND STOPPANI, G., (1961); In vitro decarboxylation of tryptophans by mammalian decarboxylase. Nature (Lond.), 189, 483. FISCHER,E.H., KENT,A. B., SNYDER, E. R., AND KREBS,E. G., (1958); Reaction of sodium borohydride with muscle phosphorylase. J. Amer. chem. SOC.,80,2906-2907. GASKELL, W. H., (1916);The Involuntary Nervous System. London, Longmans, Green and Company (p. 146). GONNARD, P., AND BOIGN~, N., (1961);R61e co-enzymatique d’hydrazones de phospho-5-pyridoxalcynureninase et hydrazides cycliques. Bull. SOC.Chim. biol. (Paris), 43, 609-617. GONNARD, P., AND NGUYEN-CHI, .I. P., (1958);Action de l’hydrazide isonicotinique et de composes de structure voisine sur la dopa decarboxylase. B u t . SOC.Chim. biol. (Paris), 40,485-495. GONNARD, P., AND NGUYEN-CHI, J. P., (1959); R6le co-enzymatique d’hydrazones de phosphopyridoxal vis-a-vis de la dopa decarboxylation. Bull. SOC.Chim. biol. (Paris), 41, 1455-1461. GONNARD, P., AND NGUYEN-PHILIPPON, L., (1961); The coenzyme role of phospho-5-pyridoxal, glutamic-aspartic transaminase and cyclic hydrazides. Bull. SOC.Chim. biol. (Paris), 43,601-607. HAGEN,P., AND COHEN,L. H., Physiological release and transport of 5-hydroxytryptamine. Biosynthesis of Indolea!kylamines. Prof. Erspamer, Editor. Heidelberg, Springer. HAGEN,P., AND WELCH,A. D., (1956);The adrenal medulla and the biosynthesis of pressor amines. Recent Progr. Hormone Res., 12, 2744. HOLTZ,P., (1959);Role of L-DOPA decarboxylase in the biosynthesis of catecholamines in nervous tissue and the adrenal medulla. Pharmacol. Rev., 11, 317-329. HUGHES,R. C., JENKINS, W. T., AND FISCHER, E. H., (1962); The site of binding of pyridoxal-5phosphate to heart glutamic-aspartic transaminase. Proc. nut. Acad. Sci. (Wash.), 48, 1615-1618. KEILIN,D., AND HARTREE, E. F., (1936);Uricase, amino acid oxidase and xanthine oxidase. f r o c . roy. SOC.B, 119, 114-140. METZLER, D. E., IKAWA, M., AND SNELL,E. E., (1954);A general mechanism for vitamin BE-catalysed reactions. J . Amer. chem. SOC.,76, 648-652. NEUBAUER, O.,(I 928); Interrnediarer Eiweissstofwechsel. Handbuch der normalen und pathologischen Physiologie. A. Bethe, G . Von Bergmann, G. Ernbden and A. Ellinger, Editors. Berlin, Springer Verlag, 5 (p. 671-989). PALM,D., (1958); Uber die Hemmung der Dopa-Decarboxylase durch Isonicotinsaurehydrazid. Naunyn-Schmiedeberg’s Arch. exp. farh. Pharmak., 234, 206-209. POLL,H., A N D SOMMER, A., (1903);Uber phaeochrome Zellen im Zentralnervensystem des Blutegels. Arch. Anat. Physiol., 549-550. SCHUMANN, H. J., AND PHILIPPU, A., (1962);The mechanism of catecholamine release by tyramine. Int. J . Neuropharmacol., 1, 179-182. SMITH,S. E., (1960); The pharmacological actions of 3,4-dihydroxyphenyl-a-methylalanine (umethyldopa), an inhibitor of 5-hydroxytryptophan decarboxylase. Brit. J . Pharmacol., 15,319-327. SNELL, E.E.,(1961);The r6le of vitamin Bs in catalysis of reactions by amino acids. The mechanism of action of water-soluble vitamins. A. V. S. De Reuck and M. O’Connor, Editors. Ciba Found. Study Group, 7 , 18-3 I. SOURKES, T., (1955); Substrate specificity of hydroxy-a-phenyl-alanine decarboxylases and related enzymes. Rev. canad. Biol., 14, 49-63. SOURKES, T., HENEAGUE, P., A N D TRANO,Y., (1952);Enzymatic decarboxylation of isomers and derivatives of dihydroxyphenylalanine. Arch. Biochem., 40, 185-193. UDENFRIEND, S., LOVENBERG, W. M., AND WEISSBACH, H., (1960); L-Amino acid decarboxylase activity in mammalian tissues and its inhibition by a-methyldopa. Fed. Proc., 19, 7. WILSON,E. M., AND SNELL,E. E., (1962);Metabolism of u-methylserine. I. a-methylserine hydroxymethyltransferase. J. biol. Chem., 237, 3 171-3179.

9

Functional Significance of Drug-Induced Changes in Brain Monoamine Levels ARVID CARLSSON Departmelit of Pharmacology, University of Giiteborg, Giiteborg (Sweden)

In the past decade numerous attempts have been made to correlate changes in monoamine levels in brain, induced e.g. by drugs, with changes in behavior and other brain functions. The results have been variable. For example, a fall in the level of one of the monoamines may coincide with sedation, excitation or no change in behavior. It is thus evident that no strict correlation exists. It would, of course, be erroneous to conclude from this that the monoamines of the brain are of no functional importance. On the contrary, we have good reasons for assuming that the catecholamines as well as 5-hydroxytryptamine (5-HT) serve as transmitters in brain. Dr. Falck will present some new histochemical evidence for this. There are also good reasons for a poor correlation. The major part of the monoamines in the brain occur in an inactive store, and the level of this store may vary independently of the level of free and active monoamines in the extracellular space near the receptor sites of the effector neurons. The situation is further complicated by the fact that the monoamine stores may consist of at least two different fractions, as demonstrated first by Hillarp (1960) on adrenal medullary granules. In addition, as Dr. Falck will show, part of the stored transmitter may be located at an appreciable distance from the effector cells. I should like to discuss first the functional significance of the monoamine stores: how much can they be reduced without impairment of function? COMPARISON BETWEEN RESERPINE A N D

a - ME T H Y L - m - T Y R O S I N E ( (I - M M T)

As is well known, there are two types of drugs which cause particularly marked depletion of tissue monoamines, namely, the Rauwolfia alkaloids and benzoquinolizines on the one hand, and the DOPA analogues a-methyl-DOPA and a-methylm-tyrosine (a-MMT) on the other. While the former group acts on the catecholamines as well as 5-HT, the latter acts fairly selectively on noradrenaline (Hess et al., 1961; Porter et a/., 1961). A common feature of reserpine and these DOPA analogues is that both cause a marked depletion of noradrenaline in central as well as peripheral noradrenergic neurons. From the technical point of view the peripheral noradrenergic References p . 25-27

10

ARVID CARLSSON

neurons have the great advantage that their function can be easily studied. As is well known, the peripheral noradrenergic neurons cease to function under the influence of reserpine, when given in doses which cause depletion of the adrenergic transmitter. We know of no data in the literature dealing with the function of the adrenergic nerves after severe depletion of the transmitter by a-methyl-DOPA or a-M MT. Stone et al. (1962), have done some studies along this line in dogs, but with the doses used the noradrenaline levels in tissues dropped only by about 50 per cent. Experiments with u-MMT in doses causing severe depletion have, therefore, been carried out in our laboratory (AndCn and Magnusson, 1964). It was found that in order to obtain almost complete depletion of noradrenaline u-M MT had to be given in large repeated doses (400 mg/kg daily for 2 or 3 days). In fact, maximum effect was obtained if, in addition, a small dose of metaraminol (0.2 mg/kg) was given intravenously about 4 h before the experiment. The action of u-MMT is largely mediated through metaraminol (Carlsson and Lindqvist, 1962a and b). Under such conditions some 97 per cent depletion of noradrenaline was obtained in brain, heart, and spleen of rats and cats and in the iris and nictitating membrane of cats, using the extremely sensitive method of Hiiggendal (1963a). In no instance has it been possible to block the noradrenergic transmission mechanism, irrespective of whether a-MMT has been given in single or repeated doses or whether it has been given alone or in combination with metaraminol. The function of the noradrenergic nerves has been studied in several ways, mostly after unilateral cervical sympathectomy. Lack of ptosis, miosis, and relaxation of the nictitating membrane on the intact side while at the same time these symptoms have been present on the side of cervical sympathectomy has been taken as indication of persistent sympathetic activity. Protrusion of the eyeball, dilatation of the pupils, and contraction of the nictitating membrane following electrical stimulation of the cervical sympathetic has been taken as evidence of an intact noradrenergic transmission mechanism. Furthermore, the rise in blood pressure following electrical stimulation of the splanchnic nerves, carotid occlusion, injection of tyramine or carbachol after atropine in adrenalectomized or demedullated animals has been used to investigate the noradrenergic transmission mechanism. The experiments have been performed in cats as well as in rats. The experiments showed conclusively that cr-MMT was unable to impair the noradrenergic transmission mechanism, even in doses which caused the virtually complete depletion of the stores of noradrenergic transmitter. There thus appears to be a fundamental difference in the mode of action of reserpine and a-MMT. As to the mode of action of u-MMT, the following 3 alternatives may be considered: (I) For the functions mentioned the sympathetic system may not be entirely dependent on the noradrenergic transmitter but may work partly through other transmitters as well. This alternative might be seriously considered if a-MMT had caused some reduction of sympathetic activity. However, the sympathetic system seemed to function quite normally. This alternative, therefore, seems unlikely. (2) The decarboxylation products of u-M MT, i.e. a-methyl-rn-tyramine and amethyl-j3-hydroxy-m-tyramine or metaraminol may be stored in the nerve endings and take over the functions of the adrenergic transmitter.

C H A N G E S I N B R A I N MONOAMINE LEVELS

11

At the First International Pharmacological Meeting in Stockholm, August 1961, we reported that the prolonged depletion of noradrenaline caused by a-methyl-DOPA and a-MMT was mediated by their respective decarboxylation products (Carlsson, 1962, cJ Carlsson and Lindqvist, 1962a and b, Fig. I). This was not generally accepted

METARAMINOL (a-METHYLNORADRENALINE)

CI -METHY L METATYROSlNE (a-METHYLDOPA)

a-METHYLMETATYRAMINE (a-METHYLDOPAMINE)

Fig. 1. a-Methyl-DOPA, u-methyl-nr-tyrosine and their decarboxylation products.

at first, but now there seems to be general agreement that this is so. It has been admitted also by Costa et al. (1 962a) as is evident e.g. from the printed, considerably revised version of their presentation at the same meeting. However, one point remains to be discussed, namely, the mechanisms by which these amines cause depletion of noradrenaline. In our first report we presented evidence to support the view that displacement had taken place: the amounts of a-methylated amines (at late intervals as 24 h or more the P-hydroxy derivatives dominated) found were of the same order as the missing noradrenaline. This has been disputed by Costa et a]. (1962a), as well as by Udenfriend and Zaltzman-Nirenberg ( 1 962). According to these investigators, the amounts of decarboxylation products formed from a-MMT are small and disappear rapidly, usually within 24 h, while the depletion of noradrenaline persists for several days. We have repeated several of the experiments reported by these investigators (see also Gessa e t a / . , 1962a and b; Brodie et a]., 1962b; Costa et al., 1962b), and in all instances we find much larger amounts of decarboxylation products than they have found. For example, in the brains of rabbits given a-MMT in a dose of 100 mg/kg intravenously, metaraminol was found in amounts corresponding roughly to the missing noradrenaline as late as 7 days after the injection (Fig. 2). According to our results displacement plays an important role in the depletion of noradrenaline by a-MMT (and a-methyl-DOPA). Other factors, e.g. inlubition of synthesis, seem, however, to contribute. This will be discussed later. The possibility that a-methyl-m-tyramine and metaraminol act as substitutes for the noradrenergic transmitter, may, therefore, be considered. Certain facts argue against this mechanism as being the only or even the chief factor, however. Metaraminol is a much weaker sympathomimetic agent than noradrenaline. This might be compensated by increased release, but this probably does not occur, since metaraminol actually appears to remain in the stores much longer than noradrenaline. Furthermore, it has been found that D-adrenaline is an efficient displacer of noradrenaline (Andtn, unpublished experiments). Even after the almost complete depletion of noradrenaline by D-adrenaline the noradrenergic nerves appear to function normally, as judged by the criteria just mentioned (Anden and Magnusson, 1964). References p . 25-27

12

ARVID CARLSSON

The physiological activity of D-adrenaline is much lower than that of L-noradrenaline. It might be argued that this is compensated by a more rapid liberation, but this is unlikely as the disappearance of L- and D-adrenaline occur at about equal rates.

t 1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

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1 -5

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I

e 25pq DA

3tK-2

- 3 - 2 - t

"

0

1

2

3

4

5

6

7

8

9

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11

1 2 1 3 1 4

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DAYS

Fig. 2. Insert. Paper chromatograms of the present laboratory demonstrating metaraminol (left) and u-methyl-m-tyramine (right) in brain stem and striatum, respectively, of rabbits injected with u-methylm-tyrosine (100 mg/kg i.v.) 7 days previously (for techniques, see Carlsson and Lindqvist, 1962a). Graph from Gessa et al. (1962b) demonstrating absence of these decarboxylation products in brain stem of rabbits under the same experimental conditions. The amount of metaraminol found on the chromatogram is of the same order as the missing noradrenaline.

(3) A third explanation as to why noradrenergic transmission appears to be intact even after the virtually complete depletion of the transmitter by a-MMT (and amethyl-DOPA and D-adrenaline) is that the major part of the store of noradrenergic transmitter is not essential for the transmitter function. If this is the correct alternative it remains to explain why reserpine blocks the noradrenergic transmission. It has long been known that the sedative action of reserpine is not strictly correlated with the monoamine levels in the brain. This is particularly true of the recovery stage, where functional recovery is reached while the monoamines are still very low. In fact, it has proved possible to keep the monoamine levels in rabbit brain very low by small daily doses of reserpine (0.2 mg/kg subcutaneously) with but slight functional impairment (Hiiggendal and Lindqvist, 1963). These animals are somewhat sedated for some 6 h after each injection, but after 24 h some very slight miosis and photophobia are the only conspicuous signs of reserpine effect, in spite of the fact that the monoamine levels in the brain even at this interval are very low (about 10 per cent of normal). It is, however, of interest to note that in these chronically treated animals the monoamine levels are significantly lower at the time of sedation (after 4 h) than after 24 h,

13

CHANGES I N B R A I N MONOAMINE LEVELS

but after a single dose rather the reverse is true. This phenomenon deserves further investigation with chemical analyses and histochemical examination in parallel. Although a direct action of reserpine on platelets 5-HT was demonstrated many years ago by Brodie and his coworkers -1 had the privilege to be one of them (Carlsson, Shore et al., 1957) - it has only recently become possible to demonstrate a direct action of the drug on specific storage granules. This has been unfortunate because the suspicion that such an effect does not exist, has caused confusion. As a result of independent work of Kirshner (1962), and Hillarp and coworkers (Carlsson, Hillarp, et al., 1962, 1963) we now know that if adrenal medullary storage granules are 0

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I

,I

'I

I

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,

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72

Fig. 3. Adrenaline level of adrenal medulla and uptake of 14C-catecholaminesby adrenal medullary 0 granules in vitro at various intervals following injection of reserpine (5 mg/kg i.v.) to rabbits. 0 = Uptake of 14C-amine, -@ . = Adrenaline level. This figure is based on data of P. Lundborg (1963).

---

incubated together with labelled noradrenaline (adrenaline, dopamine, or 5-HT) in low concentration together with some ATP and Mg ions, the granules incorporate the amine at a fairly high rate. The ATP does not seem to be incorporated in stoichiometric amounts, so it appears that the incorporation occurs primarily in the labile ATP-free fraction discovered by Hillarp (1 960). This incorporation is blocked by reserpine, when added in low concentration to the suspension medium. If adrenal medullary granules are examined in this way at different intervals following injection of a single dose of reserpine (5 mg/kg intravenously) to rabbits, incorporation of labelled amine is blocked 12 to 24 h after the injection. After about 48 h the incorporation is restored to normal in spite of the fact that the adrenal medulla is still completely depleted of catecholamines (Lundborg, 1963, Fig. 3). In other words, restoration of the storage function precedes that of ainine levels. It appears that storage function rather than amine levels is correlated with sedation and other pharmacological effects of reserpine. This suggests that the amine must be taken up by the granules before it can be released by nerve activity. When all the data are considered together, the following hypothetical picture References p . 25-27

14

ARVID CARLSSON

emerges (Fig. 4). Normally the precursor amino acidenters the cell and is decarboxylated. Alternatively the ninine enters the cell from the extracellular space. The amiire then enters the storage granule, where it is incorporated into the labile fraction. The presence of ATP and Mg ions is required for this incorporation. Part of the labile fraction may then be incorporated or converted into the stable fraction or, altermitochondrion

amine / and/or amino acid

Fig. 4. Hypothetical model of monoaminergic transmission unit.

natively, released into the extracellular space by an influence of the action potential on the storage granule. There may also be some back-leakage to the cytoplasmic sap and monoamine oxidase (MAO). Reserpine blocks the incorporation into thc labile fraction. Synthesis is going on but the amine formed is destroyed in the cell by the M A 0 of the mitochondria. Depletion of the labile fraction is delayed by replenishment from the stable fraction. Consequently, nerve transmission is still possible for some time. However, finally the labile fraction disappears and nerve trammission ceases. During recovery newly formed amine is incorporated into the labile fraction, which is immediately utilized, i.e. released by nerve activity. In other words, transmission is restored before the amines accumulate. The decarboxylation products of a-methyl-DOPA and a-MMT, as well as Dadrenaline conceivably enter the labile fraction first, and later the stable fraction. However, noradrenaline continues to be synthesized, and its incorporation into the labile fraction is not blocked. Transmission is, therefore, unimpaired. In any case it is evident that the use of drugs as tools for clarifying the functions of the monoamines may easily lead to erroneous conclusions. For example, it cannot be expected that a drug like a-MMT which causes depletion of monoamine stores without interfering with the transmission mechanism, should affect behavior in the same manner as reserpine, which is capable of blocking transmission. In the brain the situation is rendered even more difficult through the fact that, for example, noradrenaline-containingneurons occur in a variety of functionally more or less independent systems, which show marked differences in sensitivity to drugs such as reserpine. This has been revealed by histochemical work (Falck, this symposium, p. 28). Attempts to correlate monoamine levels in e.g. the whole hypothalamus, not to mention the whole brain, with functional variables may, therefore, prove hazardous.

CHANGES I N BRAIN MONOAMINE LEVELS

15

Another common mistake is to identify the noradrenergically innervated centers of the brain with sympathetic centers. This is entirely unjustified as shown by histochemical data. Data on the impulse flow in peripheral sympathetic nerves, therefore, do not permit any conclusions concerning central noradrenergic mechanisms. MONOAMINE PRECURSORS

Even if there are no absolutely reliable tools for investigating the physiological role of brain monoamines, some tools appear to be less hazardous than others. Among the relatively safe tools I should like to mention first the precursors 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylserine (DOPS), and 5-hydroxytryptophan (5-HTP). In contrast to the monoamines, their precursors are able to penetrate into the brain where they undergo decarboxylation to the respective amines i.e. dopamine (from DOPA), noradrenaline (from DOPS), and 5-HT (from 5-HTP). Injections of these precursors are accompanied by characteristic central and peripheral effects, which are strongly potentiated by monoamine oxidase inhibitors and beyond any doubt mediated by the respective monoamines. Certain objections have been raised against the use of the precursors (Gessa et al., 1962b). Their decarboxylation appears to be brought about by one and the same enzyme. This would mean, for example, that after injection of 5-HTP, 5-HT would accumulate not only at serotonergic but also at noradrenergic, dopaminergic, and perhaps adrenergic synapses. 5-HT would then be able to activate hypothetical postsynaptic receptors beyond the reach of the 5-HT formed normally. This possibility cannot be excluded at the present time. However, the syndromes produced by 5-HTP and DOPA are markedly different, indicating that different receptors are activated by their respective decarboxylation products. There is no reason to doubt that each amine when formed from the administered precursor, activates its own physiological receptors. It is also reasonable to assume that this activation forms an important feature of the characteristic syndrome of each amine, just as injection of noradrenaline produces a syndrome similar to that caused by stimulation of the noradrenergic system peripherally. 5-HTP causes tremors, convulsions, and hyperextension of limbs, suggesting that 5-HT neurons participate in the control of motor functions. It is unable to antagorize the akinesia caused by reserpine. DOPA stimulates spontaneous motility and may in suitable dosage restore reserpinized animals almost to normal (Carlsson, Lindqvist et al., 1957; Carlsson, 1959). Also more complicated functions such as the conditioned avoidance response are partially restored (unpublished experiments of this laboratory, Seiden, 1963). DOPS, which is probably not a physiological precursor of noradrenaline, is decarboxylated very slowly by the decarboxylase. It, therefore, gives rise to little accumulation of noradrenaline when given alone. After inhibition of the monoamine oxidase it causes accuinulation of noradrenaline in both brain and heart. NOWboth central and peripheral effects are observed, provided that sufficiently large doses are given, for clearcut central actions in mice 500 mg/kg of the m-form is needed. The peripheral effects are seen also after smaller doses and correspond to those seen after injection of noradrenaline. They are blocked by phentolainine (in so far as the Rejerenics p 25-27

16

A R V I D CARLSSON

a-effects are concerned). The central effects are excitatory. They persist after phentolamine pretreatment. Reserpine-treated animals are awakened and start to move around almost like normal animals. Thus the actions of DOPA and DOPS are similar.

No of Reserpine 5 m g / k Niolarnide 100rng fkg 2-(3.4-dihydroxyphenyl) pentanoyl amide 5 0 0 m g / k g DL-threo-dops lOOOrng/kg

+

+

-

+

-

-

-

+

+

+

-

+

+

-

+

+

+

-

-

- - + + + + +

+

+

+

Fig. 5. Noradrenaline in mouse brain (a) and heart (b) following treatment, alone and in various combinations, with reserpine (20 h), inhibitors of monoamine oxidase (nialamide, 2 h) and catechol0-methyltransferase [2-(3,4-dihydroxyphenyl)-pentanoylamide, I .5 h], and ~~-threo-3,4-dihydroxyphenylserine (DOPS, 1 h). All drugs were given intraperitoneally. Times above refer to intervals before sacrifice.

Incidentally, the experiments with DOPS seem to settle the problem whether monoamine oxidase may influence noradrenaline metabolism directly or only via its precursor dopamine. As noradrenaline is formed directly from DOPS, the marked potentiation by a monoamine oxidase inhibitor indicates that the former alternative is true. This appears to be the case even in peripheral tissues. An influence of catechol0-methyltransferase on the noradrenaline formed from DOPS is also apparent both in brain and heart, as indicated by increased accumulation of noradrenaline following treatment with an inhibitor of the enzyme (Fig. 5 ) . This will be further discussed later. The data on monoamine precursors available thus far suggest that dopamine, noradrenaline, and 5-HT are largely excitatory transmitters in the brain. This does not exclude the possibility that also inhibitory actions will be disclosed in the further analysis. The fact that the monoamines counteract rather than mimic the action of reserpine, supports the view that this alkaloid acts by blocking transmission mechanisms of central neurons (just as they do with peripheral neurons) rather than causing an excess of free and active transmitter, as suggested by Dr. Brodie.

CHANGES I N BRAIN MONOAMINE LEVELS

17

ENZYME INHIBITORS

Selectively acting enzyme inhibitors should belong to the relatively safe tools for studying monoamine functions in the brain. Of course themonoamine oxidase inhibitors have already proved useful. Among these agents nialamide and MO 911 appear to be most selective. They seem to give comparable results. In our laboratory we are mostly using nialamide, which became available first. When these drugs are given to mice in doses sufficient to cause the virtually complete inhibition of monoamine oxidase, the monoamines accumulate rapidly in the brain. The drugs cause central stimulation, as is well known, but again there is no strict temporal correlation with total monoamine levels. Excitation does not seem to set in until the monoamines have already reached high values. This lag may indicate that excess liberation of monoamines to postsynaptic receptors does not set in until the stores have been maximally filled. In support of this the accumulation of normetanephrine has been found to show a similar lag (Carlsson, Lindqvistetal., 1960). In any event there is good reason for the assumption that the syndrome caused by a monoamine oxidase inhibitor such as nialamide is mediated by monoamines. The syndrome does not appear if the accumulation of monoamine is prevented by the administration of agents which inhibit their synthesis (see below). Nialamide is able to counteract the sedative effect of reserpine, provided the dose of the latter is not too large. This has been observed in our laboratory in rabbits (Bertler, 1961) and mice (Carlsson, unpublished experiments) and has been confirmed with MO 911 in rabbits by Brodie and Costa (1962). Both groups of workers agree that the effect shows a better correlation with rise in noradrenaline and/or dopamine than 5-HT. However, 5-HT may very well contribute to the overall effect. In any event the data do not support the view of Brodie and coworkers that the action of reserpine is due to excess free 5-HT, since in animals treated with reserpine followed by a monoamine oxidase inhibitor 5-HT rises much more than the catecholamines. If Dr. Brodie’s hypothesis was correct this should result in aggravation rather than counteraction of the reserpine syndrome. Thus the results with (1) peripheral adrenergic nerves, (2) precursors of the monoamines, and (3) monoamine oxidase inhibitors all point in one and the same direction, namely, that the reserpine syndrome is largely caused by blockade of the transmission mechanism of monoaminergic neurons of different kinds. One further piece of evidence may be added. As is well known, reserpine causes the syndrome of parkinsonism. This may be related to the loss of dopamine (possibly also 5-HT) from the basal ganglia (Carlsson, Lindqvist et al., 1958; Bertler and Rosengren, 1959). In patients suffering from ‘spontaneous’ parkinsonism severe reduction of dopamine and 5-HT has been observed in the basal ganglia (Ehringer and Hornykiewicz, 1960; unpublished data of this laboratory). Treatment ‘of patients suffering from parkinsonism with DOPA results in alleviation of some of the symptoms, particularly the akinesia (Birkmayer and Hornykiewicz, 1961, 1962; Barbeau et al., 1962; unpublished observations of the present research group). Opinions differ greatly as to the physiological importance of catechol-0-methylRt-ft-rencesp . 25-27

18

A R V l D CARLSSON

transferase (COMT). While this enzyme seems to be mainly responsible for the breakdown of circulating catecholamines (Axelrod et al., 1958), which appears to be largely due to the high COMT activity of the liver (Axelrod, 1959; Crout et a/., 1961 ; De Schaepdryver and Kirshner, 1961 ; Carlsson and Waldeck, 1963), it has proved difficult to evaluate its role in other tissues. I n fact, Brodie and Costa (1962) find it unlikely that COMT is even essential for the degradation of circulating catecholamines, owing to the existence of alternative pathways. The study of the role of COMT has been greatly hampered by thc lack of selective inhibitors of the enzyme. Of the inhibitors available, pyrogallol has been used most extensively. However, data obtained with this agent are often difficult to evaluate, since pyrogallol is toxic and exerts many actions which are unrelated to COMT inhibition. In Goteborg a series of more selective COMT inhibitors has been synthesized by Dr. Corrodi (Carlsson, Lindqvist et al., 1962c, 1963a). The compounds are

PYROGALLOL

CATECHOL

DOPACETAMIDE SERIES

4-METHYLTROPOLONE

TROPOLONEACETAMIDE SERIES

Fig. 6 . Catechol-0-methyltransferase inhibitors of current interest. Catechol and pyrogallol have been most widely used so far. 4-methyltropolone is a potent inhibitor described by Belleau and Burba (1961). The dopacetamide and tropoloneacetamide series have been synthesized by Dr. Corrodi, Hassle Ltd., Goteborg (Sweden).

derivatives of 3,4-dihydroxyphenylacetamide(Fig. 6). In nontoxic doses these compounds cause marked inhibition of COMT in vivo (Fig. 7). Several members of the series have a disturbing but interesting ‘side-effect’ : they inhibit the synthesis of catecholamines and 5-HT in the brain. Attempts are being made to separate the two effects. Normetanephrine and 3-methoxytyramine occur in the brain normally and disappear rapidly (Fig. 8) following COMT inhibition by one of the new compounds (Haggendal, 1963b). The accumulation of dopamine and noradrenaline following administration of DOPA (Fig. 7) and DOPS (Fig. 5), respectively, is increased by COMT inhibition, resulting in potentiation of pharmacological actions of these precursors. It, therefore, seems likely that COMT is of physiological importance also in the brain. In particular, the possibility should be considered that in the brain as in the rest of the body COMT is largely responsible for the degradation of ex-

C H A N G E S I N B R A I N MONOAMINE LEVELS

19

no 22107

I

22\07 lg/kg I p

Fig. 7. Effect of L-DOPA (7.5 mg/kg,l h) on dopamine (DA), 3-methoxytyramine (MT), noradrenaline (NA), and normetanephrine (NM) levels in mouse brain following pretreatment with reserpine (25 mg/kg, 20 h), nialamide (100 mg/kg, 2 h), and a-ethoxydopacetamide (22/07, 1 g/kg, 1.5 h). All drugs were given intraperitoneally. Times above refer to intervals before sacrifice.

40

r

Ro bbit brain normal

50 40-

0'

1

Rabbit brai n 4 5 rnin after infusion for 4 5 rnin of 2-(3.4-dihydraxyphenyl)-hexanayi amide 5 0 0 m g / k g

10

20

30

40

50

Fraction No.

Fig. 8. Noradrenaline (NA), normetanephrine (NM), dopamine (DA), and 3-methoxytyramine (MT) in rabbit brain normally and after treatment with an inhibitor of catechol-0-methyltransferase. References p . 25-27

20

ARVID CARLSSON

tracellular catecholamine while M A 0 is responsible for degradation of catecholamines intracellularly near the site of synthesis and storage. Data in support of this hypothesis were put forward a number of years ago (Carlsson, Lindqvist et al., 1960; Carlsson, 1960). Later experiments with DOPA (Carlsson and Hillarp, 1962), and now DOPS lend further support to this hypothesis. In this connection it is interesting to note that while reserpine causes an increase in the concentration in the brain of acid metabolites formed via the M A 0 pathway (Roos and Werdinius, 1962; Ashcroft and Sharman, 1962), it has been found to cause a decrease in the concentration of 3-0methylated metabolites of catecholamines (Haggendal, 1963b). This seems to indicate that reserpine causes a decrease in the liberation of catecholamines into the extracellular space, and thus to receptor sites, and at the same time a net increase in the release from the granules to the cytoplasmic sap, from where they can penetrate into the mitochondria and form a substrate for the MAO. Needless to say, it would be of great theoretical and perhaps also practical interest to have efficient inhibitors of enzymes responsible for the synthesis of monoamines in the brain. A number of years ago it was generally thought that the fall in monoarnine levels caused by a-methyl-DOPA and a-MMT was brought about by inhibition of DOPA decarboxylase. Later it was found, however, that this effect could be at least partly accounted for by the release or displacement mechanism discussed carlier.

fX-METHYLMETATYROSINE

NSD 1015

NSD 1034

K-METHYLDOPA

M K 405

NSD 1024

Fig. 9. DOPA decarboxylase inhibitors of current interest. HCS acid (Clark, 1959).

HCS =

3-hydroxycinnamoyl salicylic

Today the view seems to be favored that inhibition of synthesis is of no great importance for the action of these DOPA analogues. One argument supporting this view is that we know of a series of DOPA decarboxylase inhibitors (the ‘NSD compounds’, Fig. 9) which are unable to produce a fall in monoamine levels, although they are more potent inhibitors of the enzyme (Drain et al., 1962; Brodie et al., 1962a). Although the decarboxylation of exogenous precursor is largely blocked by thcse compounds, it appears that normal synthesis is unimpaired, suggesting that the enzyme is present in large excess of the normal needs. We have reinvestigated the problem using a biochemical method in vivo, i.e., as an

CHANGES I N BRAIN MONOAMINE LEVELS

21

indicator of decarboxylase activity we have used the accumulation of 5-HT following 5-HTP administration (50 mg/kg i.p.) to mice pretreated with a M A 0 inhibitor (nialamide 100 mg/kg i.p. 30 min before the 5-HTP). The drug to be tested for decarboxylase activity was given i.p. 30 min before the M A 0 inhibitor. Both brain and kidney were examined. The results are expressed as per cent inhibition (Fig. 10). Percentage inhibition was set to zero when the accumulation of 5-HT was the same as in animals given the M A 0 inhibitor and 5-HTP only. It was set to 100 per cent if 5-HT values were the same as in untreated normal animals. Brain

fl Kidney

100

8ol

a-Methylm-tyrosine

100 4 0 0 @-Methyldopa

100 MK-485

50 100 .. ~~

NSD 1015

Fig. 10. Activity of decarboxylase inhibitors in mice in vivo.

Of the 3 DOPA analogues tested, a-methyl-DOPA was clearly more efficient than a-MMT and MK 485 (the hydrazine analogue of a-methyl-DOPA, Porter et al., 1962b), particularly in the brain. Of the NSD compounds, NSD 1015 (rn-hydroxybenzylhydrazine) was clearly more efficient than NSD 1034, i.e. its N-methyl derivative, and NSD 1024 (m-hydroxybenzyloxyamine). No data on NSD 1015 seem to have been published before. According to the present data this compound is a more potent inhibitor of the decarboxylase than a-methyl-DOPA. Unlike a-methyl-DOPA, however, it was unable to produce a decrease in the brain 5-HT. It also proved to be unable to block the fall in the brain 5-HT caused by a-methyl DOPA, although it blocked the prolonged and pronounced fall in noradrenaline, apparently by blocking the decarboxylation of a-methyl-DOPA. This suggests that the fall in 5-HT is caused by a direct action of a-methyl-DOPA rather than by its decarboxylation products. In our laboratory Roos and Werdinius (1963), have found that treatment of rabbits with a-methyl-DOPA results in a drop in both 5-HT and 5-hydroxyindoleacetic acid. Similar observations have been made by Sharman and Smith (1962). This argues against release or displacement and favors the view that a-methyl-DOPA inhibits the synthesis of 5-HT. If this inkbition cannot be accounted for by decarboxylase inhibition, as the experiments with NSD compounds suggest, then we seriously have References p . 25-27

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ARVID CARLSSON

4

Test compound 30min

Normal level

5-HT in mouse brain

-

2 - (3.4- dihydroxyphenylk pentonoyl ornide mg/kg 2 - (3.4- dihydroxyphenylb hexanoyl .ornide mg/kg Niolamide rnp/kg

-

100

-

-

-

500

500

500

300

-

100

-

300

-

500

500

50Q

500

Fig. 11. Blockade by two inhibitors of catechol-0-methyltransferase of the 5-hydroxytryptamine accumulation induced in brain by inhibition of monoamine oxidase.

inhibition of synthesis caused by a direct action of the amino acids, and (b) dkplacement caused by their decarboxylation products. Which of these effects, if any, is responsible for the fall in blood pressure and sedation caused by a-methyl-DOPA is not known, although inhibition of synthesis appears to be the more likely alternative. As already mentioned, a number of dihydroxyphenylacetamide derivatives have been found to inhibit not only COMT but also the synthesis of monoamines in the brain. There is no inhibition of the decarboxylase, so the site of attack is probably the first step in the synthesis, i.e. the hydroxylation of tryptophan on the 5 position and the hydroxylation of tyrosine on the 3 position, respectively. The evidence for inhibition of synthesis is (1) fall in 5-HT, dopamine, and noradrenaline levels in the brain, (2) block of the accumulation of monoamines in the brain caused by inhibition ; Fig. 1 l), and (3) fall i n 5-hydroxyof M A 0 (Carlsson, Lindqvist et al., 1 9 6 2 ~ 1963a; indoleacetic acid level in brain (Roos and Werdinius, 1963). The compounds have a depressant effect on the central nervous system, but it is not known if this effect is caused by the inhibition of monoamine synthesis. EXPERIMENTS WITH BRAIN LESIONS

The localization of noradrenaline, and probably dopamine and 5-HT, to neurons in the brain has prompted us to investigate the noradrenaline (Magnusson and Rosengren, 1963) and 5-HT (Carlsson, Magnusson et al., 1963b) levels of the spinal cord of rabbits following transection at the level of the second thoracic segment. Both nor-

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23

adrenaline and 5-HT were found to disappear almost entirely below the lesion but were unchanged above the lesion, indicating the existence of descending noradrenergic and serotonergic pathways in the spinal cord (Fig. 12). Intravenous injection of L-DOPA (100 mg/kg) was followed by marked stimulation of spinal reflexes below the lesion, suggesting a facilitating function of noradrenergic neurons. Likewise,

0.31 0.1

0

0

t

Above Th2

@ Below Th2

I

S.E.M.

k

k

NA

normal No.of animals 5

T

transection

5

normal 4

transection

3

Fig. 12. Noradrenaline and 5-hydroxytryptamine in rabbit spinal cord normally and after transection at second thoracic segment. (Magnusson and Rosengren, 1963; Carlsson, Magnusson et al., 1963).

injection of 5-HTP caused stimulation of spinal reflexes, although the picture appeared to be qualitatively different from that caused by DOPA. Recently Heller et al. (1962) reported that destruction of the medial forebrain bundle within the lateral hypothalamus of the rat produced a fall of 36 per cent in the brain 5-HT levels as compared with normal controls. CONCLUDING SPECULATIONS

The present data suggest that the monoamine-storing granules have a dual function, namely (a) to serve as a store of monoamines, and (b) to facilitate the transfer of monoamines from the site of synthesis to the site of liberation into the synaptic cleft. The significance of the first function is dubious, as the organism apparently can do well without the store. It may possibly be of importance under special emergency conditions, e.g. if the synthesis of transmitter or its transfer from the site of synthesis to the site of liberation into the synaptic cleft is blocked. This may possibly be the case during the early stage of reserpine action. It will also be interesting to see, if displacement of the noradrenergic transmitter by a less active analogue is accompanied by increased sensitivity to agents which inhibit the synthesis of transmitter. Preliminary observations suggest that this may be so. In rats the injection of a-methylDOPA (400 mg/kg i.p.) causes but slight sedation and ptosis. A second injection of the same dose 24 h later causes clearcut sedation and ptosis (Anden and Magnusson, unpublished experiments). T h s may be interpreted to mean that the first injection References p . 25-27

24

ARVID CARLSSON

causes inhibition of synthesis, but transmitter functions are unimpaired since they may proceed at the expense of the stores. At the time of the second injection, however, the noradrenergic transmitter has been replaced by the less active a-methyl analogue in the store. When synthesis is blocked by the second injection, the transmission mechanism loses much of its efficiency. The possibility should be considered that the store of transmitter cannot always be mobilized at sufficient rate to keep transmission intact, should the synthesis or the uptake by the granules be blocked e.g. by a drug. It is remarkable that symptoms of depression may set in before the monoamine stores are emptied. To a certain extent this is true of reserpine, but even more so of the benzoquinolizines. Actually some of the benzoquinolizines, for example, benzquinamide, have been stated to depress brain functions in doses which do not cause any decrease at all in brain monoamine levels (Weissman and Finger, 1962; Pletscher et al., 1962). In connection with the benzoquinolizines it is interesting to note that the Ihydroxyphenylacetamide derivatives mentioned before cause depression of the central nervous system and inhibition of monoamine synthesis. The symptoms of depression seem to reach their maximum before the monoamine levels have reached their minimum, which is in analogy with the benzoquinolizines. Of course, it is possible that no causal relationship exists between the biochemical and behavioral effects in the case of these two groups of drugs. The possibility cannot be excluded, however, that a small number of highly active and functionally essential neurons are almost exclusively dependent on newly synthesized transmitter, possibly because they are unable to mobilize the store at sufficient speed. It may thus be as difficult to disprove as to prove a causal relationship between biochemical and behavioral effects of drugs. SUMMARY

The actions of various agents interfering with formation, degradation, storage, and release of catecholamines and 5-hydroxytryptamine is discussed in the light of recent evidence that these monoamines serve as transmitters in the central nervous system. Depletion of transmitter stores can be brought about by different mechanisms, which are not equivalent from the functional point of view. Blockade of monoamine uptake by the storage granules produced by reserpine thus leads to depletion of the transmitter stores and to blockade of transmission. On the other hand, displacement of transmitter from the storage sites of the granules by less active transmitter analogues, e.g. metaraminol (formed from a-methyl-m-tyrosine) and D-adrenaline, does not result in any readily detectable impairment of transmission. Thus blockade of the uptake mechanism of the storage granules seems to be an essential component in the the action of reserpine. This is further supported by the better time correlation of the pharmacological actions of reserpine to the blockade of the uptake mechanism than to the tissue amine levels. The data support the view that the granules do not merely serve as stores of transmitter but are directly involved in the transmission mechanism. The usefulness and limitations of various agents, e . g . monoamine precursors and analogues, and inhibitors of enzymes responsible for the formation and degradation

C H A N G E S I N B R A I N M O N O A M I N E LEV ELS

25

of monoamines are discussed. The pitfalls arising from the lack of a direct correlation between amine levels and functions are emphasized. ACKNOWLEDGEMENTS

For generous supply of drugs I am indebted to Mr. E.M. Bavin, Smith and Nephew Research Ltd. (‘NSD compounds’), to Dr. W. G. Clark,Psychopharmacology Research Laboratories, Sepulveda, California (3-hydroxycinnamoylsalicylic acid), to Dr. H. Corrodi, Hassle Ltd., Goteborg (~~-threo-3,4-dihydroxyphenylserine), to Dr. A. M. Lands, Sterling-Winthrop Research Institute (D-adrenaline), to the Swedish Ciba Ltd. (Serpasil) and to the Swedish Pfizer Ltd. (Niamid). This work has been supported in part by the Directorate of Life Sciences, AFOSR, Office of Aerospace Research United States Air Force, monitored by the European Office, Office of Aerospace Research under Grant No. AF-EOAR-61-44. REFERENCES ANDEN,N.-E., AND M AGNUSSON, T., (1964); Functional significance of noradrenaline depletion by a-methyl-m-tyrosine, metaraminol and D-adrenaline. Symposium on Cholinergic and Adrenergic Transmission. A. Carlsson, G. B. Koelle, and W. W. Douglas, Editors. London-New York, Pergamon Press. ASHCROFT, G. W., AND SHARMAN, D. F., (1962); Drug-induced changes in the concentration of 5-OR indolyl compounds in cerebrospinal fluid and caudate nucleus. Brit. J. Pharmacol., 19, 153-160. AXELROD, J., (1959); The metabolism of catecholamines in vivo and in vitro. Pharmucol. Rev., 11, Part 11, 402-408. J., INSCOE, J. K., SENOH,S., AND WITKOP,B., (1958); 0-Methylation, the principal pathway AXELROD, for the metabolism of epinephrine and norepinephrine in the rat. Biochim. biophys. Acta (Amst.), 27, 210-211. BARBEAU, A., SOURKES, T. L., AND MURPHY,G. F., (1962); Les catkcholamines dans la maladie de Parkinson, Monoamines et Systbme nerveux central. J. De Ajuriaguerra, Editor. Symposium Bel-Air, Geneva, 1961. Geneva, Georg & Co. (pp. 247-262). BELLEAU, B., AND BURBA,J., (1961); Tropolones: a unique class of potent noncompetitive inhibitors Biochim. biophys. Acta (Amst.), 54,195-1 96. of 5-adenosyl-methionine-catechol-methyltransferase. A., (1961); Effect of reserpine on the storage of catecholamines in brain and other tissues. BERTLER, Acta physiol. scand., 51, 75-83. A,, AND ROSENGREN, E., (1959); Occurrence and distribution of dopamine in brain and BERTLER, other tissues. Experientiu (Busel), 15, 10. BIRKMAYER, W., AND HORNYKIEWICZ, O., (1961); Der ~-3,4-Dioxyphenylalanin(DOPA)-Effekt bei der Parkinson-Akinese. Wen. klin. Wschr., 73, 787-788. O . , (1 962); Der L-Dioxyphenylalanin (L-DOPA)-Effekt beim BIRKMAYER, W., AND HORNYKIEWICZ, Parkinson-Syndrom des Menschen : Zur Pathogenese und Behandlung der Parkinson-Akinese. Arch. Psychiat. Nervenkr., 203, 560-574. BRODIE,B. B., AND COSTA,E., (1962); Some current views on brain monoamines. Monoamines et Systbme nerveux central. J. De Ajuriaguerra, Editor. Symposium Bel-Air, Geneva, 1961. Geneva, Georg & Co. (pp. 1349). BRODIE,B. B., GESSA,G . L., A N D COSTA,E., (1962b); Association between reserpine syndrome and blockade of brain serotonin storage process. Life Sciences, 10, 551-560. BRODIE,B. B., KUNTZMAN, R., HIRSCH,C. W., AND COSTA,E., (1962a); Effects of decarboxylase inhibition on the biosynthesis of brain monoamines. Life Sciences, 3, 81-84. CARLSSON, A., (1 959); The occurrence, distribution and physiological role of catecholamines in the central nervous system. Pharmacol. Rev., 11, 490493. CARLSSON, A,, (1960); Discussion remark. Ciba Symposium on Adrenergic Mechanisms. J. R. Vane, G. E. W. Wolstenholme, and M. 0. O’Connor, Editors. London, Churchill (pp. 558-559). CARLSSON, A., (1962) ; Discussion remark, Pharmacological Analysis of central nervous Action. Vol. 8.

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W. D. M. Paton and P. Lindgren, Editors. Proceedings of the First International Pharmacological Meeting. Oxford, Pergamon Press (pp. 71-74). CARLSSON, A., AND HILLARP, N.-A., (1962); Formation of phenolic acids in brain after administration of 3,4-dihydroxyphenylalanine.Acta physiol. scand., 55, 95-1 00. CARLSSON, A., HILLARP,N.-A., AND WALDECK, B., (1962); A Mg++-ATP-dependent storage mechanism in the amine granules of the adrenal medulla. Med. exp. (Basel), 6, 47-53. CARLSSON, A,, HILLARP, N.-A., AND WALDECK, B., (1963); Acta physiol. scand., Suppl. 215, 59, 1-38. M., (1962a); DOPA analogues as tools for the study of dopamine CARLSSON, A., AND LINDQVIST, and noradrenaline in brain. Monoamines et Systdme nerveux central. J. De Ajuriaguerra, Editor. Symposium Bel-Air, Geneva, 1961. Geneva, Georg & Co. (pp. 89-92). CARLSSON, A,, AND LINDQVIST, M., (1962b); In vivo decarboxylation of a-methyl-DOPA and amethyl-m-tyrosine. Acta physiol. scand., 54, 87-94. CARLSSON, A., LINDQVIST, M., AND CORRODI,H., (1963a); Synthese von Catechol-0-methyl-transferase-hemmenden Verbindungen. In den Catecholaminmetabolismus eingreifende Substanzen. 2. Mitteilung. Helv. chim. Acta, 46, 2271-2282. A., LINDQVIST, M., FILA-HROMADKO, S., AND CORRODI,H., (1962~);Synthese von CateCARLSSON, chol-0-methyl-transferase-hemmendenVerbindungen. In den Catecholaminmetabolismus eingreifende Substanzen. 1. Mitteilung. Helv. chim. Acfa, 45, 270-276. CARLSSON, A., LINDQVIST,M., AND MAGNUSSON, T., (1957); 3,4-Dihydroxyphenylalanine and 5hydroxytryptophan as reserpine antagonists. Nature (Lond.), 180, 1200. CARLSSON, A., LINDQVIST, M., MAGNUSSON, T., AND WALDECK, B., (1958); On the presence of 3-hydroxytyramine in brain. Science, 127, 471. CARLSSON, A., LINDQVIST, M., AND MAGNUSSON, T., (1960); On the biochemistry and posilble functions of dopamine and noradrenaline in brain. Ciba Sympovium on Adrenergic Mechanisms. J. R. Vane, G. E. W. Wolstenholme and M. O’Connor, Editors. London, Churchill (pp. 432-439). CARLSSON, A., MAGNUSSON, T., AND ROSENGREN, E., (l963b); 5-Hydroxytryptamine of the spinal cord normally and after transection. Experientia (Basel), 19, 359. CARLSSON, A., SHORE,P. A., AND BRODIE,B. B., (1957); Release of serotonin from blood platelets by reserpine in vitro. J. Pharmacol. exp. Ther., 120, 334-339. CARLSSON, A., AND WALDECK, B., (1963); On the role of liver catechol-0-methyltransferase in the metabolism of circulating catecholamines. Acta pharmacol. toxicol., 20, 47-55. CLARK, W. G., (1959); Studies on inhibition of L-DOPA decarboxylase in vitro and in vivo. Pharmacol. Rev., 11, 330-349. COSTA,E., GESSA,G. L., KUNTZMAN, R., AND BRODIE,B. B., (1962a); The effect of drugs on storage and release of serotonin and catecholamines in brain. Pharmacological Analysis of central nervous Action. Vol. 8. W. D. M. Paton and P. Lindgren, Editors. Proceedings of the First International Pharmacological Meeting. Oxford, Pergamon Press (pp. 43-71). COSTA,E., GESSA,G. L., KUNTZMAN, R., AND BRODIE,B. B., (1962b); A differential action of reserpine on brain dopamine stores in rabbit. Life Sciences, 11, 599-604. CROUT,J. R., CREVELING, c. R.. AND UDENFRIEND, S., (1961); Norepinephrine metabolism in rat brain and heart. J . Pharmacol. exp. Ther., 132, 269-277. DE SCHAEPDRYVER, A. F., AND KIRSHNER, N., (1961); The metabolism of ~ ~ - a d r e n a l i n e - 2 -in l ~ the c cat. 11. Tissue metabolism. Arch. int. Pharmacodyn., 131, 433-449. M., LAZARE, R., AND POULTER, G . A,, (1962); The effect of a-methylDRAIN,D. J., HORLINGTON, DOPA and some other decarboxylase inhibitors on brain 5-hydroxytryptamine. Life Sciences, 3, 93-97, EHRINGER, H., AND HORNYKIEWICZ, O., (1960); Verteilung von Noradrenalin und Dopamin (3Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr., 38, 1236-1239. GESSA,G. L., COSTA,E., KUNTZMAN, R., AND BRODIE,B. B., (1962a); On the mechanism of norepinephrine release by a-methyl-m-tyrosine. Life Sciences, 8, 353-360. GESSA,G., COSTA,E., KUNTZMAN, R., AND BRODIE, B. B., (1962b); Evidence that the loss of brain catecholamine stores due to blockade of storage does not cause sedation. Life Sciences, 9, 441-452. HAGGENDAL, J., (1963a); An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues. Acta physiol. scand., 59, 242-254. HAGGENDAL, J., (1963b); The presence of 0-methylated noradrenaline (normetanephrine) in normal brain tissue. Acta physiol. scand., 59, 261-268. HAGGENDAL, J., AND LINDQVIST, M., (1963) ; Behaviour and monoamine levels during long-term administration of reserpine to rabbits. Acta physiol. scand., 57, 431436.

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HELLER, A., HARVEY, J. A., AND MOORE, R. Y . ,(1962); A demonstration of a fall in brain serotonin following central nervous system lesions in the rat. Biochem. Pharrnacol., 11, 859-866. HESS,S. M., CONNAMACHER, R. H., OZAKI,M., AND UDENFRIEND, S., (1961); The effects of a-methylDOPA and a-methyl-m-tyrosine on the metabolism of norepinephrine and serotonin in vivo. J . Pharmacol. exp. Ther.,l31, 129-138. HILLARP, N.-A., (1960); Different pools of catecholamines stored in the adrenal medulla. Acfaphysiol. scand., 50, 8-22. KIRSHNER, N., (1962); Uptake of catecholamines by a particulate fraction of the adrenal medulla. Science, 135, 107-108. LUNDBORG, P., (1963); Storage function and amine levels of the adrenal medullary granules at various intervals after reserpine treatment. Experientia (Basel), 19, 479. MAGNUSSON, T., AND ROSENGREN, E., (1963); Catecholamines of the spinal cord normally and after transection. Expevientia (Basel), 19, 229. PLETSCHER, A., BESENDORF, H., STEINER, F. A., A N D GEY,K. F., (1962); The effect of 2-hydroxybenzoquinolizines on cerebral 5-hydroxytryptamine, spontaneous locomotor activity, and ethanol hypnosis in mice. Med. exp. (Basel), 7 , 15-20. PORTER, C. C., TOTARO, J. A., AND LEIBY,C. M., (1961); Some biochemical effects of a-methyl-3,4dihydroxyphenylalanine and related compounds in mice. J . Pharmacol. exp. Thev., 134, 139-145. PORTER, C. C., WATSON, L. S., TITUS,D. C., TOTARO, J. A., AND BYER,S . S., (1962b); Inhibition of DOPA decarboxylase by the hydrazino analogue of a-methyl-DOPA. Biochem. Phavmacol., 11, 1067-1077. Roos, B.-E., AND WERDINIUS, B., (1962); Effect of reserpine on the level of 5-hydroxyindoleacetic acid in brain. Life Sciences, 3, 105-107. Roos, B.-E., AND WERDINIUS, B., (1963); The effect of a-methyl-DOPA on the metabolism of 5hydroxytryptamine in brain. Life Sciences, 2, 92-96. L. S., AND CARLSSON, A., (1963); Temporary and partial antagonism by L-DOPA of SEIDEN, reserpine-induced suppression of a conditioned avoidance response. Psychopharmacologia ( B e d . ) , 4, 418423. SHARMAN, D. F., AND SMITH,S. E., (1962); The effect of a-methyl-DOPA on the metabolism of 5-hydroxytryptamine in rat brain. J . Neurochem., 9, 403406. STONE,C. A., Ross, C. A., WENGER, H. C., LUDDEN,C. T., BLESSING, J. A., TOTARO, J. A., AND PORTER, C . C., (1962); Effect of a-methyl-3,4-dihydroxyphenylalanine(methyl-DOPA), reserpine and related agents on some vascular responses in the dog. J. Pharmacol. exp. Ther., 136, 80-88. UDENFRIEND, S., AND ZALTZMAN-NIRENBERG, P., (1962); On the mechanism of norepinephrine release produced by a-methyl-m-tyrosine. J. Pharmacol. exp. Ther., 138, 194-199. WEISSMAN, A., AND FINGER,K. F., (1962); Effects of benzquinamide on avoidance behaviour and brain amine levels. Biochem. Pharmacol., 11, 871-880.

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Cellular Localization of Monoamines B E N G T FALCK Depavtnient of Histology, University of Lund, Lund (Sweden)

To interpret the functions of monoamines it is unquestionably necessary to be able to localize accurately their tissue storage sites. The hitherto available histochemical methods have one feature in common: their sensitivity is low and permits the demonstration of monoamines only in cells which store them in high concentrations e.g. adrenal medullary cells and enterochromaffin cells. There may be several causes for this low degree of sensitivity, the most obvious one being that the histotechnical procedures involve the use of fluid fixatives and reaction solutions which cause diffusion or extraction of a certain amount of monoamines from the cellular stores before the desired histochemical reaction has reached its endpoint. When Carlsson et al. (1962) introduced EhrlCn’s (1948) fluorimetric determination method for catecholamines into histochemistry these difficulties were partly overcome. They oxidized the catecholamines in sections from freeze-dried adrenals in a weak solution of iodine in benzene, whereafter the sections were treated in benzene saturated with ammonium or methylamine. This alkaline rearrangement of the oxidation products gave rise to an intense fluorescence in the medullary cells. However, in spite of a high sensitivity and specificity it has as yet not been possible to utilize this technique for the visualization of noradrenaline for example in noradrenergic nerves; again, probably, because the extraction and diffusion problems were not sufficiently solved. THE PRESENT METHOD

A more promising degree of progress was obtained with the finding that certain catecholamines, included in a dry protein film, are transformed into products with an intense green to yellow-green fluorescence on exposure to formaldehyde gas (Falck et at., 1962). Investigations on the chemical nature and specificity of this reaction have been performed with model systems (Corrodi et at., 1962). There is ample evidence that the primary amines, such as dopamine (DA) and noradrenaline (NA), condense with formaldehyde to 1, 2, 3, 4-tetrahydroisoquinoline derivatives, which - provided protein is present -are then rapidly transformed into fluorescent 3,4-dihydroisoquinolines.The 3-OH group is essential for the first step in the reaction whereas only amines with hydroxyl groups at both positions 3 and 4 yield products with a very intense fluorescence. Secondary amines, such as adrenaline (A), also

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easily condense with formaldehyde to tetrahydroisoquinolines, but the second step -in this case yielding 3,4-dihydroisoquinolineswith a quaternary nitrogen - requires more time and a higher temperature than needed for primary catecholamines. The necessity of proteins for the second step in the reaction cannot as yet be explained. Certain tryptamines -e.g. 5-hydroxytryptamine (5-HT) and 5-methoxytryptamine react with formaldehyde in a similar way to yield 3,4-dihydronorharman derivatives with a n intense yellow to sometimes green-yellow fluorescence. Based on this principle a histotechnical procedure has been devised, the details of which have been dealt with in a previous paper (Falck, 1962). Only a brief description

Fig. 1. Stretch-preparation from the dilator muscle of the rat iris. The noradrenergic varicose terminals running in the autonomic ground-plexus exhibit a high fluorescence, especially the varicosities. Magnification: 375 X . References p . 43/44

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of the technique will be given here. Freeze-dried tissue pieces are treated at 80" for 1-3 h in closed glass vessels containing paraformaldehyde. In this dry milieu the catecholamines and 5-HT are transformed into their fluorophores without any diffusion from their storage sites. The fluorophores are not extracted with hot paraffin or organic solvents such as xylene. The preparations can therefore be embedded in paraffin and the sections deparaffinized for example in xylene. After mounting, the sections are studied in a fluorescence microscope equipped with a dark-field condensor system. The exciting light (Osram HBO 200 high-pressure lamp) is filtered through 3-5 mm Schott BG 12 and the emitted light through a secondary filter in the tube with a high absorption below 480 mp. Thin tissue sheets, such as iris or mesentery from smaller animals, can be spread across slides, dried for a short time at room temperature in the air or in VUI'LIO, and, after treatment in formaldehyde gas, directly analyzed in the fluorescence microscope (Fig. 1). The primary catecholamines react readily with formaldehyde and are transformed into intensely green to sometimes yellow-green fluorescent compounds within one hour's treatment. The same fluorescence can be obtained from secondary catecholamines, such as A, but only if the formaldehyde treatment is prolonged to 2-3 h. Tryptamines, such as 5-HT, react as readily as the primary catecholamines. The fluorophore of 5-HT, however, emits an intense yellow fluorescence. In this way the method offers a possibility to differentiate between primary and secondary catecholamines as well as between catecholamines and tryptamines. The fluorescence that develops on formaldehyde treatment has been studied in a large number of different tissues from both vertebrates and invertebrates. So far the reaction conditions for the development of such fluorescence and the properties of the condensation products have shown the same characteristics as are valid for biogenic monoamines in model systems (pure substances included in dry protein films). Up to now it has been possible to identify the fluorescent substances in many tissues by parallel histochemical and chemical analyses in combination with denervation and treatment with drugs causing a selective monoamine depletion. The only compounds detected so far in tissues are 5-HT and the catecholamines A, NA and DA. It seems that other biogenic compounds which may react with formaldehyde to form fluorescent products either do not develop a fluorescence in the green to yellow range, or are present in too small amounts to emit a light which interferes with the localization of monoamines. There are thus strong reasons for believing that the specificity of the method is very high. The sensitivity of the method is likewise high and has proved sufficient for the demonstration of intraneuronally located monoamines, which will be further discussed below. To illustrate the degree of sensitivity, it may be of value to mention some other types of monoamine depots that can be demonstrated by means of this technique. The 5-HT in normal mast cells from rat and mouse shows an intense yellow fluorescence, which is clearly seen to be confined to granules. Tndividual granules from disrupted mast cells also exhibit a high fluorescence. Tn blood smears from some species the platelets fluoresce because of their content of 5-HT. Cells showing a specific

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fluorescence have been demonstrated in tissues where monoamines were not earlier known to occur. Such cells are present in for example the pancreatic islets in several species (Falck and Hellman, 1963). In the guinea-pig these fluorescent cells are identical with the @-cellsand in this case the monoamine probably belongs to the tryptamine group (Falck and Hellman, 1964). O C C U R R E N C E O F M O N O A M I N E S I N P E R I P H E R A L NERVES

In many peripheral mammalian tissues a specific intense green fluorescence, typical of primary catecholamines, develops in fine nerve fibres having the same characteristic morphology and topography as those of the axons running in the autonomic groundplexus (cf. Hillarp, 1959). These fibres are constantly found in organs where monoaminergic nerves are known to be present (e.g. heart, iris, submaxillary gland, vas deferens and blood vessels in several organs); in the iris they are distributed mainly to the dilator muscle, while the sphincter muscle, which has a cholinergic innervation, contains only few fluorescent fibres, most of which are distributed along the vessels. In some of the investigated tissues (e.g. heart and vas deferens) only NA is known to occur in significant amounts and the only fluorescent structure to be found is nerve fibres. The effect of reserpine on the fluorescence of the nerves has been studied in several tissues. In dose-response and time-dose experiments, as well as in recovery experiments, a close correlation has been found between the disappearance and reappearance, respectively, of N A in tissues and the fluorescence of the nerve fibres (Falck, 1962). The fluorescence is further caused to disappear more or less by administration of drugs such as m-tyrosine, a-methyl-mefa-tyrosine, aramine and guanethidine, which deplete the tissues of their monoamines. After bilateral cervical sympathectomy (rat) the fluorescence in the iris nerves remains essentially unchanged during the first 20-25 h, thereafter a dramatic reduction occurs. Moreover, 30 h postoperatively, only a few, weakly fluorescent fibres remain, which have completely disappeared after 48 h. The findings agree with the observations on the disappearance of N A in tissues after postganglionic denervation (Furchgott, 1960; Sidman et al., 1962; Weiner et a]., 1962). After preganglionic denervation, which according to Rehn (1958) does not alter the organ content of NA, no change in the fluorescence of the iris nerves can be registered. These findings conclusively prove that the fluorescent nerves are monoaminergic and that in some tissues they contain NA. However, it must be stressed that the technique offers no direct possibilities to differentiate between primary catecholamines in the fluorescence microscope. The differentiation between these amines must therefore be based on other criteria, e.g. their content in the tissues. According to Schumann (1956) noradrenergic nerves contain both NA and DA in equal quantities. However, later experiments of Bertler and Rosengren (1959) showed that appreciable quantities of DA occur in peripheral tissues only in ruminants and did not confirm Schiimann’s findings (1958, 1959) concerning the occurrence of DA in dog tissues. Strong evidence has been provided that DA in ruminants is stored References p. 43/44

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in a widely distributed system of granular chromaffin cells (Falck et al., 1959a, b ; Bertler et al., 1959). Further investigations (Falck, Nystedt and Stenflo, unpublished observations) have demonstrated that, when tissues from ruminants are treated according to the present fluorescence method, the granules of these cells show an intense fluorescence characteristic of primary catecholamines, and that the cells in fact are mast cells. Their distributional pattern is typical of mast cells, for they are metachromatic and stain with Astrablue (at pH 0.2-0.3), which is a selective stain for mast cells (Bloom and Kelly, 1960). This agrees with the finding of Coupland and Heath (1961) that at least part of the chromaffin cells in cow liver capsule and gut stain as mast cells. Tt is of importance to note that these cells may be present in nerves. In the sciatic and splenic nerves they occur in a number that well explains the amount of DA found in these nerves (Schumann, 1956; Von Euler and Lishajko, 1957; Bertler et al., 1959). In a current investigation on the mammalian pineal body (Bertler et al., 1963) evidence has accumulated that in some species (rat, mouse, guinea-pig, dog) the pineal nerves store a monoamine that may belong to the tryptamine group, judging from the characteristics of the fluorescence reaction. However, differences between the species exist. Thus, the fluorescence reaction in the pineal nerves of cat and rabbit indicates the presence of a primary catecholamine. The pineal body of the rat and mouse differs from that of the other animals in that a specific, intense yellowish fluorescence develops not only in the nerves, but also in the parenchymal cells. Correspondingly, the rat pineal body was found to contain large amounts of 5-HT (0.07-0.09 pglgland). The pineal nerves enclose the vessels with a dense network of delicate varicose fibres. From this vascular plexus, fibres issue forming a network which enmeshes the pineal cells. It has not been made clear whether this arrangement represents a true parenchymal innervation or anastomosing strands between the vascular plexuses. The identity of the intraneuronal monoamine has not been established with certainty, but results of investigations on the rat pineal body indicate that the amine is 5-HT. Five to seven days after bilateral cervical sympathectomy- which causes the pineal nerves to degenerate (Kappers, 1960) -the pineal 5-HT decreases to about 50 % of the normal value. This is also the case 24 h after administration of reserpine in a dose of 5 mg/kg. Remarkably, however, it is not possible to bring about a further decrease when reserpine is given once daily in a dose of 1 mg/kg, a treatment which has a more pronounced effect on such relatively reserpine-refractory depots as the rat mast cells than has a high single dose. The fluorescence reaction in the pineal nerves is completely abolished after denervation and reserpine treatment as above ; also a slight decrease in the fluorescence intensity of the pineal cells seems to occur. However, it has not as yet been established whether this represents an actual decrease in the pineal cells or merely an unmasking of their normal fluorescence due to the disappearance of the fluorescent nerves. The fluorescence of the nerves is lost after a very small dose of reserpine (0.1 5 mg/kg -animals killed after 24 h) and very rapidly within 1.5 h-after a single dose of 5 mg/kg; the parenchymal fluorescence shows no certain change in either case. These doses also reduce the pineal 5-HT by almost 50%.

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It thus seems that about half of the 5-HT is easily released with reserpine, whereas the other half is remarkably refractory to reserpine. Wolfe et a / . (1962) injected labelled DL-NAinto rats and found with an autoradiographic technique a n uptake of N A in the pineal nerves. This finding has been confirmed, utilizing the present histochemical method: in pineals excised 45 min after administration of DL-NA (5 mg/kg intraperitoneally) all the nerves exhibit an intense green fluorescence, characteristic of primary catecholamines. Concomitantly, a considerable reduction to about 50 % of the pineal 5-HT was found to occur. It is obvious from these experiments that about half of the pineal 5-HT must be stored in the pineal cells, but it is not immediately evident whether the rest, partly or wholly, is stored in the nerves. However, the existence of two depots in the pineal cells, sharply differing in their sensitivity to reserpine, does not seem likely; it is more probable that the various factors which extinguish the yellow fluorescence in the nerves, and concomitantly cause a release of about the same quantity of 5-HT, deplete a store of 5-HT located in the nerves. The results do not exclude the possibility that the pineal nerves also contain NA, whose fluorescence is masked by the yellowish fluorescence. Various drugs have been tested for their depletion effects of pineal 5-HT. Unexpectedly, the most effective agent found so far is aramine. Spectrophotofluorimetric determinations on rat pineal bodies excised 5-6 h after a single injection of 25 mg/kg aramine demonstrated that the 5-HT was reduced to almost insignificant amounts. The classical experiments of Loewi showed that A was released from the frog heart during sympathetic stimulation, and Loewi concluded that A served as a transmitter in this animal. After Von Euler found that the adrenergic transmitter in mammals is NA, Loewi’s findings began to be neglected. In a recent review on autonomic neuroeffector transmission Von Euler (1960) called attention to the lack of direct evidence that A acts as an adrenergic transmitter in any animal. However, when freeze-dried frog hearts (Rana temporaria) are exposed to dry formaldehyde gas, a green fluorescence develops only slowly in the monoaminergic nerves and its maximum intensity is reached only after an exposure time which is more than double the reaction time needed for primary catecholamines (Falck et a/., 1963). In serial sections from whole hearts this green fluorescence has been found in an abundance of nerve bundles and varicose fibres and also in some ganglion cells located in Bidder’s ganglia, but in no other structures, Spectrophotofluorimetric determinations showed that A was present in a concentration (1.7 ,ug/g) corresponding well with the fluorescence microscopic findings, whereas only small amounts of N A (0.01 ,ug/g) and little if any DA and 5-HT could be found. This content of N A cannot be responsible for the fluorescence in the nerves, but may well represent the N A that should be present as precursor of A. The same fluorescence reaction has also been demonstrated in nerves in other peripheral organs (kidney, urinary bladder and mesentery), indicating that A serves as transmitter not only in the heart, but perhaps in the whole peripheral monoaminergic part of the sympathetic system. It is well known that nervous tissue in some invertebrates, especially molluscs, contains high amounts of 5-HT (cf. Welsh and Moorhead, 1960). Dahl eta/. (1962 and References p. 43/44

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unpublished observations) demonstrated that in mollusc ganglia not only 5-HT, but also DA is present in high concentrations, and that these amines are localized to neurons. Thus, 4 monoamines have so far been shown to occur in neurons: NA, A, DA and 5-HT. To avoid confusion it seems more appropriate to reserve the term “adrenergic” for structures which in fact contain A. The term adrenergic should be replaced by “monoaminergic” and the different neurons in the monoaminergic system should be called noradrenergic, dopaminergic, serotonergic and adrenergic. In all species examined so far, from mammals to lower invertebrates, the different neurons in which these monoamines are located have proved to have some important qualities in common: when the axons reach their innervation structures, they are transformed into the varicose fibre type, i.e. rounded or elongated enlargements of the fibres appear. Furthermore, this part of the neuron always exhibits the strongest fluorescence. The other part of the neuron, including the cell body, shows a weaker fluorescence: sometimes a fluorescence can be detected in this part of the neuron only after an increase in the amine content has been induced, e . g . with monoamine oxidase inhibitors. Obviously the monoaminergic cell body and the first part of its process contain only small amounts of monoamines as compared with the varicose part. This agrees well with the findings of Von Euler (1956), which strongly indicate that NA in noradrenergic nerves is accumulated in the terminal part of the neuron and in a concentration that may well be of the same order as the catecholamine content in the adrenal medullary cells. In the terminal part, the varicosities fluoresce most intensely, while the part of the axon between the varicosities is usually very thin and has a fainter sometimes barely visible fluorescence. This suggests that most of the NA in the terminal ramifications is accumulated in the varicosities (Fig. 1). Investigations on the construction of the monoaminergic innervation apparatus in different tissues have demonstrated that monoaminergic fibres are often transformed into the varicose type already at a certain distance from the effector cells. For example, the nerve bundles in the adventitial layer of the vas deferens contain many varicose fibres which issue into the autonomic ground-plexus of the musculature. The portions of the varicose fibres located apart from the effector cells also exhibit a strong fluorescence, although the intensity is in some cases somewhat lower than that displayed by the axonal parts which lie in contact with the effector cells. This indicates that part of the peripheral neuronal NA is not immediately available for transmission. Finally, it should be mentioned that green fluorescent varicose terminals enclosing non-fluorescent nerve cells synaptically have been found in some ganglia, e.g. the superior cervical ganglion, the ganglia in the cat pancreas and the intestinal Auerbach‘s ganglia (Falck, 1963; Norberg, 1963; unpublished observations). In the intestines these fibres may be inhibitory. The varicose fibres in the intestinal wall are mainly distributed to the vessels and to Auerbach’s ganglia while only few fibres can be found in the muscular layer. From this distributional pattern it seems likely that the adrenergic inhibitory function on the intestinal wall is exerted in the ganglia and not directly on the muscle cells.

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F L U O R E S C E N T S T R U C T U R E S I N T H E C E N T R A L N E R V O U S S Y S TEM

The investigations on the cellular localization of monoamines in the central nervous system have so far mostly been concentrated on the mammalian hypothalamus. Below a survey will be given of the results of this work (Carlsson et al., 1962) and of some current studies on other brain areas. First, I will discuss hypothalamic structures showing a fluorescence characteristic of primary catecholamines. In the hypothalamus there occur a large number of intensely green to yellow-green fluorescent fibres possessing the same characteristic

Fig. 2. Numerous intensely fluorescent terminals in the supraoptic nucleus. No fluorescence in the optic chiasma (left). At the basal surface of the brain below chiasma to the left an artery surrounded by fluorescent nerves. The internal elastic membrane of the artery shows autofluorescence. Magnification: 150 x .

appearance as the terminal varicose parts of noradrenergic neurons in peripheral tissues. They have an uneven distribution and are especially concentrated to 4 bilaterally symmetrical areas: (a) a large area i n the preoptic region just below, partlli medial and lateral to and above the anterior commissure; (6) the supraoptic nucey (Fig. 2); (c) the paraventricular nuclei (Fig. 3); and ( d ) an area in the walls of the third ventricle (the periventricular nuclei) (Fig. 4). Fairly dense accumulations are also found in other places, for example in the preoptic region just above the optic nerves, and in a large area just posterior to the paraventricular nuclei. Only a small amount of scattered fibres occurs in the posterior hypothalamus and these fibres are entirely missing in some regions, such as the suprachiasmatic nuclei and optic nerves, chiasma and tracts. The thickness of the fibres varies. Generally their caliber is less than that of norReferences p . 43/44

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Fig. 3. Numerous fluorescent terminals in the paraventricular nucleus. Magnification: 150 x .

adrenergic nerves in peripheral tissues and part of them seems even to be submicroscopic and observable only because of the intense fluorescence of their varicosities. A small population of strikingly thick fibres is present, chiefly in the anterior hypothalamus (Fig. 5). These hypothalamic fibres run to nerve cells which they enclose - and on which they sometimes can also be seen to terminate -in a synaptic arrangement (Fig. 6). The nerve cells on which the fibres are superimposed do not show a fluorescence due to the treatment with formaldehyde, but may contain autofluorescent granules emitting a brown-yellow to red-brown light. Practically none of the intracerebral fluorescent fibres seem to innervate blood vessels. No observable changes of the fluorescent

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Fig. 4. Numerous fluorescent terminals in the periventricular nucleus of the anterior hypothalamus. Magnification: 150 x

.

structures are found after bilateral cervical sympathectomy and, except for the pineal body, fluorescent fibres associated with intracerebral vessels are seldom seen. The fluorescence of the nerve plexuses surrounding e.g. the arteries at the basal surface of the brain as seen in Fig. 2 is, of course, absent after bilateral cervical sympathectomy. The hypothalamic fluorescent fibres appear already at a certain distance from the effector cells; this part of the fibres is usually very short, an exception being the abovementioned thick fibres, which may reach a considerable length. No fluorescence in the preterminal part of the neurons has been observed either in the hypothalamus or in other hitherto investigated brain areas. Under certain experimental conditions (see below) which enhance the amount of monoamines in the preterminals these, however, can be visualized. In the brain of some non-mammalian species, such as the pigeon (Fuxe and Ljunggren, unpublished observations) and in teleosts (Falck and Mecklenburg, unpublished observations) preterminal fibres exist which exhibit a weak but clearly observable fluorescence. There thus seem to be good possibilities for tracing the whole neuron directly in serial sections of different brain regions. References p. 43144

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Convincing evidence exists that the green fluorescent terminals contain a monoamine which is NA, although it cannot be excluded that they contain also D A or that some of them exclusively store D A : (a) the high specificity of the fluorescence method for certain catecholamines and tryptamines has already been discussed ; (6) after administration of reserpine the disappearance and the recovery as a function of

Fig. 5. Thicklfluorescent varicose terminals in the anterior hypothalamus. Magnification: 250 x .

dose and time agree very well with those of the catecholamines (Carlsson et a/., 1957; Brodie, 1958; Brodie et a / . , 1961); (c) the properties of the fluorescent product and the reaction conditions under which it is formed indicate that a primary catecholamine is demonstrated ; (d) of compounds that yield intensely fluorescent condensation products with formaldehyde oiily A, NA, DA and 5-HT have been found in significant amounts in the hypothalamus; (e) when a-methyl-m-tyrosiue is administered in a way that causes little if a n y change in the hypothalamic 5-HT and DA, the green fluorescence is completely abolished except in one area - the median eminence (see below); and ( f ) finally the distribution of NA - but not 5-HT and DA - in the different parts of the hypothalamus (Bertler, 1961) corresponds with that of the green fluorescent fibres. From the foregoing it is evident that the preterminal part of these noradrenergic neurons has a low content of NA as compared with the terminal part. In this respect and in their general appearance, they are quite similar to the peripheral noradrenergic

L O C A L I Z A T I O N O F MONOAMINES

39

nerves. This fact strongly supports the view that NA also serves as a transmitter in the brain. A green to yellow-green fluorescence, which histochemically seems to be derived

Fig. 6 . Fine fluorescent varicose fibres at the lateral border of the paraventricular nucleus. The fibres enclose nerve cells in the nucleus (left) and part of the nerve cells outside the nucleus. The innervated nerve cells are non-fluorescent. Magnification: 250 X .

from a primary catecholamine, develops in the median eminence (Carlsson et al., 1962). Preliminary investigations (Haggendal, unpublished data) on the monoamines in the median eminence suggest a high DA content. After depletion of the hypothalamic N A the fluorescence of the median eminence is unchanged or only slightly decreased, a n observation which also might suggest the presence of DA. The neurohypophysis of the mouse is being studied a t present by the fluorescence technique (Enemar and Falck, unpublished observations). The neurohypophysis consists of the median eminence and the neural lobe. The latter is attached to the caudal part of the eminence via a slender neck, the infundibular stem. The fluorescent structures of the neurohypophysis are strictly confined to the median eminence and to the infundibular stem. Within these areas they are found close to or in the immediate surroundings of the blood vessels. The latter constitute the so-called primary plexus of the hypophysial portal system. In the mouse (as in most mammals) this plexus is distributed over the whole eminence and the infundibular stem. Thus the fluorescent Rrfcrencrs p . 43/44

40

BENGT FALCK

structures are found in that part of the neurohypophysis in which blood vessels are exclusively drained towards the sinus system of the pars distalis of the adenohypophysis via the portal vessels. The neural lobe has always appeared completely dark in the fluorescence microscope. The fluorescent structures of the neurohypophysis can be classified into 3 types: (1) varicose fibres; (2) a diffuse fluorescence; (3) distinct and fairly large droplets. The varicose fibres have the same appearance as the noradrenergic terminals in the hypothalamus. They occur in a relatively small amount and are almost exclusively located close to the capillaries of the primary plexus. In independent investigations Fuxe (unpublished observations) has found that in many mammals (e.g. cat, guinea-pig) the fluorescence in the eminence is confined to varicose terminals associated with the capillaries of the primary plexus. The diffuse fluorescence (type 2 above) is mainly restricted to the outer layer (the zona externa) of the eminence and the infundibular stem. This layer is richly vascularized by the vessels of the primary plexus. Here it consists of a close capillary network adhering to the surface of the organs and giving rise to shallow capillary loops penetrating the tissue of the zona externa. The fluorescent material is concentrated around the vessels and in places it seems to be condensed to form indistinct granules attached to the wall of the capillaries. The cellular localization of this material is not yet established. The diffuse nature of the fluorescence does not necessarily indicate that the amine is not stored intraneuronally, but may well be caused by very closely packed fine axons. The distinct and fairly large droplets (type 3 above) are mainly found around the so-called deep vessels of the eminence and the infundibular stem. These vessels, which are often dilated or sinusoidal in appearance, are chiefly distributed between the ependymal border of the infundibular recess and the fibre layer formed by the tractus hypophyseus. They are drained towards the pars distalis and thus belong to the primary plexus. In the adult mouse these deep vessels are often surrounded by masses of neurosecretory material in the form oflarge spherical or pear-shaped droplets or bodies (Herring bodies). The fluorescent droplets, which are usually smaller, show exactly the same arrangement. In a number of instances 2 successive sections have been compared after which one of them has been treated for demonstration of neurosecretory material and the other prepared for fluorescence microscopy. According to these preliminary investigations the fluorescent droplets are not identical with those stained by chromic haematoxylin or paraldehyde fuchsin. Those parts of the vessels of the primary plexus lacking contact with the tissue of the median eminence or the infundibular stem are not provided with fluorescent material. There is no fluorescence in the tissues contiguous to the portal vessels which run separated from the wall of the eminence. The blood supply of the primary plexus is carried by the infundibular arteries. The latter are distinctly provided with fluorescent varicose nerve fibres which to a certain extent follow the arterial branches on theiraway over the eminence before they are broken up into the primary capillaries. Although the cellular localization of the fluorescence is not completely revealed,

LOCALIZATION OF MONOAMINES

41

its intimate relation to the hypophysial portal system suggests the presence of a monoaminergic mechanism playing a role in the humoral regulation of the pituitary functions. A group of small nerve cells that develops a fairly weak fluorescence is present in the lateral walls of the third ventricle above the median eminence. A stronger green fluorescence develops in the bodies and the first part of the processes of big nerve cells, which form 2 large groups situated laterally to the mammillary region (Fig. 7).

Fig. 7. Green fluorescent nerve cells in the posterior hypothalamus.

These groups are in fact the anterior parts of two elongated masses of fluorescent cells which extend down into the pons. Such nerve cells are also found in circumscribed areas in other regions of the central nervous system, e.g. the medulla oblongata. Varicose fibres with a green-yellow to yellow fluorescence appear in several regions in the hypothalamus, e.g. the suprachiasmatic nucleus, and in the brain stem, as well as in other brain regions, where they enclose non-fluorescent nerve cells synaptically. The appearance of this fluorescence is prevented by reserpine but not by m-tyrosine or a-methyl-m-tyrosine. These fibres are very fine and often just visible. When nialamide is administered, both varicose and smooth fibres, many of which do not show any fluorescence in the normal brain, appear with a fairly strong yellow fluorescence. This treatment also elicits a yellow fluorescence in the perikarya and processes of nerve cells localized to circumscribed areas in the posterior hypothalamus and in the brain stem. The yellow fluorescence also develops upon administration of nialamide after depletion of the monoamines with reserpine. The above data favour the Refeiiwces p. 43/44

42

BENGT FALCK

view that 5-HT is stored in these neurons. It is of interest that not only yellow but also green fluorescent smooth fibres are visible after administration of nialamide. This indicates that M A 0 may be present in the whole monoaminergic neuron. The several fluorescent areas in the hypothalamus exhibit a remarkably different sensitivity to reserpine. In time-dose as well as in dose-response experiments the supraoptic nuclei, for example, were found to be very sensitive, whereas the fluorescence in the paraventricular nuclei requires higher doses or more time to be completely abolished. Most resistant to reserpine are the green fluorescent cell bodies. It is interesting to note that these are the first to recover and they do so within a much shorter time than the terminals after treatment with reserpine. In sections at different levels of the spinal medulla (so far only the mouse and rat have been investigated) no fluorescent nerve cells have been observed. Preliminary observations (Carlsson, Falck, Fuxe and Hillarp, unpublished experiments), however, have demonstrated the presence of many fine varicose fibres in the gray matter exhibiting a fluorescence characteristic of primary catecholamines. These fibres are synaptically associated with a large number of non-fluorescent nerve cells. The majority of the large motor cells in the anterior horns do not seem to have a contact with monoaminergic fibres; an intimate connection has been observed between only some of the large motor cells and green fluorescent terminals, but whether this may be considered as a true synapse has not as yet been established. In the autonomic intermediolateral column of the gray matter there occur numerous yellow and green fluorescent terminals, some of which are barely visible, which superimpose on the nerve cells in synaptic fashion. The terminals originate from bundles of fibres which apparently descend in the lateral funiculus. These fibres show a weak fluorescence and can only to a limited extent be detected in the normal medulla, but after injection of nialamide they display a relatively strong fluorescence and considerably more yellow fibres appear than are seen in normal animals. The increase in yellow fluorescence is very pronounced and seems to agree well with the increase of spinal 5-HT that has been found in spectrophotofluorimetric determinations after administration of nialamide: from 0.43 pg/g in normal animals to 2.9 pg/g in nialamide-treated animals (Carlsson, personal communication). The results thus seem to confirm the findings of the investigations on the hypothalamus and brain stem that the intraneuronal content of 5-HT is low normally, especially in the preterminals, and further that M A 0 is probably present in the whole neuron. After transection at the level of the second thoracic segment no fluorescent fibres and practically no NA or 5-HT (Carlsson, this symposium) can be demonstrated below the lesion, which strongly supports the view that descending noradrenergic and serotonergic pathways are present in the spinal cord. Dopamine has been detected histochemically in the caudate nucleus but the cellular localization has not as yet been positively established. The fluorescence is on the whole rather diffuse, except in some areas, such as its very medial part, where extremely fine green fluorescent terminals are seen to surround nerve cells. No fluorescent nerve cells are present in the nucleus. It is quite improbable that the diffuse fluorescence depends on a diffuse distribution of DA, or that the fluorescence accumulates in the

LOCALIZATION OF MONOAMINES

43

glia throughout the nucleus. It is more probable that the amine is mainly localized to submicroscopic structures belonging to, for instance, the neuropil. Finally it should be mentioned that between the inner plexiform layer and the inner nuclear layer in the retina of the rat there occur fluorescent cell bodies and varicose terminals which are accumulated around non-fluorescent cell bodies (Malmfors, 1963). The fluorescence disappears after treatment with reserpine or m-tyrosine, but not after opticotomy or bilateral excision of the cervical sympathetic chain. The characteristics of the fluorescence reaction indicate the presence of a primary catecholamine. The findings seem to demonstrate the presence of intraretinal monoaminergic neurons forming synapses with nerve cells and it must perhaps be taken into consideration that the eye symptoms such as photophobia which develop on administration of reserpine may have a retinal component.

ACKNOWLEDGEMENTS

This work has been supported by grants from the United States Public Health Service ( N B-02854) ; the Swedish Medical Research Council ; AB Ferrosan, Malmo, Sweden and the Directorate of Life Sciences, AFOSR, Office of Aerospace Research, United States Air Force, monitored by the European Office, Office of Aerospace Research (Grant No. AF-EOAR-61-44). For generous supplies of drugs I am indebted to the Swedish Ciba Ltd. (serpasil) and to the Swedish Pfizer Ltd. (niamide). Fig. I is from B. Falck: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method, Acta physiol. scand., 1962, Suppl. 197 and Figs. 2-6 are from A. Carlsson, B. Falck and N.-A. Hillarp: Cellular localization of brain monoamines, Acta physiol. scand., 1962, Suppl. 196. These figures are reproduced with the kind permission of the Editorial Office, Acta physiologica scandinavica.

REFERENCES BERTLER, A., (1961); Occurrence and localization of catechol amines in the human brain. Acra physiol. scand., 51, 97-107. BERTLER, A,,FALCK,B., HILLARP,N.-A., ROSENGREN, E., AND TORP,A,, (1959); Dopamine and chromaffin cells. Acta physiol. scand., 47, 251-258. BERTLER, A., FALCK,B., AND OWMAN, CH., (1963); Cellular localization of 5-hydroxytryptamine in the mammalian pineal gland. To be published. BERTLER, A., AND ROSENGREN, E., (1959); Occurrence and distribution of dopamine in brain and other tissues. Experientia (Basel), 15, 10. BLOOM,G., AND KELLY,J. W., (1960); The copper phtalocyanin dye “Astrablau” and its staining properties, especially the staining of mast cells. Hisrochenlie, 2, 48-57. BRODIE,B. B., (1958); Storage and release of 5-hydroxytryptamine. 5-Hydroxytryptarnine. G . P. Lewis, Editor. London, Pergamon (p. 64-83). BRODIE,B. B., MAICKEL, R. P., AND WESTERMAN, E. O., (1961); Action of reserpine on pituitaryadrenocortical system through possible action on hypothalamus. Regional Neurocheniistry. S. S. Kety and J. Elkes, Editors. Oxford, Pergamon (p. 351-361). CARLSSON, A,, FALCK, B., A N D HILLARP, N.-A., ( I 962); Cellular localization of brain monoamines. Acta physiol. .scad., 56, Suppl. 196, 1-27. CARLSSON, A,, ROSENGREN, E., BERTLER, A,, A N D NILSSON,J., (1957); Effect of reserpine on the metabolism of catcchol amincs.Psychotropic Drugs. S. Garattini and V. Ghetti, Editors. Amsterdam, Elsevier (p. 363-372).

44

BENGT FALCK

CORRODI,H., FALCK,B., A N D HILLARP,N.-A., (1962); Sensitive fluorescence methods for histochemical demonstration of catecholamines at the cellular level. Report at the Meeting for Scandinavian Pharmacologists, August 1962 (Giiteborg, Sweden). COUPLAND, R. E., AND HEATH, J. D., (1961); Chromaffin cells, mast cells and melanin. 11. J . Endocr., 22,11-76. DAHL,E., FALCK,B., LINDQVIST, M., AND VON MECKLENBURG, C . , (1962); Monoamines in mollusc neurons. Kungliga Fysiografiska Sallskapets i Lirnd F6rhancllingar, 32,89-92. EHRL~N I.,, (1948); Fluorimetric determination of adrenaline. TI. Farni. Rev. (Stockh.), 47,242. FALCK, B.,(1962); Observations on the possib es of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand., 56,Suppl. 197, 1-25. J., AND OWMAN, CH., (1963); The localization of adrenaline in adrenergic FALCK,B., HAGGENDAL, nerves in the frog. Quart. J . exp. Physiol., 48, 253-257. B., (1963a); Evidence for the presence of biogenic amines in pancreatic FALCK,B., AND HELLMAN, islets. Experientia (Basel), 19, 139-140. B., (1964); A fluorescent reaction for monoamines in the insulin producing FALCK, B., A N D HELLMAN, cells of the guinea-pig. Acta Enhcr., 45, 133-1 38. FALCK,B., HILLARP, N.-A., THEME,G . , A N D TORP,A., (1962); Fluorescence of catecholamines and related compounds condensed with formaldehyde. J . Histochem. Cytochem., 10, 348-354. FALCK,B., HILLAKP, N.-A., AND TORP,A,, (1959a); A new type of chromaffin cells, probably storing dopamine. Nature (Lond.), 183,261-268. N.+f., AND TORP,A., (1959b); Some observations on the histology and histoFALCK,B., HILLARP, chemistry of the chroniaffin cells probably storing dopamine. J . Histocheni. Cytocheni., 7,323-328. R. F., (1960); Adrenergic Mechanisnis. J. R. Vane, G . E. Wolstenholme and M. FURCHGOTT, O’Connor, Editors. Ciba Foundation Symposium. London, Churchill (p. 353). N.-A., (1959); The construction and functional organization of the autonomic innervation HILLARP, apparatus. Acta physiol. scand., 46,Suppl. 157, 1-38. J . A., (1960); The development, topographical relation and innervation of the epiphysis KAPPERS, cerebri in the albino rat. Z. Zellforsch., 52, 163-215. T., (1963); Evidence of adrenergic neurons with synaptic terminals in the retina of rats MALMFORS, demonstrated with fluorescence and electron microscopy. Acta physiol. scand., 58, 99-1 00. REHN,N. O., (1958); Effect of decentralization on the content of catecholamines in the spleen and kidney of the cat. Acta physiol. scanrl., 42,309-312. SCHUMANN, H. J., (1956); Nachweis von Oxytyramin (Dopamin) in sympatischen Nerven und Ganglien. Naunyn-Schiiiiedeberg’s Arch. exp. Path. Pharrnak., 227,566-573, SCHUMANN, H. J., (1958); Uber den Hydroxytyramin- und Noradrenalingehalt der Lunge. NaunynSchmiedeherg’s Arch. exp. Path. Pharmak., 234, 262-290. SCHUMANN, H. J., (1959); uber den Hydroxytyramingehalt der Organe. Naunyn-Schmiedeberg’s Arch. exp. Path. Pharnrak., 236,474-482. SIDMAN,R. L., PERKINS,M., AND WEINER,N., (1962); Noradrenaline and adrenaline content of adipose tissues. Nature (Lond.), 193,36-37. VON EULER,U. S., (1956); Noradrenaline. Springfield, Ill., Ch. C . Thomas. VONEULER,U. S., (1960); Autonomic neuroeffector transmission. Handbook ofphysiology. I. Neurophysiology. J. Field, Editor. Baltimore, Waverly (p. 21 5-237). F., (1957); Dopamine in mammalian lung and spleen. Acta VON EULER,U. S., AND LISHAJKO, physiol. pharniacol. neerl., 6,295-303. WEINER, N., PERKINS, M., AND SIDMAN, R. L., (1962); Effect of reserpine on noradrenaline content of innervated and denervated brown adipose tissue of the rat. Nature ( L o n d ) , 193,137-138. M., (1960); The quantitative distribution of 5-hydroxytryptamine in WELSH,J. H., AND MOORHEAD, the invertebrates, especially in their neurons system. J . Neurochem., 6, 146-169. K. C., AND AXELROD, J., (1962); Localizing tritiated WOLFE,D. E., POTTER,L. T., RICHARDSON, norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138, 440442.

45

Accumulation of Exogenous Monoamines in Brain in vivo and its Alteration by Drugs A . P L E T S C H E R , K . F. G E Y

AND

E. K U N Z

Medical Research Department, F. Hoffiiann-La Roche and Co. Ltd., Bade (Switzerland)

Storing and metabolism of exogenous (e.g. 14C-or 3H-labelled) catecholamines in the intact animal have mainly been investigated in peripheral organs like heart, sympathetic nerve endings, etc. (Axelrod eta/., 1961a, b ; Hertting et a/., 1961a; Potter et al., 1962). In experiments with brain, precursors, e.g. amino acids like 3,4-dihydroxyphenylalanine (dopa), have to be used, because the amines, except in a few areas, hardly penetrate the blood-brain barrier. Amino acids show, however, a more complex metabolism than the amines (e.g. decarboxylation, transamination), which makes the interpretation of the experimental results relatively difficult. Nevertheless, the study of amine storing and amine metabolism by using precursors instead of amines might have some theoretical advantage. Thus, the amines derived from injected precursors are probably formed at the same sites as the endogenous amines, whereas injected amines might also enter tissue compartments normally not containing endogenous amines (e.g. by adsorption). In the work to which the present paper refers the fate of ~ ~ - 2 - l ~ C - d in o pthe a brain was investigated by measuring the total radioactivity in the fractions of the amino acids, the amines and the metabolic end-products (mainly phenolcarboxylic acids). The following measurements were carried out : (a) 14C-Content in the 3 fractions of whole brain as compared to heart and blood of normal rats. (b) 14C-Content in the fractions of various brain regions and blood of normal rabbits. (c) Effect of various drugs on the 3 fractions in rat brain. ( d ) Effect of hypothermia on amine penetration in rat brain. In part of the experiments, tryptamine instead of 1%-dopa was used. M E T H 0D S

After a fasting period of 16 h, rabbits (2-3 kg) as well as Wistar rats (70-130 g) received 1.5 and 20 mg/kg ~ ~ - 2 - l ~ C - d orespectively, pa, at various intervals before distortion of the neck (rabbits) or decapitation (rats). HC104 extracts from blood and tissue were incubated under Na a t 37” for 2 h with “glusulase” (enzyme mixture References p . 52

46

A. P L E T S C H E R , K. F. G E Y A N D E. K U N Z

containing glucuronidase and sulphatase; Endolab). The extract was fractionated on 2 microcolumns of Dowex-50-X4 (pH = 6.5 and H f form) into 3 fractions: amino acids, amines and metabolic end-products (mainly phenolcarboxylic acids). The total radioactivity of the whole extract (overall radioactivity) as well as of each fraction was measured in a Packard liquid scintillation spectrometer and expressed per g tissue or ml blood respectively in per cent of the radioactivity injected per g of body weight. 2-14C-Dopa, 2-14C-3-hydroxytyramine (dopamine), 2-14C-norepinephrine, and 1-14C-phenolcarboxylic acids added to the deproteinized HC104 extract could be recovered to 95-100%. Aliquots of the fractions of amino acids and amines were further analysed by paper chromatography. The radioactivity of the paper strips cut in small sections was counted in a Packard liquid scintillation spectrometer [Gey and Pletscher, 1963). TABLE I AVERAGE COMPOSITION O F THE AMINE

6 0 min 20mg/kg D L - 2 1 4 C - D O P A ( 2 ,fLC/,UllloleS) Separation of the amines by paper chromatography in n-butanol/0.5 N HCI (24 h; 22").

FRACTION OF NORMAL R A T BRAIN AFTER S.C. I N J E C T I O N O F

Amine

Dopamine 3-Methoxytyramine Norepinephrine

"/, of total amines 47 15

25

The analysis of paper chromatograms showed that the amino acid fraction consisted mainly of 2-1%-dopa. The composition of the amine fraction is shown in Table I. RESULTS A N D DISCUSSION

( I ) In normal rats the l4C-dopa-induced increase of the overall radioactivity as well as of the radioactivity in the 3 fractions is much more marked in the heart and blood than in the brain (Fig. 1). According to these results, the brain probably takes up less 14C-dopa than the heart. This does, however, not explain entirely why the radioactivity in the amine fraction of the brain shows only very little increase as compared to the heart. It is conceivable that in the brain the amines formed from l4C-dopa are stored to a relatively small extent, but are rather quickly metabolized, which then results in a considerable increase of the phenolcarboxylic acids. The heart, however, possibly stores a greater amount of the newly formed amines than the brain or takes even up a part of the amines from the blood. (2) Various regions ?f the brain of the normal rabbit differ in their pattern of radioactivity after i.v. injection of 14C-dopa. The radioactivity of the amines and phenol-

47

EXOGENOUS MONOAMINES

carboxylic acids is higher in the medulla oblongata, mesencephalon, diencephalon, and caudate nucleus than in the cerebellum and cerebrum. The activity of the amino acid fraction, however, rises more markedly in the cerebellum and cerebrum than in the other regions of the brain. The activity of the amines is highest in the caudate

'1.

Brain 10

wI

0

;

i 'h

3

30

Blood

'\

Heart 20

iQ

p :+-. ..y

;

'I

1

-a...

I-----.----.-.-.- ..-.-...__ .-._ ~ - .

0

0

I:?

1

3

Fig. 1. Increase of radioactivity of various tissue fractions in brain, heart and blood after administration of ~ ~ - 2 - l ~ C - d oOrdinate: pa. Total activity per g of tissue in per cent of administered radioactivity per g of body weight. Abscissa: Time in h after i.p. administration of 10 mg/kg ~ ~ - 2 - l ~ C - d o p a (1.5 pC/,umoles). - - - - Overall radioactivity (sum of the 3 fractions); -__Fraction of amino acids; . . . . . . Fraction of amines; -. -. -. -. -. Fraction of phenolcarboxylic acids. Each point represents an average of 2-3 experiments with standard error.

.

nucleus, whereas that of the phenolcarboxylic acids increases most in the di- and mesencephalon (Pletscher and Gey, 1962) (Fig. 2). The differences in the various brain regions cannot be the consequence of unequal penetration of 14C-dopa through the blood-brain barrier, because, after 5 min, the sum of the radioactivity of the 3 fractions (overall radioactivity) is similar in all brain areas. These differences might, however, be rather due to variations in the metabolism of dopa and in the storing of the amines formed from "T-dopa. (3) Various drugs influence the radioactivity of the 3 fractions in a different way (Table 11). References p. 52

48

A. P L E T S C H E R , K. F. G E Y A N D E. K U N Z

T

.&:

CAUDATE NUCLEUS

DIENCEPHALON

& ..a , 16

16

Fig. 2. Characteristic differences of ~ ~ - 2 - l ~ C - d metabolism opa in some brain areas and in blood of the rabbit. Ordinate: Radioactivity per g of tissue and ml blood resp. in per cent of the total ~ ~ - 2 - I ~ C - d o p a administered per g of body weight. Abscissa: Time in h after i.v. injection of 1.5 mg/kg ~ ~ - 2 - l ~ C - d o p a (2.5-3.2 pC/p moles). - - - - - - Overall radioactivity (sum of the 3 fractions); Fraction of amino acids;. . . . . . . Fraction of amines; -.-.-.-.-. Fraction of phenolcarboxylic acids and other oxidation products. The points with vertical lines represent averages and standard error of 3-6 experiments. The points without vertical lines are single values (Pletscher and Gey, 1962).

( a ) Monoamine releasers (e.g. the benzoquinolizine derivative Ro 4-1284*, Pletscher et a]., 1962) diminish the radioactivity in the amine fraction, but increase that of the phenolcarboxylic acids. This effect might be explained by a decrease in the storing capacity in the tissue for monoamines. Therefore, the 14C-amines formed from 1%-dopa accumulate less than in controls, but undergo oxidative deamination and further metabolism, e.g. to the phenolcarboxylic acids, which show an increase. (b) The M A 0 inhibitor iproniazid markedly raises the content of exogenous amines and in consequence decreases the level of their metabolic end-products. The increase of the radioactivity in the amine fraction and its decrease in the fraction of phenolcarboxylic acids seem to be due to M A 0 inhibition or/and enhanced monoamine storing. (c) The neuroleptics chlorpromazine and chlorprothixene, though not influencing the total content of endogenous amines in the brain, decrease the radioactivity some-

* R o 4-1284 = 2-hydroxy-2-ethyl-3-isobutyl-9,IO-dirnethoxy-l, 2, 3, 4, 6, 7-hexahydro-11 bHbenzo [a] quinolizine.

49

EXOGENOUS MONOAMINES

T A B L E I1 INFLUENCE OFVARIOUS D R U G S O N THE D L - 2 - ' 4 C - D O P A - I N D U C E D I N C R E A S E OFTHE OVERALL

R A D I O A C T I V I T Y A S W E L L A S OF T H E R A D I O A C T I V I T Y O F T H E

3

FRACTIONS (AMINO ACIDS,

AMINES, PHENOLCARBOXYLIC ACIDS) IN RAT BRAIN

The absolute overall activity per g of fresh brain is indicated in per cent of the administered total radioactivity per gof body weight. Eachfigure represents an average of 3 4 experiments (overall activity: 19 experiments) with standard error. 1.p. injection of 10 mg/kg ~ ~ - 2 - ' ~ C - d o(1.5 p a pC/pmoles) 60 min before decapitation and 60 min after the drugs S.C.

Drug

of

Controls RO 4- 1284 lproniazid Chlorpromazine Chlorprothixene Imipramine Amitriptyline

% of overall activity

Overall activity

7.35 5 0 . 0 2 7.62 i 0.39 8.96 10.69 5.95 & 0.47 7.08 & 0.68 6.34 10.43 6.44 1.02

"/, controls

100 f I 104 & 5

122 19 81 + 6 96 & 9 86 16 88 14

Amino acids

Amines

Phenolcarbonic acids

51 1 3 46 & 5 33 13 43 9 40 t 3 51 1 9 51 1 9

6.5 & 0.0 2.0 & 0.0 37.5 t 6.0 4.5 0.05 5.0 0.05 5.5 t 0.05 5.5 0.05

41 1 2 54 11 29 & 1 56 & 5 53 * 6 43 9 43 17

*

*+ *

*

what in the amine fraction and markedly increase that in the fraction of phenolcarboxylic acid. Imipramine and amitriptyline, on the other hand, have no marked effect on the distribution of the radioactivity in the 3 fractions. Chlorpromazine and chlorprothixene do not seem to decrease the storing capacity for endogenous monoamines, since (unlike reserpine and benzoquinolizine derivatives) they are unable to diminish endogenous 5-hydroxytryptamine (5HT) and catecholamines in the brain (Gey and Pletscher, 1961). Their action on 1%-amines and 14C-phenolcarboxylicacids might, however, be explained by a decreased penetration of the amines to the storing sites. In consequence, the accumulation of 14C-amines would be diminished, but the content of phenolcarboxylic acids increased. Interference of chlorpromazine with the permeation of dopa into the brain as well as with that of catecholamines and 5HT into peripheral tissues or brain slices has been shown by T A B L E I11 EFFECT OF C H L O R P R O M A Z I N E O N T H E T R Y P T A M I N E I N C R E A S E OF B R A I N , H E A R T A N D B L O O D D U E T O INJECTION OF T R Y P T A M I N E I N R A T S

15 mg/kg tryptamine were injected i.p. 1 h after 20 mg/kg chlorpromazine S.C. Decapitation 0.5 h after tryptamine. All the animals had been treated with 155 mg/kg iproniazid phosphate i.p. 16 h prior to tryptamine. Each figure represents an average of I I experiments with standard error.

Brain Heart Blood

Controls

Chlorpromazine

P

5.01 & 0.17 11.57 10.63 2.70 31 0.21

1.22 10.07* 10.98E! 0.68 2.69 & 0.1 3

<0.01 >0.1 >0.1 -

*

Corrected figures; in the brain higher doses of chlorpromazine (10-20 mg/kg) interfere somewhat with the fluorescence of tryptamine (Pletscher et al., 1963). Rejerences p . 52

50

A. PLETSCHER, K . F. G E Y A N D E. K U N Z

Axelrod et al., 1961a, b, Dengler et al., 1961, Gey and Pletscher, 1961, 1962, Hertting et al., 1961b, Long and Lessin, 1962, and Stacey, 1961. Recently, it could also be demonstrated that the uptake of tryptamine by brain of intact animals was markedly decreased by chlorpromazine and chlorprothixene, whereas the drugs had no effect on the uptake of tryptamine by the heart (Pletscher et al., 1963) (Table 111). It has to be considered whether the effect of chlorpromazine and chlorprothixene on the permeation of amines into and within the brain is due to hypothermia caused by these drugs when given to animals kept at room temperature (18-20"). Hypothermia might, for instance, inhibit transport mechanisms. This possibility was investigated with rats kept at elevated environmental temperature (3 1-35'), which prevents chlorpromazine from inducing hypothermia. Under these conditions (i.e. in normothermic animals) chlorpromazine caused only a weak increase of the radioactivity in the fraction of phenolcarboxylic acids (Table IV). This demonstrates that T A B L E IV EFFEC T O F C H L O R P R O M A Z I N E O N T H E T O T A L R A D I O A C T I V I T Y O F A M I N O A C I D S , A M I N E S A N D M ETABOLIC E N D - P R O D U C T S I N R A T B R A I N 6 0 Illin AF T E R D L - 2 - ' 4 c - D O P A Chlorpromazine (20 rng/kg i.p.)was administered 30 min prior to 20 rng/kg l*C-dopa (2pC/,umoles) S.C.

Experimental

conditions Absolute (body f e ~ ") w ~( N O . of expts.)

Controls (37.0f 0.1)

%, of overall activity

Overall activity

9.1

+ 0.2

'% of cotrirols

Amino acids

100 I 2

50

36+ I'

7fl

56 t 3 *

+2

7+1

46 1 2

10+

I

Phenolcarbonic acids

41 1 2

(17)

Chlorpromazine hypothermic (31.040.4)

I I .6 i 0.5 (13)

128

Chlorpromazine normothermic (37.6 & 0.3)

13.4 f 0.45 (4)

147 F 5*

*

t2

Amines

6*

47

p <0.01 in comparison to controls.

the action of chlorpromazine on amine permeation is probably in part due to hypothermia. Experiments with tryptamine suggest that the effect of chlorpromazine on the permeation of this amine is also at least in part due to hypothermia. Thus, the chlorpromazine-induced decrease of tryptamine penetration into the brain could be counteracted if body temperature was kept normal. Furthermore, chlorisondamine (EcolidB), which causes a similar degree of hypothermia as chlorpromazine, but probably enters the brain only to a small extent, also decreased the tryptamine penetration into the brain (Pletscher et al., 1963) (Table V). It remains to be elucidated whether the chlorpromazine-induced decrease of the tryptamine uptake by brain is exclusively due to hypothermia or whether other mechanisms (e.g. a direct effect of the drug on biological membranes) are also involved.

51

EXOGENOUS MONOAMINES

TABLE V E F F E C T O F H Y P O T H E R M I A I N T H E D R U G - I N D U C E D D E C R E A S E OF TRYPTAMINE UPTAKE OF RAT BRAIN

10 mg/kg chlorpromazine or 25 mg/kg chlorisondamine were administered S.C. 2 h before 15 mg/kg tryptamine i.p. Decapitation 0.5 h after tryptamine. All the animals were pretreated with 155 mg/kg iproniazid phosphate i.p. 14 h prior to tryptamine. Measurement of body temperature in the rectum with a thermocouple immediately before tryptamine injection. Each figure represents an average of 9-12 experiments with standard error.

Environmental temperature

Drug

("I ~~

Controls Controls Chlorpramazine Chlorpromdzine Chlorisondamine

*

Body temperature (")

~

18-20 30-3 I 18-20 30-3 1 18-20

37.3 t 0.2 37.7 f 0.1 30.7 i 0.4 37.9 0.1 31.1 i 0.2

*

Tryptamine content ( %)

* *

100 9 107 i 3 25 2* 113 k 5* 44 2

+

Corrected figures (see Table 111).

SUMMARY A N D CONCLUSIONS

After injection of 14C-dopa, the radioactivity of the fractions of amino acids, amines and metabolic end-products (mainly phenolcarboxylic acids) was measured in the brain. Furthermore, the penetration of tryptamine into the brain was investigated. The following results were obtained: (I) In the whole brain of rats the increase of all 3 fractions and especially of the amines is much less pronounced than in heart and blood. (2) I n various regions of rabbit brain the sum of radioactivity of the 3 fractions is about the same shortly after 14C-dopa injection; the 3 fractions behave, however, differently. The caudate nucleus as well as the mes- and diencephalon, for instance, show a marked rise of the amines and the phenolcarboxylic acids, respectively, whereas this is not the case in the cerebellum and the cerebrum. (3) The monoamine oxidase inhibitor iproniazid increases the radioactivity of the amine fractions and decreases that of the phenolcarboxylic acids, whereas the monoamine releaser Ro 4-1284 (benzoquinolizine derivative) and the neuroleptics chlorpromazine and chlorprothixene decrease the amines and increase the phenolcarboxylic acids. Chlorpromazine also diminishes the penetration of tryptamine into the brain. ( 4 ) The effects of chlorpromazine can partly be reversed if hypothermia is prevented by increase of the environmental temperature. Hypothermia induced by chlorisondamine also decreases tryptamine penetration into the brain. In conclusion, the metabolism of dopa and the storing of monoamiiies seem to differ in various organs and brain regions. Furthermore, the effect of monoamine oxidase inhibitors and monoamine releasers can probably be explained by increased and decreased monoamine storing respectively, whereas chlorpromazine and chlorReferences p . 52

52

A. PLETSCHER, K. F. GEY A N D E. K U N Z

prothixene might interfere with amine permeation into storing organelles. The latter effect seems to be connected with hypothermia. REFERENCES

AXELROD, J., HERTTING, G., AND PATRICK, R. W., (1961a); Inhibition of H3-norepinephrine release by monoarnine oxidase inhibitors. J . Pharmacol. exp. Ther., 134, 325-328. AXELROD, J., WHITBY,L. G., AND HERTTING,G., (1961 b); Effect of psychotropic drugs on the uptake of H3-norepinephrine by tissues. Science, 133, 383-384. DENGLER, H. J., SPIEGEL, H. E., AND TITUS,E., (1961); Effects of drugs on uptake of isotopic norepinephrine by cat tissues. Nature (Land.), 191, 816-817. GEY, K. F., AND PLETSCHER, A., (1961); Influence of chlorpromazine and chlorprothixene on the cerebral metabolism of 5-hydroxytryptamine, norepinephrine and dopamine. J . Pharmacol. exp. Ther., 133, 18-24. GEY,K . F., AND PLETSCHER, A , , (1962); Interference of chlorpromazine with the metabolism of aromatic amino acids in rat brain. Nature (Land.), 194, 387-389. GEY,K. F., A ND PLETSCHER, A., (1963); In preparation. HERTTING, G., AXELROD, J . , KOPIN, I. J., AND WHITBY, L. G., (1961a); Lack Of uptake Of catecholamines after chronic denervation of sympathetic nerves. Nature (Land.), 189, 66. HERTTING, G., AXELROD, J., AND WHITBY,L. G., (I961b); Effect of drugs on the uptake and metabolism of H3-norepinephrine. J . Pharmacol. exp. Ther., 134, 146-1 53. LONG,R. F., AND LESSIN,A . W., (1962); Inhibition of 5-hydroxytryptamine uptake by platelets it1 vitro and in vivo. Biochem. J., 82, 4 ~ - 5 ~ , PLETSCHER, A., AND GEY,K. F., (1962); Topographical differences in the cerebral metabolism of ~ ~ - 2 - ’ ~ C - 3 , 4hydroxyphenylalanine. -di Experientiu (Basel), 18, 5 I 2-5 16. PLETSCHER, A,, BROSSI,A,, AND GEY, K. F., (1962); Benzoquinolizine derivatives: a new class of monamine decreasing drugs with psychotropic action. I n f . Rev. Neurohiol., 4, 275-306. PLETSCHER, A., KUNZ, E., A N D GEY,K. F., (1963); In preparation. POTTER, L. T., AXELROD, J., AND KOPIN,I. J., (1962); Differential binding and release of norepinephrine and tachyphylaxis. Biochern. Phartnacol., 11, 254-256. STACEY, R . S., (1961); Uptake of 5-hydroxytryptarnine by platelets. Brit. J . Phurmacol., 16, 284-295.

53

The Effect of Central Nervous System Lesions in the Rat on Brain Serotonin A L F R E D H E L L E R , JOHN A . H A R V E Y

AND

ROBERT Y. MOORE

Departments of Pharmacology, Psychology and Anatomy, The University of Chicago, Chicago, Ill. (U.S.A.)

Studies on the distribution of brain serotonin have demonstrated that this biogenic amine has a non-uniform distribution within the central nervous system (Bogdanski, Weissbach and Udenfriend, 1957; Kuntzman et al., 1961 ; Paasonen, Mac Lean and Giarman, 1957). In addition, many workers have shown that those areas of brain, generally containing high levels of serotonin, are important in autonomic function. This localization of serotonin to areas of the brain important in autonomic function has indeed been given as part of the evidence for ascribing a central neurohumoral role to this amine (Costa et al., 1962). The relationship of the physiological and behavioral effects of ablation or stimulation of these autonomic structures to changes in brain serotonin has, however, not been demonstrated. To investigate this problem we have studied the effects of central nervous system lesions on total brain serotonin levels (Heller, Harvey and Moore, 1962). Bilateral electrolytic lesions were placed in the brains of 83 to 85-day-old male albino rats. Serotonin brain levels were determined 35 days later by the spectrofluorimetric method of Bogdanski et al. (1956). As can be seen in Table 1, destruction of the medial forebrain bundle within the lateral hypothalamus produced a 36 % fall in brain serotonin levels as compared with normal controls. Destruction of the septa1 region, dorsomedial midbrain tegnientum, and ventral midbrain tegmentum (areas contributing fibres to this region of the medial forebrain bundle) produced decreases of 12-15%. Destruction of areas not related to this region of the medial forebrain bundle or destruction of other fibre tracts did not result in a significant fall in brain serotonin levels. It would appear from these findings that the fall in brain serotonin is a result of damage to one particular system of fibres, the fibres of the medial forebrain bundle within the lateral hypothalamus. In a subsequent study we have shown that the time-course of serotonin fall following bilateral medial forebrain bundle lesions is in good agreement with the known time-course for degeneration of such small central nervous system fibres. In addition, it was found that the effect of a unilateral lesion on total brain serotonin level was approximately half of that produced by a bilateral lesion. When the brains of unilateral lesioned rats were sectioned longitudinally into Referenrcs p. 55

54

A. H E L L E R

et al.

TABLE T EFFECT O F C E N T R A L N E R V O U S SYSTEM L E S I O N S I N T H E MALE A L B I N O RAT ON B R A I N SEROTONIN LEVELS

Expevinient a/ group

Normal control Sham-operated Lateral hypothalamus (medial forebrain bundle) Ventral midbrain tegmentuni Dorsomedial midbrain tegmentum Septa1 region Medial hypothalamus Habenula Hippocampus Cortex Caudate Lateral pontine tegrnentum

( H E L L E Ret al., 1962)

N

Mean brain serotonin level (pglg)

Change f r o m normal ( %)

8 7

0.59 0.61

-i-3

8 8

0.38 0.50 0.51 0.52 0.55 0.55 0.56 0.58 0.59 0.63

-36 -15

P*

- 0.005 0.01

-14

-12

*

0.01 0.01

-7 -7 -5 -2 0 t7

* P values were obtained from a 2 x 2 contingency table using the pgglg value obtained from each animal. P value of 0.01 indicates only one overlap between the experimental and control group. P value of 0.005 indicates no overlap. two hemi-brains and assayed separately, it was found that the fall in brain serotonin was entirely restricted to the hemi-brain containing the lesion. No fall in serotonin could be observed in the hemi-brain contralateral to the lesion. The percentage decrease of brain serotonin on the lesioned side of the brain was, therefore, the same as that seen with bilateral lesions (Harvey, Heller and Moore, 1963). These findings are of interest since Guillery (1956) has demonstrated that the majority of medial forebrain bundle fibres are uncrossed and if the fall is secondary to damage to such fibres the effect should be restricted to the side of lesion. We have interpreted the results described above to indicate that the fall in brain serotonin is related to the degeneration of medial forebrain bundle fibres which normally produce serotonin. These results are of course similar to the reported fall in acetylcholine in the superior cervical ganglia following section of its preganglionic fibres (Brown and Feldberg, 1936; MacIntosh, 1938), and the fall in tissue catecholamines seen following section of adrenergic nerves (Cannon and Lissak, 1939; Goodall, 1951 ; Von Euler and Purkhold, 1951). This indication of a possible neural origin for at least part of the brain serotonin is of interest in view of the suggestion made by a number of authors that serotonin may have a neurohunioral function in the brain (Amin, Crawford and Gaddum, 1954; Woolley and Shaw, 1954; Brodie and Shore, 1957). I n this regard, Brodie and Shore (1957) have speculated on the existence of “serotonergic” fibres in the central nervous system. Our findings, showing that central nervous system lesions lead to a fall in brain serotonin, may represent evidence for serotonergic fibres within the medial forebrain bundle. It is clear, however, that

RRAlN SEROTONIN LEVELS

55

the demonstration of a neural origin for serotonin does not provide any direct evidence for ascribing to this amine a function in either axonal or synaptic transmission.

SUMMARY

Bilateral electrolytic brain lesions in the rat destroying either the medial forebrain bundle within the lateral hypothalamus or areas contributing fibres to this region of the medial forebrain bundle produced a significant fall in total brain serotonin levels. Unilateral medial forebrain bundle lesions also produced a fall in brain serotonin which was restricted to the side of the brain containing the lesion. In addition, the time-course of serotonin fall following a bilateral medial forebrain lesion was found to be in good agreement with the known time-course for degeneration of such small central nervous system fibres. These results indicate the presence of serotonin producing fibres within the medial forebrain bundle of the rat. REFERENCES AMIN,A. H., CRAWFORD, T. B. B., A N D GADDUM, J. H., (1954); The distribution of substance P and 5-hydroxytryptamine in the central nervous system of the dog. J . Physiol. (Lond.), 126, 596-618. BOGDANSKI, D. F., PLETSCHER, A., BRODIE,B. B., AND UDENFRIEND, S., (1956); Tdentification and assay of serotonin in brain. J. Pharmacol. exp. Ther., 117, 82-88. BOGDANSKI, D. F., WEISSBACH, H., AND UDENFRIEND, S., (1957); The distribution of serotonin, 5-hydroxytryptophane decarboxylase and monoamine oxidase in brain. J . Neurochem., 1, 272-278. BRODIE,B. B., AND SHORE,P. A., (1957); A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N . Y. Acad. Sci., 66, 631-641. BROWN,G . L., AND FELDBERG, W., (1936); The action of potassium on the superior cervical ganglion of the cat. J . Physiol. (Lond.), 86, 290-305. CANNON, W. B., AND LISSAK,K., (1939); Evidence for adrenaline in adrenergic neurones. Amer. J . Physiol., 125, 765-777. COSTA,E., GESSA,G. L., HIRSCI-I, C., KUNTZMAN, R., A N D BRODIE,B. B., (1962); On current status of serotonin as a brain neurohormone and on action of reserpinelike drugs. Ann. N . Y . Acad. Sci., 96, 118-131. GOODALL, McC., (1951); Studies of adrenaline and noradrenaline in mammalian heart and SUprarenals. Acfa physiol. scand., 24, Suppl. 85. GUILLERY, R. W., (1956); Degeneration in the hypothalamic connexions of the albino rat. J . Anaf. (Lond.), 90, 350. HARVEY, J. A., HELLER, A., AND MOORE,R. Y., (1963); The effect of unilateral and bilateral medial forebrain bundle lesions on brain serotonin. J . Pharmacol. exp. Ther., 140, 103. HELLER, A., HARVEY, J. A., A N D MOORE, R . Y., (1962); A demonstration of a fall in brain serotonin following central nervous system lesions in the rat. Biochem. Pharmacd., 11, 859-866. KUNTZMAN, R., SHORE, P. A,, BOGDANSKI, D.,AND BRODIE,B. B., (1961); Microanalytical procedures for fluorometric assay of brain dopa-5 HTP decarboxylase, norepinephrine and serotonin, and a detailed mapping of decarboxylase activity in brain. J . Neurochem., 6 , 226-232. F. C., (1938); L'effet de la section des fibres preganglionnaires sur la teneur cn acetylMACINTOSH, choline du ganglion sympathique. Arch. inf. Physiol., 47, 321. P. D., AND GIARMAN, N. J., (1957); 5-Hydroxytryptamine content of PAASONEN, M. K., MACLEAN, structures of the limbic system. J . Neurochem., 1, 326-333. VONEULER,U. S., A N D PURKHOLD, A,, (1 951); Effect of sympathetic denervation on the noradrenaline and adrenaline content of the spleen, kidney and salivary glands i n the sheep. A c f aphysiol. scand., 24, 212--217. WOOLLEY, D. W., A N D SHAW,E., (1954); A biochemical and pharmacological suggestion about certain mental disorders. Proc. naf. Acad. Sci. (Wash.), 40, 228-231.

56

The Hydroxylation of Tryptophan by Pigeon Brain in vivu EMERY M. G A L

AND

FINLEY D. MARSHALL, J R .

Nerrrocketiiical Research Division, Departtnent of Psychiatry, College of Medicine, State University of Iowa, Iowa City, Iowa (U.S.A.)

The manner in which 5-hydroxytryptamine (5-HT) in a two-step process is derived from tryptophan is well established. Recent evidence (Freedland et al., 1961; Cooper and Melcer, 1961) gave first demonstration of an in vitro hydroxylation of tryptophan (TP) to 5-hydroxytryptophan (5-HTP) by rat liver and intestinal mucosal cells respectively. The enzyme responsible for this hydroxylation in the liver was shown to be phenylalanine hydroxylase (Renson et al., 1962). These authors, however, questioned the physiological significance of the hydroxylation of tryptophan by the phenylalanine hydroxylase as the real contributing factor to the biosynthesis of 5-HTP in the body. Studies on the turnover of 5-HT in the brain (Udenfriend and Weissbach, 1958) suggest a rapid formation and release of it by this organ. Furthermore, the results on the 5-HT content of the brain of total gastro-enterectomized rats (Bertaccini, 1960) strengthened the likelihood of an independent biosynthesis of 5-HTP by the brain. The results presented herein are summarized from some experiments which are but a part of a larger program aimed at studying the in vivo and in vitro hydroxylation o f tryptophan by the brain tissue of various species. Pigeons of about 400 g weight kept on standard pigeon feed were divided into 2 groups. One group served as control while the pigeons of the other group received intraperitoneally 3.57 lO-5/M/kg p-phenylisopropylhydrazineHCl (PIH) (Lakeside Laboratories) 2 h prior to injection of radioactive tryptophan (Tracerlab, Inc.). Some animals of the control and PIH-treated groups were intraperitoneally given 1.5 pmoles of ~~-tryptophan-[2-~~C],'while others received subarachnoidally 0.085 prnoles of ~ ~ - t r y p t o p h a n - [ 3 - ~ The ~ C ] animals . were killed at various time intervals and their brain (average 2.0 g weight) was immediately removed and homogenized in ice-cold 0.01 N HCI. Aliquots of the homogenates were taken for determination of total radioactivity of the brain, while the remainder was extracted for 5-HT content by a modified technique of the butanol extraction method (Bogdanski et al., 1956). Following spectrophotofluorometric measurement of the 5-HT content, the aqueous extracts were adjusted to pH 4-5 and lyophilized to dryness. The dry residues were extracted with 80% acetone and the acetone extracts were concentrated to 0.2-0.5 ml volume. The concentrates were spotted for paper chromatography. Air-dried paper 1

57

THE H Y D R O X Y L A T I O N O F T R Y P T O P H A N

chromatograms were used for radioautography. The 5-HT spots were identified by their Rpcompared to a 5-HT [I4C] (New England Nuclear Corp.) marker run alongside. The radioactive spots corresponding to 5-HT were cut out and were counted in a liquid scintillation counter (Nuclear-Chicago Corp.) according to the immersion technique recommended by Takahashi et al. (1961). In many instances, the radioactive 5-HT spots were eluted and rerun. The radioactive 5-HT was also identified by it5 picrate salt in the presence of added carrier 5-HT. This 5-HT was recovered as a hydrochloride salt from its picrate and counted again. We surmized that the high 5-HT levels of the avian brain were suggestive of a fair hydroxylating ability of this organ and that this hydroxylation might be fairly rapid. In order to test this, one must ascertain that intracerebrally administered radioactive tryptophan, after its efflux and following its hydroxylation in the liver or in the intestine, is not returned to the brain as radioactive 5-HTP in sufficient amounts to account for the radioactive 5-HT recovered from that organ. TABLE I APPEARANCE OF RADIOACTIVITY I N PIGEON BRAIN F O L L O W I N G I N T R A P E R I T O N E A L I N J E C T I O N OF T R Y P T O P H A N

1 . 5 pmoles of ~ ~ - t r y p t o p h a n - [ 2 - ~(I~.2C .] lo6 d.p.m.) was injected into each animal. PTH (B-phenylisopropylhydrazine) 3.57 . lO-5/kg was administered 2 h before the injection of tryptophan.

Time from injection (nlin)

20 60 120

PIH cl.p.m./g

5-HT

Control d.P.dK

lwig

d.p.m.

550 1289 1674

1.28 1.27 0.86

None None None

488 1429 1790

5-HT IdK

d.p.m.

3.27 3.95 3.48

None None None

TABLE I1 LEVELS O F R A D I O A C T I V I T Y A N D C O N V E R S I O N OF S U B A R A C H N O I D A L L Y A D M I N I S T E R E D T R Y P T O P H A N T O 5-HT I N P I G E O N B R A I N

0.085 pmoles of o~-tryptophan-[3-~~C] (8.9.103 d.p.m.) was injected. PIH was administered intraperitoneally 2 h before injection of tryptophan. Time

d.p.ni. total

(min)

.lo5

2 20

7.50 6.45 5.37

40

5-HT

% of total 84 72 60

Pg/g

d.p.rn./,ug

1.88 2.13

572 507

S. A . Radioactivity 5-HT in 5-HT ,iiC/nimole ( %)

45.9 40.7

0.26 0.23

The results given in Table I clearly show that after intraperitoneal administration of about 20 times larger amounts of radioactive tryptophan than that administered intracerebrally, the total radioactivity i n the brain did not exceed 0.3% of all the labeled tryptophan administered. In no instance could radioactive 5-HT be detected References p . 60

58

E. M. G A L A N D F. D. M A R S H A L L , JR.

Fig. 1. Radiochromatography of the butanol extract of pigeon brain following subarachnoidal injection of DL-[l ‘C]-tryptophan. Solvent =isopropanol : N H IOH : water (20 : 1 : 2). Components: I , tryptamine; 2, 5-hydroxytryptamine; 3, tryptophan. Exposure time, 2 weeks. Animal sacrificed at 20 min.

THE H Y D R O X Y L A T I O N OF TRYPTOPHAN

59

either in the absence or in the presence of PIH. It is of interest to note that administration of intraperitoneal PIH, while leading to the expected rise of cerebral 5-HT did not appreciably alter the penetration of tryptophan into the central nervous system. In Table 11, a few examples of the conversion of subarachnoidally injected tryptophan to 5-HT are given. This group of animals was pretreated with intraperitoneal PIH. The recovery of the label in 5-HT within a period of 40 min was fairly constant in presence of the monoamine oxidase inhibitor. From the data assembled, there is every evidence that the pigeon brain is able to hydroxylate tryptophan to 5-HTP. Radioautographic record presented in Fig. 1 demonstrates the presence of labeled 5-HT and tryptamine obtained from the brain extract of a pigeon sacrificed 20 min after intracerebral injection of radioactive tryptophan. (Incidentally, from this figure, it is also apparent that butanol extraction, even after repeated washings, fails to eliminate about 1-5 % of the labeled tryptophan administered.) The percentage of total radioactivity of tryptophan removable as 5-HT as well as the calculation of the specific activity of 5-HT is based on several assumptions. Among these, the foremost is the one that at zero time, there is an actual 8.9 * 105 d.p.m. of tryptophan present in the brain. It is obvious that if one is to calculate both the specific activity and the percentage recovery of the labeled 5-HT on the basis of the total radioactivity found in the CNS at the time of sacrifice, both values will be significantly higher. Furthermore, the total radioactivity injected represents both D- and L-forms. It is not yet clear whether D-tryptophan can be hydroxylated to 5-HTP or to what degree its presence is inhibitory to the hydroxylation of the L-isomer. Therefore, it was tentatively accepted that both isomers were equally hydroxylated to 5-HTP. Of course, the extent to which D-tryptophan is either contributory or inhibitory to the hydroxylation of L-tryptophan to 5-HTP will influence the actual specific activity and the percentage recovery of the label in 5-HT at any given time. In addition, the inhibition of monoamine oxidase by PIH may have a “feed-back’’ effect on tryptophan hydroxylase. In all, an accurate quantitative assessment of 5-HTP synthesis in the brain cannot be calculated at this time. Mindful of the aforesaid assumptions, the best we can say at present is that based on the recovery of radioactive 5-HT, 0.2-1.0% of tryptophan is hydroxylated to 5-HTP in pigeon brain within 10 min of its intracerebral administration. In spite of the monoamine oxidase inhibition, a noticeable decrease of the label in both 5-HT and in tryptophan takes place after an hour while in 2 h time, the presence of radioactive 5-HT is no longer detectable. In some experiments, besides the labeled metabolites shown in Fig. 1 , radioactive 5-HTP, N-acetyl-tryptophan and a yet unidentified radioactive substance could be isolated from some of the brain extracts. In conclusion, these results established the direct hydroxylation of tryptophan to 5-HTP in the pigeon brain tissue, thus implying the presence of tryptophan hydroxylase. This fact and the lack of evidence for phenylalanine hydroxylase in the CNS is taken as a further support for the existence of 2 separate hydroxylating enzymes, one for tryptophan and the other one for phenylalanine respectively. Full details of this work will be published elsewhere. References p . 60

60

E. M. G A L A N D F. D. MARSHALL, JR.

SUMMARY

It was established that intracerebrally injected radioactive tryptophan was hydroxylated to 5-hydroxytryptophan by the brain tissue. Evidence for this was obtained by isolating radioactive 5-hydroxytryptamine from the brain of animals sacrificed within 20 to 30 min after the intracerebral injection of radioactive tryptophan. Intraperitoneal injection of 20 times larger amounts of labeled tryptophan did not lead to the appearance of the label in the 5-hydroxytryptamine isolated from the brain. It is concluded that the brain tissue is capable of hydroxylation of tryptophan. ACKNOWLEDGEMENTS

The authors gratefully acknowledge the grant support from the State of Iowa Mental Health Research Fund, and the conscientious technical assistance of Merrily Poczik and Earl M. Boulton. REFERENCES BERTACCINI, G., (1960); Tissue 5-hydroxytryptamine and urinary 5-hydroxyindoleacetic acid after partial or total removal of the gastrointestinal tract in the rat. J. Physiol. (Lond.), 153, 239-249. BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B., AND UDENFRIEND, S., (1956); Identification and assay of serotonin in brain. J. Pharmacol. exp. Ther., 117, 82-88. COOPER, J. R., AND MELCER, I., (1961); The enzymatic oxidation of tryptophan to S-hydroxytryptophan in the biosynthesis of serotonin. J. Pharrnacol. exp. Ther., 132,265-268. FREEDLAND, R. A., WADZINSKI, I. M., AND WAISMAN, H. A., (1961); The enzymatic hydroxylation of tryptophan. Biochern. biophys. Res. Conimun., 5, 94-91. RENSON, J., WEISSBACH, H., AND UD~NFRIEND, S., (1962); Hydroxylation of tryptophan by phenylalanine hydroxylase. J . biol. Chem., 237, 2261-2264. TAKAHASHI, H., HATTORT, T., AND MARUO,B., (1961); Liquid scintillation counting of I4C paper chromatograms. Analyt. Biochem., 2, 447-462. UDENFRIEND, s., AND WEISSBACH, H., (1958); Turnover of 5-hydroxytryptamine (serotonin) in tissues. Proc. SOC.exp. Biol. ( N . Y.), 97, 148-151.

61

Circadian and Estrous Rhythms in Pineal and Brain Serotonin* W. B . Q U A Y University of California, Berkeley, Calif. ( U S .A . )

The mammalian pineal organ is a derivative of the central nervous system and contains specialized parenchymal cells and an extremely high concentration of serotonin (5-hydroxytryptamine). Pineal serotonin is significant, at least in part, as the precursor of the more pineal-specific compound, melatonin (N-acetyl-5-methoxytryptamine). The metabolic relations of these and related pineal compounds have been reviewed and developmental and pharmacological changes in pineal serotonin of rats have been described previously (Quay and Halevy, 1962). With the aid of an improved and more specific extraction procedure (Quay, 1963) and spectrophotofluorometry, the serotonin content of adult rat pineals has been studied further. Of the 5-hydroxy- and 5-methoxyindoles in the pineals, serotonin is present in the greatest amount, but it exhibits a 9-fold fluctuation during the course of a daily or circadian cycle correlated with the periods of light and darkness (Fig. 1). TABLE I P I N E A L S E R O T O N I N A T D I F F E R E N T TIMES D U R I N G THE E S T R O U S C Y C L E

I N L O N G - E V A NR S A T S 13-20 W E E K S O L D A T S A C R I F I C E Nanograms serotonin base/pineal; means f standard errors of the means (number of samples) are listed.

Day of estrous cycle

04:OO-05:00 1 1 :30-13:OO 17 :OO-18 :00 21 :OO-21:30

I (proestrus)

56.9 t 5.2 (10) 79.7 t 5.2 (16) 62.0 & 3.9 (13) 31.5 Sr 3.3 (6)*

2

51.1 f 2.7 (16)* 77.4 5.3 (19) 62.8 f 3.3 (14) 23.3 & 4.5 (6)

3

65.4 3.9 (14)* 70.2 i 3.8 (21) 65.6 i 3.1 (14) 25.6 & 1.2 (7)

4

63.4 & 2.7 (10) 69.8 rt 3.4 (15) 61.7 4.0 (11) 15.4 f 1.3 (5)*

~

* Probability that the means of these populations are actually equal or different in the opposite direction is
*

A report on these studies will be published in extenso in Gen. comp. Endocr.

References p . 63

62

W. R . Q U A Y

Time (clock h)

Fig. 1. Circadian rhythm in pineal serotonin of adult male Long-Evans rats. Means (= dots) (twelve samples/solid dot, average of 5 samples/open dot) are plotted according to time of sampling in the daily periods of darkness (stippled) and light (clear). Vertical lines extend two times the standard error on each side of the means.

monitored by daily vaginal smears and selected for 4-day cycles) (Table I). In additional studies it has been shown that the daily fall in pineal serotonin depends upon the occurrence of darkness within a critical period of several hours. If the onset of darkness is delayed until midnight (24:00,standard photoperiod = 04:OO-18:OO) the nocturnal fall in pineal serotonin is inhibited. If the lights are turned on much earlier (at 22:OO) than usual, the pineal serotonin content rises precociously, but the midday level remains about the same. Available information suggests that the circadian rhythm in pineal serotonin may be an intrinsic one, since serotonin injection does not significantly increase pineal serotonin and since a circadian rhythm, at least of this amplitude and relation to photoperiod, has not been found in the central nervous system. Little has been published on circadian variation in blood or brain serotonin. Albrecht et al. (1956) reported a fluctuation of about 50% in mouse brain, with higher values around noon than after 16:30 (photoperiod = 06:OO-18:OO). I have attempted (with Dr. A. Halevy) to demonstrate circadian differences in anterior and posterior hypothalamic serotonin in adult female rats that were sacrificed at 05 :OO-06:30,12:OO-13 :OO, and 2O:OO-21:30,times at which under identical conditions pineal serotonin shows pronounced differences. However, no differences could be found in the hypothalamic areas at these times.

RHYTHMS I N PINEAL A N D BRAIN SEROTONIN

63

It was shown previously (Quay and Halevy, 1962) that injection of beta-phenylisopropylhydrazine and 5-hydroxytryptophan caused increases in late morning levels of pineal serotonin significantly beyond the usual maximum occurring at this time of day. Injection of reserpine (3 mg/kg intraperitoneally to adult male rats) in the evening (l7:00-18:00) produces decrease in pineal serotonin of about 20% (P<0.02) when measured at 07:OO-09:OO the following morning. However, this level is still far higher than the untreated pineal’s nocturnal minimum level of serotonin. Thus, interpretation of the dynamics of pineal serotonin level in terms of either metabolism or binding must take into account the profound and regular circadian pattern. The possibility remains that circadian or other types of physiological variation in serotonin levels within distinct areas of the central nervous system may occur and may provide critical evidence concerning the central significance(s) of this compound. S U M M A RY

Differential extraction and spectrophotofluorometry of pineal serotonin in rats demonstrated a 9-fold circadian rhythm which was shown to be dependent in its timing on the periods of light and darkness and to be slightly modified by the estrous cycle. A circadian change in the serotonin content of anterior and posterior areas of the hypothalamus could not be demonstrated. The interpretation of the dynamics of serotonin level in pineal, and possibly some areas of the central nervous system, in terms either of metabolism or binding, must take into account the occurrence or possibility of a circadian rhythm. ACKNOWLEDGEMENT

This investigation was supported by a research grant (RG-5219) from the National Institutes of Health, U.S. Public Health Service. RE FE RE N C E S

P.,VISSCHER, M. B., BITTNER, J. J., A N D HALBERG, F., (1956); Daily changes in 5- hydroxytryptamine concentration in mouse brain. froc. Soc. exp. Biol. (N. Y . ) , 92, 703-706. QUAY,W. B., (1963); Differential extractions for the spectrophotofluorometric measurement of diverse 5-hydroxy- and 5-methoxyindoles. Analyt. Biocheni., 5, 51-59. QUAY, W. B., AND HALEVY, A,, (1962); Experimental modification of the rat pineal’s content of serotonin and related indole amines. fhysiol. Zool., 35, 1-7.

ALBRECHT,

64

Action of Serotonin, Allied Compounds and Monoamine Oxidase Inhibitors on Peripheral Nerve Fibers V I N C E N Z O D1 CARLO* Thuriichurn Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. ( U.S.A.)

In this meeting the importance of nerve endings as binding sites of neurohormones has been emphasized. This is certainly justified: the ending of a neuron is the part that secretes the chemical transmitter and where vesicles have been found. However, I think it cannot be excluded that a comparable importance as binding sites of neurohormones should be attributed to other parts of a neuron. Indeed, no evidence was so far produced that neurohormones are not present along the whole length of nerve fibers, whether contained in vesicles or not, and that they are not involved in conduction as well as in transmission of nerve impulses. Particularly as regards this last point, some 30 years of research by many investigators have accumulated a large amount of data that support the hypothesis of chemical conduction. Nachmansohn, in a review of these data, including many remarkable contributions of himself and of his coworkers, reached such conclusions on the role of acetylcholine in nerve conduction (Nachmansohn, 1959). A certain indirect support to the thesis of chemical conduction and the evidence that a neurohormone different from acetylcholine (and norepinephrine) has, if not binding sites, at least receptors in peripheral nerve fibers may be provided by the results of a pharmacological research project on which I began working in 1958. Here are such results (V. Di Carlo, 1958; V. Di Carlo and R. Di Carlo, 1961; V. Di Carlo, 1964a, b). (1) Serotonin antagonists, such as LSD and BOL, are powerful local anesthetics (rabbit cornea) and nerve blocking agents (isolated frog sciatic nerve). The effects on rabbit cornea were tested by the method of Regnier, slightly modified (Di Carlo, 1958). The cornea was kept covered by abundant volumes of the experimental solutions for fixed periods of 3 min in each instance. The degree of anesthesia was measured by the number of stimuli, rhythmically applied by means of a suitably calibrated hair, required to provoke the winking reflex. The results on the isolated frog sciatic nerves were evaluated by measuring changes in the voltages of the action potentials. For comparison, experiments with the classic local anesthetics were performed by both techniques.

*

Present address: L. B. Mendel Research Laboratory, Elgin State Hospital, Elgin, Ill.

ACTION O F DRUGS ON NERVE FIBRES

65

(2) Serotonin and 5-methoxytryptamine are able to counteract the local anesthetic effect on rabbit cornea of drugs such as procaine, cocaine and percaine, when they too are applied on the cornea (either before the anesthetic, or with the anesthetic, or after the anesthetic), as well as when they are given by intravenous route. For example, a 1 % solution of 5-hydroxytryptamine creatinine sulfate was able to inhibit completely the anesthetic effect of 2 % procaine hydrochloride, whether applied before, or after, or with procaine hydrochloride. Similarly, 10 mg/kg of 5-hydroxytryptamine creatinine sulfate, given by the intravenous route, inhibited markedly the anesthetic effect of 1 % cocaine hydrochloride administered 2 h later. (3) A number of hydrazine monoamine oxidase inhibitors and the non-hydrazine monoamine oxidase inhibitors pargyline (MO-9 I 1) and tranylcypromine, applied on the rabbit cornea before well-known local anesthetics, or given by intravenous route, are able to inhibit local anesthesia. (4) 5-Hydroxytryptophan, given in high dose by intraperitoneal route, is able to counteract the effect of local anesthetics on rabbit cornea. (5) In the guinea-pig, iproniazid and 5-hydroxytryptophan, given in high doses by intraperitoneal route, are able to counteract the effect of local anesthetics injected into the skin. The technique of Biilbring and Wajda (1945) was employed for the evaluation of intradermal anesthesia in these experiments. (6) On the isolated frog sciatic nerve, iproniazid, pargyline and 5-hydroxytryptophan are able to increase the voltage of the action potential and to antagonize the block by cocaine. These effects have at least some specificity, as for example, isoniazid and tryptamine do not exert any anti-anesthetic action (whilst iproniazid and 5-hydroxytryptamine do). Moreover, tryptamine even enhances local anesthesia. On the basis of these results it can be tentatively suggested that in peripheral nerve fibers, or at least in peripheral sensory nerve fibers, there is a physiological mechanism on which serotonin, drugs similar to serotonin and drugs able to increase the serotonin (and other amines) content of tissues can act; that this mechanism is important for nerve function; that its ‘stimulation’ is able to increase the voltage of nerve action potential as well as to counteract the effect of local anesthetics; that its ‘depression’ (as in the case of tryptamine action) is able to potentiate the effect of local anesthetics. It is also interesting that drugs capable of antagonizing serotonin, as LSD and BOL, are also effective local anesthetics. The presence of receptors for serotonin in peripheral axons, or at least in peripheral sensory axons, and the lack of evidence that serotonin or similar compounds are not contained in those axons (Florey and Florey, 1953; V. Di Carlo and Cilento, 1959; Falck, 1963) give some support to the hypothesis that serotonin (or a similar compound) might play a physiological role in peripheral nerve fibers, at least in the sensory ones (V. Di Carlo, 1958). SUMMARY

Serotonin, 5-methoxytryptamine, 5-hydroxytryptophan and several MAO-inhibitors References p . 66

66

V I N C E N Z O DI C A R L 0

are able to inhibit local anesthesia in vitro and in vivo (by local application as well as by general route administration). Some of these drugs are also able to increase the voltage of the action potentials in the isolated frog sciatic nerve. On the other hand, serotonin antagonists, such as LSD and BOL, are powerful local anesthetics and nerve blocking agents. REFERENCES BULBRING, E., AND WAJDA.I., (1945); Biological compariscn of local anesthetics. J . Pharnracol. e x p . Ther., 85, 78-84. Dr CARLO,V., (1958); Ricerche sul meccanismo di azione degli anestetici locali. X Congress0 della Societa Italizna di Farmacologia, Napoli. Published in Arch. ital Sci. farmacol., 1961, 11, 1-12. Dr CARLO,V., (1964a); Further in\estigations on the interference of serotonin, allied compounds and monoamine oxidase inhibitors with local anesthesia. In preparation. DI CARLO,V.. (1964b); Antagonism of the blocking effect of cocaine on frog sciatic nerve by serotonin drugs and monoamine oxidase inhibitors. In preparation. DI CARLO,V., A N D CILENTO,A,, (1959); Ricerche sulla libcrazione di istamina e di 5-ossi-triptamina da stimolazione di un nervo misto. Boll. Sac. ital. Biol. sper., 35, 207-209. Dr CARLO,V., A N D Di CARLO,R., (1961); L’effctto antianestetico locale degli inibitori della monoamino-ossidasi. XI Congrcsso della Societa ltaliana di Farmacologia, Castellamare di Stabia. Arch. ital. Sci.farmacol., 12, 207-212. FALCK, B., (1963); This Symposium. FLOREY,E., AND FLOREY,E., (1953); U t e r die Bedeutung von 5-Hydroxytryptamin als nervose Aktionssubstanz bei Cephalopoden und Dekapoden Crustaceen. Naturwissenschafren, 40, 4 13. NACHMANSOHN, D., ( I 959); Chemicaland Molecular Basis of Nerve Aciivity. New York and London, Academic Press.

67

General Discussion *

COSTA:Dr. Carlsson states that his results with a-MMT agree neither with ours nor with those of Udenfriend. Explicitly, we fail to find measurable amounts (0.05 ,ug/g) of either a-methyl-metatyramine or of rnetaraminol in rabbit brain 10 days after giving a-MMT (100 mg/kg i.v.). I suggest that no real discrepancy might really exist. Dr. Carlsson presents no quantitative data on the actual amount of metaraminol in rabbit brain 10 days after giving a-MMT but shows chromatographic spots representing the metaraminol present. These spots indicate that trace amounts of araminol are found in the brain stem of his rabbits when large amounts of tissue are processed. Of course larger amounts of this amine will be found if larger amounts of a-MMT are given. By the same token much smaller amounts of araminol would be expected after giving 20 mg/kg i.v. of a-MMT which also completely depletes brain NE. Dr. Carlsson has superimposed his qualitative results onto the quantitative data of our published figure. Such a presentation implies in a rather dramatic way that he is right after all. But this is a little like trying to claim a gold strike on the basis of a spot-test for gold. I believe that a note or two about the assay of metaraminol and a-methyl-metatyramine is in order. In our method we used the same extraction procedure used for NE. The organic phase-to-water partition ratios of the a-MMT amines are considerably greater than that of NE. It is not surprising therefore to find that the 2 amines are completely extracted from brain homogenates. As a result the procedure is accurate and exquisitely sensitive. Another point of interest is that the amount of amines formed from (I-MMT does not seem to be related to the condition of the catecholamine binding sites. Thus, in animals pretreated with reserpine, a-M M T produces the same amount of amines in the brain as in aninlals not so pretreated. I n contrast, after pretreatment of rats with reserpine DOPA produces a much lower level of dopamine than in control animals. Finally, we are happy to see that Dr. Carlsson concludes that inhibition of monoamine uptake rather than its depletion is the important factor in explaining reserpine action. We agree with him (Pharmacol. Rev., 1959, 11, 548). Speaking of granules, does Dr. Carlsson think that data obtained with granules from the adrenal medulla can be extrapolated to those in nerve endings? I mention this because small doses of a-MMT that deplete catecholamines from brain and peripheral nerve endings do not affect those from adrenal medulla.

* This discussion refers to the papers of Dr. H. Blaschko, Dr. A. Carlsson, Dr. B. Falck and Dr. A. Pletscher.

68

DISCUSSION

CARLSSON: The discrepancy between our data and those of Dr. Costa and Dr. Udenfriend is probably to be sought in different analytical techniques. Perhaps the most probable explanation is that for some reason metaraminol (aramine) is not so easily extracted from the tissues as, for example, noradrenaline, and that the comparably mild extraction used by Dr. Costa and Dr. Brodie and Dr. Udenfriend is insufficient. There is no doubt that we are dealing with a true discrepancy. Tn the experiment of Fig. 2 (this Symposium), 5 g of brain stem were processed. The metaraminol spot indicates at least l pg. After correction for recovery (50%) we thus obtain at least 0.4 pg/g or 2.4 10-19 moles/g. According to Fig. 2 the loss of noradrenaline is 2.7 * lO-lg moles/g. There is thus reasonably good agreement between the metaraminol found and the missing noradrenaline. We get similar results when measuring metaraminol quantitatively, combining our extraction and ion-exchange chromatography with Udenfriend’s colorimetry (AndCn, to be published). To Dr. Costa’s statement that we are not publishing our data quickly enough, my only defense is that we are more anxious to publish correctly than quickly. However, the quantitative data demanded by Dr. Costa will be published shortly. For obvious reasons T do not think that Dr. Costa can take his data on a-methylmetatyramine in the caudate nucleus as evidence that his method does not fail to detect metaraminol in the brain stem. We have also observed that reserpine does not cause any marked decrease i n a-methyl-metatyramine or metaraminol in brain. This is not surprising in view of their resistance to M A 0 and COMT. It certainly cannot be taken as evidence that the storage granules are normally unable to accumulate these amines. Dr. Costa apparently finds it impossible to believe that metaraminol in a dose 0.2 pg/g i.v. can accumulate in heart to a concentration of 1 pg/g, but he underestimates the power of adrenergic nerve endings to concentrate the transmitter and its analogs. For example, Whitby, Axelrod and Weil-Malherbe (.I. Pharmacol. exp. Ther., 1961, 132, 193) have observed that 3H-noradrenaline injected intravenously to cats in a dose of 0.025 pg/g accumulates in heart within 2 h to a concentration of 0.20 pg/g, i.e. an eight-fold concentration. Consequently a five-fold concentration of metaraminol in Dr. Costa’s experiment is by no means unreasonable. Certainly lack of stoichiometry is not ‘proved’ by this experiment. The reason why the metaraminol does not readily show up under the fluorescence microscope remains to be discovered. The a-methyl group has been found to delay though not prevent the fluorophore formation. In contrast to reserpine, metaraminol has been found not to block amine uptake by adrenal medullary granules (Carlsson, Hillarp and Waldeck, to be published). Tt is true that transport in the granules of nerve endings has not been demonstrated to date. But to my knowledge no experiments to this end have been carried out so far*. The effects of reserpine on amine storage in the adrenal medulla and in the nor1

* Recently Von Euler and Lishajko (personal communication) have demonstrated ATP-dependent monoamine uptake by granules of noradrenergic nerves. Thc uptake is blocked by reserpine in low concentrations. This observation emphasizes the close similarity between these granules and those of the adrenal medulla.

69

DISCUSSION

adrenergic nerve endings are remarkably similar. Experiments in our laboratory (Carlsson and Waldeck, to be published) have shown that the blockade by reserpine of uptake of labelled noradrenaline by nerve endings in vivo is relatively short-lasting and more closely correlated with functional effects than the long-lasting depletion of monoamine levels. Similarly, monoamine uptake by adrenal medullary granules in vivo recovers more rapidly from blockade by reserpine than the catecholamine levels (Carlsson, Jonasson and Rosengren, to be published), thus confirming the in vitro experiments reported in my paper. Dr. Costa refers to the fact that the adrenal medulla is not depleted by single doses of a-MMT. This is not surprising in view of the slow turnover of the adrenal medullary amines and cannot be taken to indicate that the processes going on in the granules of adrenal medulla and adrenergic nerve endings are qualitatively different. Dr. Costa’s last point is confusing. Our present view is in full agreement with our conclusion of 1957, ‘that lack rather than excess of biogenic amines in active form should be considered as the cause of some pharmacodynamic effects of reserpine’ (Carlsson et al., (1957), Psychotropic Drugs, S. Garattini and V. Ghetti, Editors, Amsterdam, Elsevier, p. 372). This view is contrary to Dr. Brodie’s concept, according to which reserpine-induced block of storage leads to an increase in the level of free and active 5-HT in the brain. AXELROD: Dr. Carlsson intimated that the granule in the nerve endings is similar to the granule in the adrenal medulla. Dr. Potter and I (Nature (Lond.), 1962, 194, 581-582) have provided evidence showing that they are not. The adrenal medullary granules are more dense than mitochondria whereas the granules in nerve are less dense than the mitochondria. The granules are surrounded by a membrane which easily ruptures. There are some similarities between the nerve granules or vesicles and the adrenal medullary granules. Both appear to contain ATP. HOLTZ:I should like to add a point to the question put forward by Dr. Carlsson

n

p 20 10

I H

Adr.

Sp1.N

Ggl.

Fig. 1 . Release of catecholamines from granules of different tissues by calcium. Adr. = Adrenal medulla (Cattle); Ca 5 ,umol/ml; H = Heart (guinea-pig), Ca 50 pmol/ml; Sp1.N = Splenic nerves (Cattle). Ca 50 pmol/ml; Ggl. = Stellate ganglion (Cattle), Ca 50 pmol/ml. Incubation: 1 h. = Spontaneous; 0 = Calcium.

70

DISCUSSION

and by Dr. Axelrod concerning the similarity between the heavier granules in the adrenal medulla and the lighter granules in the sympathetic nerves. The physiological liberator of the amines in the adrenal medulla - acetylcholine - as first shown, I suppose, by Dr. Blaschko, is unable to release the hormones from the isolated chromaffin granules. For an acetylcholine-induced relcase from the chromaffin cell calcium is required as shown by Douglasand Rubin (J.Physiol. (Lond.), 1961,159,40). Calcium is active also on the isolated granule. Thus, acetylcholine, probably by depolarization of the cell membrane, will facilitate the cntrance of calcium into the cell, Calcium in this way becomes the real ‘liberator’ of the amines. My colleagues Schumann and Philippu (Nuunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., in press) in Frankfurt were able to demonstrate, as shown in the Fig., that calcium which enhances the spontaneous release of catecholamines from the isolated granules of the adrenal medulla, is entirely inactive even in 10 times higher concentrations on the isolated granule of splenic nerves, of stellate ganglia and of the heart (Fig. 1). Thus, the catecholamine containing granules from different tissues are also functionally different in regard to the calcium-induced release of amines. MCCAMAN: Has Dr. Falck made any observations with his method concerning the localization of fluorescent substances in the choroid plexus? FALCK: In the rat, mouse and cat, parts of the choroid plexus have been observed in connection with investigations on different brain areas and in those cases we have not found any evidence for a storage site of monoamines in this organ. COSTA:I would like to add a suggestion. Rabbit platelets contain a very high amount of 5-HT, therefore, before deciding whether or not the choroid plexus contains 5-HT, one must eliminate the possible contamination by the 5-HT present in platelets. BLASCHKO: I have one very short question for Dr. Falck: What about the area postrema? Has it been studied by this method? FALCK:Yes, in a current investigation (Fuxe and Owman, unpublished data), the area postrema (cat, rabbit and guinea-pig) has been found to contain several nerve cells, of which some show a green and others a yellow fluorescence. There are also fluorescent nerve fibres but only in small amounts. GIARMAN : Speaking about the anomalous structures in the nervous system and in the brain, 1 would like to review some early studies that Gaddum and I did on sympathetic ganglia. In pooled stellate and cervical ganglia, we found the highest decarboxylase activity of any neural structure studied, and yet we were not able to find any serotonin in excised ganglia. Later we did some perfusion experiments similar to those that Gaddum and Feldberg had done with acetylcholine, and observed that if we reinforced the perfusing fluid with an MA01 and/or 5-HTP we could then find serotonin coming out in the perfusate from the superior cervical ganglion in siiu; and we concluded that the presence of the decarboxylase might not have been just fortuitous. 1 wonder if Dr. Falck was able to distinguish between serotonin and catecholamines in the superior cervical ganglion. FALCK: We have so far no evidence for a store of 5-HT in the superior cervical ganglion. In the rat we have found 3 types of fluorescent structures in this ganglion: ganglia cells with a rather weak green fluorescence, a sparse population of small cells

DISCUSSION

71

with processes showing an intense green-yellow fluorescence and finally green fluorescent synaptic terminals. The emitted light from the small intensely fluorescent cells seems to indicate that these cells store a catecholamine rather than 5-HT. BLASCHKO: The state of the adrenergic mediator at nerve endings is of interest in connection with the ideas on the quanta1 release of the mediator. In 1960 at the Ciba Foundation Symposium on Adrenergic Mechanisms, this question was discussed. Whereas we and others have had good evidence of the occurrence of catecholamine in cell particles in the chromaffin cells, e.g., of the adrenal medulla, the evidence from nervous tissue was less complete. The observations of Von Euler and Schiimann (Ciba Foundation Symposium on Adrenergic Mechanisms, 1960) left open how much of the total amine in nervous tissue was sedimentable. SOURKES: I want to add a point to the chairman’s historical introduction. I think that if one is talking about stereochemistry of the decarboxylation of the amino acids, one should recall that in 1954 Mandeles, Koppelman and Hanke, at the University of Chicago, published a paper (J. biol. Chem., 1954,209,327-336) in which they showed that the a-hydrogen of lysine, tyrosine, and glutamic acid, respectively, is not involved in the decarboxylation. If the a-C-H bond is unaffected during the catalysis, the same could be expected of the a-C-CH3 bond in a-methyldopa (Sourkes, Rev.canad. Biol., 1955, 14, 49-63). The demonstration of the decarboxylation of para-tyrosine and of a-methyldopa in mammalian tissues was achieved as soon as methods more sensitive than those of conventional manometry were introduced. But the definitive demonstration of the non-involvement of the a-hydrogen (and its linkage) came from the work of Mandeles et al.

72

Drug-induced Changes in the Subcellular Distribution of Serotonin in Rat Brain with Special Reference to the Action of Reserpine NICHOLAS J . GIARMAN, DANIEL X. FREEDMAN AND SAUL M. SCHANBERG Department of Pharmacology and Psychopharmacology Laboratory, Department of Psychiatry, Yale University School of Medicine, New Haven, Conn. ( U . S . A . )

While no precise physiological role can yet be ascribed to any of the various neurohumors in the brain, it has become useful to think of these substances in terms of their presence in particulate and non-particulate sub-structures of brain cells, i.e., as a reflection of whether they are ‘bound’ and inaccessible to active sites or ‘free’ and accessible. The terms ‘bound’ and ‘free’ are meant in this paper to have no physicochemical significance: the ‘free’ material, e.g., may be associated in some way with soluble protein, a possibility which has not been investigated. Operationally in this work, ‘bound‘ amine refers to that found associated with particulate sub-fractions of the cell, while the free amine is that found in the supernatant fraction. At first it was heuristically convenient to consider these subcellular forms in the construction of intellectual models and hypotheses. Later, it seemed necessary to adopt this way of thinking in order to accommodate many experimental findings. In 1957, for example, Weil-Malherbe and Bone found about one-half of the catecholamines in the brain bound to a mitochondria1 fraction of the homogenate. This, of course, was entirely expected after the early demonstration of catecholamines in granular material of the adrenal medulla by Blaschko and Welch (1953) and Hillarp ef al. (1953). In 1958 Hebb and Whittaker showed that 60% of the acetylcholine (ACh) in brain resides in granules; and in 1959 Giarman and Schanberg demonstrated that more than 70% of endogenous serotonin (5-HT) is associated with the particulate fraction of rat brain. Similar results with respect to 5-HT were reported by Walaszek and Abood (1 959). In this context, it is now generally acceptable to view the cerebral life cycle of an amine like 5-HT in the following simplified manner. (1)Active uptake of the precursor amino acid by the brain (Schanberg and Giarman, 1960) ; ( 2 ) Synthesis of the amine by decarboxylation of the precursor in the cytoplasm or on a granule-surface;

THE ACTION OF RESERPINE O N SEROTONlN

13

(3) Uptake and storage of the amine in membrane-bound granules; ( 4 ) Release of the amine in a free form in response to various stimuli; ( 5 ) Action of the amine at a receptor site; (6) Simultaneous or subsequent degradation of the amine by one or more enzymes, or by non-specific adsorption to 'sites of loss' in the immediate environment. Such a series of events leads to a consideration of the various subcellular forms of a cerebral amine like 5-HT in Fig. 1. STORAGE

I""'D""-I ACTIVE

BOUND

A

f

PRECURSOR

\\

UNBOUND ( " F R E E 'I )

METABOLITES

Fig. 1. Possible subcellular forms of brain neurohumors.

There are a number of points of interest in this scheme relative to subsequent discussion. Several 'bound' forms of the amine may be considered, with the largest amount of the amine probably existing bound in granules in a storage form. The active form of the amine is that which is complexed with the receptor, not the 'free' material, which is a transition state, potentially active, potentially storable, and potentially destructible. Two major paths of dissipation are open to the free material: one is an enzymatic means of inactivation and the other is binding to non-specific protein in the environment, called by some, 'sites of loss'. Presumably, steady-state equilibria exist between the 'free' material and that bound in all of the sites of binding in the scheme, with the exception of the inactivator enzyme(s). After the early observations of Shore et al. (1955) and Paasonen and Vogt (1956) that reserpine led to a gradual decline in brain levels of total 5-HT, it became compelling to accept the view of the former investigators that reserpine caused a release of 5-HT from its storage form, leading to a prolonged presence of 'free' amine in the tissues. From such a consideration, it occurred to us that certain other neuroactive drugs might act to alter the equilibria governing the subcellular forms of 5-HT, and in this way perhaps elicit their characteristic neuropharmacologic responses. It was at this point that my colleagues and I decided that it might be fruitful to investigate alterations in the subcellular distribution of 5-HT in brain induced by certain neuropharmacologic agents (Schanberg and Giarman, 1962; Freedman and Giarman, 1962). Our hope was that such investigations would provide more meaningful correlates of drug-induced behavioral change than had been provided by our studies on drug-induced changes in levels of total 5-HT. References p . 80

74

N. J. G I A R M A N , D. X . FREEDMAN A N D S. M. S C H A N B E R G

It had become apparent to us in our early studies (Giarman and Schanberg, 1959) that brain particles containing 5-HT are much more labile than similar particles in gastrointestinal mucosa, and tend to loose the amine when subjected to an elegant separation of subcellular components. We, therefore, decided to sacrifice elegance of separation for what we believed to be as accurate an estimate as possible of particulate and non-particulate amine by simply subjecting homogenates of rat brain in isotonic sucrose to centrifugation at 100,000 x g for 20 min. Our homogenizing medium consists of 0.25 M sucrose, 0.002 M p-tolylether of choline (an inhibitor of monoamine oxidase) and 0.0015 M edathamil (‘Versene’). At the end of the centrifugation, the particulate and supernatant fractions are extracted with alkaline butanol (after the method of Bogdanski et al., 1956), and the 5-HT content is estimated spectrophotofluorometrically. All preparative operations are carried out in a refrigerated laboratory. This technique has allowed us to establish the subcellular distribution of 5-HT in the brain of the unmedicated rat and the influence of certain pertinent drugs on this distribution (Table I). TABLE I SUBCELLULAR D I S T R I B U T I O NOF SEROTONIN

(5-HT)

IN T H E RAT BRAIN AND

CHANGES PRODUCED B Y CERTAIN CHEMICAL AGENTS

None Reserpine (5 mg/kg, 24 h) Tranylcypromine ( 5 mg/kg, 2 h) 5-Hydroxytryptophan (100 mg/kg, 20 min) 5-Hydroxytryptamine (20 mg/kg, 10 min) LSD-25 (1.3 mg/kg, 40 min)

* **

150

649 f 46** 498 f 48** 151

+ 21**

23

-

18

170

112

58

34

-=0.01

18

1262

I003

259

20

>0.05

18

1090

760

330

30

<,0.01

12

83 1

684

147

18

<0.01

12

752

623

129

17

-,0.001

Represents level of significance from control, calculated by means of Student’s ‘t’ test. Mean f standard deviation.

Several important points come to light in these data. It would appear that, under these conditions, about 3.5 times as much endogenous 5-HT is bound to particulate cell components as is unbound in the supernatant fraction. The 2 3 % in the supernatant fraction is probably a maximal value in terms of the absolute amount that is ‘free’ under in situ conditions and, probably, largely represents the amount that can be ‘skinned off’ particles by our centrifugation procedure. It was of great interest to us, therefore, that Michaelson and Whittaker (1 963), working under very similar conditions, have recently reported a value of 26% for 5-HT in the supernatant fraction.

THE A C T I O N O F R E S E R P I N E O N S E R O T O N I N

75

These data also indicate that reserpine, at the peak of its pharmacologic action causes a greater depletion of particulate 5-HT (77 %) than of supernatant 5-HT (60 %), the net effect being an increase in the per cent of the total 5-HT found in the supernatant fraction. This is what might be expected from current speculations on the action of reserpine; and it is the same kind of result one obtains with the synthetic reserpine-like drug, tetrabenazine. It is of some interest that the psychotomimetic drug, LSD-25, seems to exert quite the opposite effect on the subcellular distribution of 5-HT. With this drug, at a time when maximal behavioral change and autonomic effects are seen, there is an increase in total 5-HT, which can be accounted for almost entirely by an increase in particulate 5-HT. Thus, with reserpine there is a significant increase in the proportion of 5-HT which is unbound; while with LSD-25 there is both an absolute and proportionate increase in the bound amine. Another interesting finding concerns the effect of inhibitors of MAO. Schanberg and Giarman (1962) have shown that iproniazid leads to an increase of 5-HT in both cell compartments in the same proportion that exists in the unrnedicated animal. This has been suggested as evidence that an equilibrium does exist between synthesis, uptake and binding of the amine on the one hand, and its release and destruction (or dissipation) on the other. Essentially the same finding with iproniazid has been reported by Michaelson and Whittaker (1963). Another, more rapidly acting and more potent inhibitor of MAO, tranylcypromine, has been found to behave similarly (Table I). In view of these findings the speculation that M A 0 inhibitors cause an increase in the absolute amount of unbound 5-HT is probably correct, but it must be qualified to take cognizance of the fact that the proportion which is unbound is not altered. One final point of interest has to do with the difference in distribution which follows administration of exogenous 5-HT compared to that following the administration of 5-HTP, leading to endogenous formation of the amine. A significant increase in the proportion of unbound amine is seen after 5-HTP. On the other hand, administration of a large dose of 5-HT exogenously leads to an increase in the proportion of the amine which is bound. Such a marked difference may be due at least in part to the extreme stress imposed upon the animal by the massive dose of exogenous 5-HT. One of the most intriguing facets of this problem to us has been the question of whether these drug-induced changes in subcellular distribution of 5-HT correlate at all with concomitant drug-induced behavioral change. An obviously simple way to approach this question is to observe the change in subcellular distribution of 5-HT as it develops in time after a single moderate dose (2.5 mg/kg) of reserpine. WeilMalherbe (1959) has already suggested that one may learn more about the mode of action of reserpine by making observations as its effects are developing rather than at a time when some steady state has been reached. When such observations are made it becomes clear that reserpine has a temporally triphasic action with respect to subcellular distribution of 5-HT (Fig. 2). During the early stage of this action (from 15 min to 2 h), when little, if any, diarrhea, ptosis or reduced spontaneous activity are seen, depletion of the supernatant fraction occurs at a more rapid rate than that of the particulate fraction of 5-HT. A similar finding was reported by Weil-Malherbe References p. 80

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N . J. G I A R M A N , D. X. F R E E D M A N A N D S. M. S C H A N B E R G

et al. (1958, 1961) with respect to the early action of reserpine on catecholamines in the brain stem of rabbits. The expected result with reserpine of greater depletion of bound than unbound 5-HT is seen when the characteristic pharmacologic effects of the drug are fully developed, i.e., from 4 to 48 h (also cf. Table I). Following this stage, i.e., during the repletion phase, particularly from 96 h to 20 days, the rate of return to normal levels is more rapid in the bound than in the unbound fraction. Thus, the effect of reserpine on the unbound fraction is first to appear and last to disappear. A very similar pattern of action was observed with tetrabenazine (Fig. 3). The only difference with this compound is that the entire time-course of its action is compressed /TOTAL ,*PARTICULATE J N SUPERNATANT /’

00

80

n 60

40

20

\I i 72

0.25

0.5

1

2

4

24

48

240

TIME (HOURS)

Fig. 2. Time-course

-

-1

the t :ct of a single dose of reser ne (2. mg/kg, intraperitoneally)on the subcellular distribution of serotoni in the rat brain.

1

,TO TA L PART ICU LAT E S U PER N A T A N T

0

a: 8 0 Iz

0

0 LL

0

-z -I

w

60

40

1

w

_1

I-

I

20

I (D

5

10

30

120

240

TIME (MINUTES)

Fig. 3. Time-courseof the effect of a single dose of tetrabenazine (5 mg/kg, intraperitoneally)on the subcellular distribution of serotonin in the rat brain.

THE ACTION O F RESERPINE ON SEROTONIN

77

within 1 day rather than 20 days; but the early predominant effect of tetrabenazine is clearly on the unbound fraction. With this compound also the effect on the bound fraction tends to disappear more rapidly than that on the unbound fraction. In view of these findings it would appear to be a rather oversimplified concept of reserpine's action to think of it simply as a transfer of amines from the particulate to the soluble fraction. The question now is how to explain this more complex action of reserpine and related compounds. First, one must assume that at the cellular level (within the scheme shown in Fig. 1) it is possible for a drug to have multiple sites of action, by virtue of similar molecular configurations at these sites. Once this assumption is made, one can suggest at least 3 sites at which reserpine may act (Fig. 4).

CELL WALL / STORAGE GRANULE

MA0 - C O N T A I N I N G PART I CLE

IDECA R BOX Y LASE1

!$'

FAClL ITATED TRANSPORT

Fig. 4. Possible sites of action of reserpine at the subcellular level.

Site I

- Cell wall The effect expected here would be an increased outward permeability, and an initial action at this site before other sites are affected by reserpine would obviously lead to the more rapid rate of decline of supernatant 5-HT seen with reserpine during its early stage of action. Meltzer, Barrnett and Carlini (1963) have recently obtained electron micrographs of amine-containing mastocytoma cells treated in tissue culture with 10-7 M reserpine, an amount which causes 100% depletion of the 5-HT in the cells. One sees clear evidence of cytoplasmic granules leaving via the apparently intact cell wall. Reproduction of these cells (i.e.,as a reflection of generationtime and growth curves) is not altered by reserpine (Giarman, Carlini and Fischer, 1963). There is also evidence that reserpine induces the formation of new structures which morphologically resemble lysosomes. Since these organelles are known to contain a variety of enzymes capable of degrading proteins, nucleic acids and mucopolysaccharides (DeDuve, 1959), it is tempting to link these reserpine-induced lyso-

References p . 80

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N . J. G I A R M A N , D. X. FREEDMAN A N D S. M. S C H A N B E R G

somes with the reserpine-induced changes in cell wall permeability and depletion of the amines. Mast cells are, obviously, not brain cells; and we are aware that such reserpineinduced phenomena may possibly be seen only in cells that have an active transport for 5-HT a t the cell membrane (mast cells and platelets). Schanberg (1963) found that no such transport exists in brain slices. Site 2 - MAO-containing particle At this site one can conceive of reserpine’s exerting one of two possible actions : to permit the movement of the enzyme out of the particle into the cytoplasm; or to promote the accessibility of 5-HT to the enzyme within the particle. In either case, the net result would be an increased destruction of the unbound amine. An early and persistent effect of reserpine at this site could also explain the triphasic changes which were described above. It is clear from Table 11, however, that reserpine does not cause an increased destructive activity of 5-HT by the particle-free cytoplasm either early (1 h) or later (24 h) in the action. T A B L E I1 INFLUENCE OF RESERPINE O N DESTRUCTIVE CAPACITY OF S U P E R N A T A N T F R A C T I O N O F R A T B R A I N FOR S E R O T O N I N

Homogenate of brain from

__

Untreated rats (a) Supernate (b) Particulate** Reserpinized rats (2.5 mg/kg, I h) (a) Supernate (b) Particulate Reserpinizcd rats (2.5 mg/kg, 24 h) (a) Supernate (b) Particulate

* **

5-HT added 5-HT recovered* (MgJ ( I d

3 30

2.81 15.20

6.0 49.3

3 30

2.8 I 16.00

6.0 46.1

3 30

2.90 15.38

3 .O 48‘7

Corrected for loss in reagent control. 100,000 x g for 20 min.

The possibility that M A 0 acts intracellularly on reserpine-released amines finds some analogy in the interpretation that Kopin and Gordon (1962) have placed on results of their studies on the fate of reserpine-released tritiated noradrenaline. These workers feel that reserpine depletes tissues of catecholamines by releasing the granulebound amines which are then metabolized within the tissue predominantly by deamination. Site 3 - Storage granule There is a possibility of three mechanisms in the storage granule that may be

THE A C T I O N O F R E S E R P I N E O N S E R O T O N I N

79

affected by reserpine, if we assume that the granule is membrane bound and has active processes going on in its membrane. (a)Reserpine may affect the membrane to increase outward permeability of 5-HT. (b) Reserpine may in some way cause an unbinding of the amine from the intragranule binding substance. This displacement is not likely, however, because molar concentrations of reserpine to amines liberated do not indicate it; and also because reserpine will not act at 4". (c) Reserpine may block an active-transport process for 5-HT in the membrane. Our tendency at this time is to attempt to explain our findings as follows: in addition to a possible increase in the outward movement of 5-HT through the cell wall, the first effect of reserpine may be an action on M A 0 by virtue of a greater access of the substrate (5-HT) to the enzyme as described above. This would lead to a more rapid rate of decline in unbound than bound 5-HT. Later, reserpine's effect on the storage granule may be developed and the picture will change to the conventional one of greater depletion of bound than unbound 5-HT. Finally, the effects of reserpine disappear in reverse order so that repletion is seen to occur more rapidly in the bound than in the unbound form of 5-HT. It is clear to us now that in order to strengthen these ideas we must measure the rate of turnover of labeled 5-HT in both particulate and supernatant fractions, and also investigate the rate of formation of 5-hydroxyindoleacetic acid in the two subcellular fractions. C O N C L U D I N G REMARKS A N D SUMMARY

We have concluded from these studies that the mode of action of reserpine (and tetrabenazine) on 5-HT in the brain is more complex than a simple shift of the amine from a bound to a n unbound form. It is felt that other agents which influence the metabolism of monoamines in the brain may have multiple sites of action on the subcellular level, and that studies of their influence on the subcellular distribution of the amines may reveal similar complexities in their action. It is well known, for example, that a-methyldihydroxyphenylalaninenot only inhibits the decarboxylase, but also leads to a release of 5-HT. We have confirmed this with studies similar to those described here (Schanberg and Giarman, 1962). We have similarly presented evidence that certain inhibitors of M A 0 not only inhibit that enzyme, but also exert an action on the storage granule which is manifested in an antagonism to the 5-HT releasing action of reserpine (Schanberg and Giarman, 1962). ACKNOWLEDGEMENTS

This investigation was supported in whole by Public Health Service Research Grants M H 03363-0431 and NB 00940-08 from the National Institute of Mental Health and the National Institute of Neurological Diseases and Blindness, respectively. The authors gratefully acknowledge the competent technical assistance of Catherine Doolittle and Elizabeth Caruso. References p . 80

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N . J. G I A R M A N , D. X . FREEDMAN A N D S. M. S C H A N B E R G

REFERENCES

H., AND WELCH,A. D., (1953); Localization of adrenaline in cytoplasmic particles of BLASCHKO, the bovine adrenal medulla. Naunyn-SchniiedeSerg’s Arch. exp. Path. Pharmak., 219, 17-22. BOGDANSKI, D. F., PLETSCHER, A., BRODIE,B. B., AND UDENFRIEND, S., (1956); Identification and assay of serotonin in brain. J. Pharmacol. exp. Ther., 117, 82-88. DEDUVE,C., (1959); Lysosomes, a new group of cytoplasmic particles. Subcellular Parricles. T. Hayashi, Editor. New York, Ronald Press (p. 127-159). FREEDMAN, D. X., AND GIARMAN, N. J., (1962); LSD-25 and the status and level of brain serotonin. Ann. N . Y. Acad. Sci., 96, 98-106. GIARMAN, N. J., AND SCHANBERG, S., (1959); The intracellular distribution of 5-hydroxytryptamine (5-HT, Serotonin) in the rat’s brain. Biochem. Pharmacol., 1, 301-306. G. R. S., A N D FISCHER, G., (1963); Unpublished data. GIARMAN, N. J., CARLINI, HEBB,C. O., AND WHITTAKER, V. P., (1958); Intracellular distributions of acetylcholine and choline acetylase. J . Physiol. (Lond.), 142, 181-196. HILLARP,N.-,k, LAGERSTEDT, S., AND NILSON,B., (1953); The isolation of a granular fraction from the suprarenal medulla, containing the sympathomimetic catecholamines. Acta physiol. scand., 29, 251-255. KOPIN,I. J., AND GORDON, E. K., (1962); Metabolism of norepinephrine-H3 released by tyramine and reserpine. J. Pharmacol. exp. Ther., 138, 351-359. MELTZER, H. Y., BARRNETT, R., AND CARLINI,G., (1963); Unpublished observations. MICHAELSON, I. A., AND WHITTAKER, V. P., ( I 963); The subcellular localization of 5-hydroxytryptamine in guinea-pig brain. Biochem. Pharmacol., 12, 203-212. PAASONEN, M. K., AND VOW, M., (1956); The effect of drugs on the amounts of substance P and 5-hydroxytryptamine i n mammalian brain. J . Physiol. (Lond.), 131, 617-626. SCHANBERG, S., (1963); A study of the transport of 5-hydroxytryptophan and 5-hydroxytryptainine (Serotonin) into brain. J. Pharmacol. exp. Ther., 139, 191-200. SCHANBERG, S., A N D GIARMAN, N. J., (1960); Uptake of 5-hydroxytryptophan by rat brain. Biochim. biophys. Acta (Amst.), 41, 556-558. SCHANBERG, S., AND GIARMAN, N. J., (1962); Drug-induced alterations in the subcellular distribution of 5-hydroxytryptamine. Biochem. Pharmacol., 11, 187-194. SHORE,P. A., SILVER,S. L., AND BRODIE,B. B., (1955); Interaction of reserpine, serotonin and lysergic acid diethylamide in brain. Science, 122, 284-285. WALASZEK, E. J., AND ABOOD,L. G . , (1959); Fixation of 5-hydroxytryptamine by brain mitochondria. Proc. SOC.exp. Biol. ( N . Y . ) , 101, 3740. WEIL-MALHERBE, H., (1959); The effect of rcserpine on the intracellular distribution of catecholamines in the brain stem of the rabbit. Biochemistry of the Central Nervous System. F. Briicke, Editor. Proc. Fourth Intern. Congr. Biochem., 3. New York and London, Pergamon Press (p. 190-195). H., AND BONE,A. D., (1957); Intracellular distribution of catecholamines in the WEIL-MALHERBE, brain. Nature (Lond.), 180, 1051. WEIL-MALHERBE, H., AND BONE,A. D., (1958); Effect of reserpine on the intracellular distribution of catecholamines in the brain stern of the rabbit. Nature (Lond.), 181, 1474-1475. WEIL-MALHERBE, H., POSNER,H. S., AND BOWLES,G . R., (1961); Changes in the concentration and intracellular distribution of brain catecholamines: the effects of reserpine, B-phenyl-isopropylhydrazine, pyrogallol and 3,4-dihydroxy-phenylalanine,alone and in combination. J. Pharmacol. exp. Ther., 132, 278-286,

81

The Uptake and Release of Catecholamines and the Effect of Drugs JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland (U.S.A.)

During the past few years our laboratory has been engaged in studies on the metabolism, uptake and release of noradrenaline and adrenaline and the effect of drugs on these processes. In these investigations, we used radioactive catecholamines of high specific activity. The availability of these radioactive compounds not only made it possible to work with physiological amounts of the hormones but also enabled us to isolate and characterize several major and minor metabolic products and investigate in a more precise manner the fate of circulating and bound catecholamines in the body. The following is an account of some of this work.

UPTAKE O F CIRCULATJNG CATECHOLAMINES

Cats were given 3H-noradrenaline or 3H-adrenaline intravenously and the uptake of these amines was measured immediately after injection and for various time intervals thereafter (Axelrod et al., 1959; Whitby et a/., 1961). Within 2 min after their administration, both amines were taken up in unequal amounts by various tissues. The greatest quantities of the radioactive catecholamines were found in the heart, spleen and glandular tissues; skeletal muscle took up the least. High levels of catecholamines in the heart, spleen and adrenal glands were maintained for many hours which indicated that these hormones can be held in tissue in a physiologically inactive form for long periods of time until they are released. The large amounts of circulating catecholamines bound in the heart suggest that the adrenal gland could supply the heart with adrenaline and noradrenaline. Binding serves to protect the catecholamines from attack by enzymes until they are released. The uptake and retention of circulating noradrenaline and adrenaline by tissues may be an important mechanism for the physiological inactivation of these hormones. Within minutes after the administration of radioactive noradrenaline or adrenaline, large amounts of 3H-normetanephrine or 3H-metanephrine, the 0-methylated metabolites, were found in most tissues. About 90 % of the injected radioactivity was accounted for as unchanged catecholamines or their corresponding 0-methylated metabolites. These findings indicate that O-methReferences p . 87-89

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JULIUS AXELROD

ylation plays an important role in terminating the action of the circulating hormones. After the intravenous administration of radioactive adrenaline and noradrenaline, only small quantities of these compounds were present in the brain (Weil-Malherbe et al., 1959; Weil-Malherbe et al., 1961). The hypothalamus took up a small amount of the catecholamines whle the pituitary gland and the pineal body contained larger quantities presumably because there is no blood-brain barrier in these structures. The brain contains relatively large amounts of endogenous noradrenaline (Vogt, 1954). Catecholamines in the brain are probably formed from precursors that are capable of crossing the blood-brain barrier such as dopa. Using a broken cell preparation, monoamine oxidase activity was shown to be higher in the rat brain than catechol-0-methyltransferase (Crout et al., 1961). As a result of these observations, it was postulated that monoamine oxidase was mainly involved in the inactivation of noradrenaline in the brain. After the administration of pyrogallol, a catechol-0methyltransferase inhibitor (Axelrod and Laroche, 1959), into the lateral ventricle of the rabbit, there was a threefold elevation in the endogenous noradrenaline in the brain (Masami et al., 1962). The administration of 14C-noradrenaline into the lateral ventricle of cats resulted in the formation of normetanephrine and other 0-methylated products as major metabolites (Mannarino et a/., 1962). From these observations it would appear that 0-methylation is a n important enzymatic process in the metabolism of noradrenaline in nervous tissue. It still remains to be established whether binding, monoamine oxidase or catechol-0-methyltransferase or a combination of these mechanisms are involved in the initial inactivation of noradrenaline in the brain. The isolated-perfused rat heart also can take up and retain SH-noradrenaline (Axelrod et al., 1962a). The bound catecholamine is then released in a multiphasic fashion which indicates that there are several types of binding (Kopin et al., 1962). In rat hearts, noradrenaline was found to be inactivated principally by binding. The released noradrenaline in the isolated-perfused heart is metabolized by 0-methylation and deamination. The changes in the released metabolites with time suggest that 0methylation is the main pathway for the metabolism of the easily releasable noradrenaline and deamination is the main pathway for the tightly bound catecholamines. Although noradrenaline and adrenaline are taken up, stored, and metabolized in a similar manner, there are quantitative differences in their disposition (Whitby et a]., 1961). More 3H-noradrenaline was taken up in tissues and held for longer periods of time. There were more 0-methylated metabolites found after adrenaline than after noradrenaline. These findings indicate that binding is quantitatively a more important mechanism for the inactivation of noradrenaline while enzymatic 0methylation is more important for adrenaline. The higher levels of circulating endogenous noradrenaline might be explained in terms of the differences in the degree of binding of these hormones rather than in terms of a greater release of noradrenaline into the circulation. The subcellular localization of catecholamines was studied in heart, salivary and adrenal glands (Potter and Axelrod, 1962). About 80% of the circulating 3H-noradrenaline and 3H-adrenaline was taken up by a particulate fraction associated with

CATECHOLAMINES A N D T H E EFFECT O F D R U G S

83

the microsomes of the heart and salivary gland. Endogenous catecholamines were also found in this fraction of the cell. 3H-Dopamine, 3H-dopa, and 3H-normetanephrine, on the other hand, were present only in the cell sap. In the adrenal medulla, 3H-noradrenaline was found in the microsomal fraction while 3H-adrenaline, 3Hdopamine, and endogenous catecholamines were localized in the chromafin granules. 3H-Dopa was present in the supernatant fraction of the adrenal medulla and in the ‘pinched-off nerve endings’ of the brain described by Whittaker (1959). Using brain slices, 3H-noradrenaline was also found to be taken up in this layer. It was demonstrated that circulating noradrenaline and adrenaline were taken up by sympathetic nerve endings (Hertting et a/., 1961a). When sympathetic nerves were destroyed, they were unable to retain the catecholamines. Supersensitivity to noradrenaline results partly because the sympathetic nerves cannot inactivate the catecholamines by binding. The uptake of radioactive noradrenaline by sympathetic nerves made it possible to visualize sympathetic nerves by radioautographic techniques (Wolfe et a/., 1962). Electron microscopy showed a striking localization of radioactive grains only within sympathetic nerves associated with dense core vesicles 40 to 50 mp thick. These experiments provide conclusive evidence that noradrenaline can be taken up from the circulation into sympathetic nerves and stored there until it is released. RELEASE OF N O R A D R E N A L I N E A N D A D R E N A L I N E

After the administration of noradrenaline and adrenaline, these catecholamines disappeared from the whole animal in two phases (Axelrod et al., 1959; Whitby et al., 1961). In the first few minutes, there was a rapid decline followed by a slow disappearance of the catecholamines. The initial phase is due to enzymatic destruction of the catecholamines and their uptake and binding by tissues; the second to slow release and metabolism. In the first few minutes, about two-thirds of the adrenaline and one-half of the noradrenaline were metabolized. Almost all of the catecholamines that disappeared in the first few minutes could be accounted for as 0-methylated metabolites, normetanephrine and metanephrine. There was a slower decline of noradrenaline as compared with adrenaline during the second phase which indicated that noradrenaline is more tightly bound and more slowly released. Bound noradrenaline was released from the heart over a period of days and the half-life of noradrenaline became progressively longer (Axelrod et al., 1961a). These observations indicated that noradrenaline became more tightly bound with time. Tyramine has been shown to act by releasing noradrenaline (Burn and Rand, 1958). Using tyramine as a tool, it was demonstrated that noradrenaline is bound with different degrees of tenacity (Potter et al., 1962). After the first injection, tyramine released about 30% of both endogenous and 3H-noradrenaline. With repeated injections of tyramine, the amount of endogenous and exogenous noradrenaline liberated from the heart became less and less. There was no further release of noradrenaline and no blood pressure elevation after the third or fourth injection of tyramine. Yet considerable quantities of the catecholamines were still present in the heart. It was apparent from these Rejerentes p . 87-89

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JULIUS AXELROD

experiments that noradrenaline is bound in two pools, one which is easily released and another which is more firmly held. The 3H-noradrenaline recently taken up by the heart can be released by tyramine more easily. With the passage of time, tyramhe releases less and less 3H-noradrenaline. The longer the noradrenaline remained in the heart the more firmly bound it became. This is due to the different rates in which the pools were turning over. The more easily releasable pool was found to have a half-life of several hours and the more tightly bound pool has a half-life of about one day. In contrast to the finding with tyramine, reserpine releases noradrenaline from both stores equally well (Potter and Axelrod, 1963). It has been shown that the noradrenaline released by reserpine is metabolized by inonoamine oxidase while the noradrenaline released by tyramine is metabolized by catechol-0-methyltransferase (Kopin et al., 1962). Although reserpine releases large amounts of the catecholamine, it produces only a slight physiological effect while tyramine releases much less noradrenaline and produces a marked physiological response. The noradrenaline released by reserpine appears to be metabolized by monoamine oxidase within the nerve and it leaves the nerve as an inactive deaminated metabolite. Tyramine discharges noradrenaline in a physiologically active form and once it is liberated the neurohumor is inactivated by 0-methylation. It has been demonstrated that inhibition of monoamine oxidase does not prolong the physiological actions of noradrenaline (Griesemer et a]., 1953). Consequently, this enzyme appears to act within the sympathetic nerves, and metabolizes the neurohumor before it becomes physiologically active. Once noradrenaline leaves the nerve, monoamine oxidase plays a negligible role in the subsequent fate of the catecholamine. Because sympathetic nerves are capable of taking up and retaining radioactive noradrenaline it was possible to introduce 3H-noradrenaline into these nerves and study its fate when the neurohumor is discharged (Hertting and Axelrod, 1961). Cats were given 3H-noradrenaline and the spleen which contained large amounts of the radioactive catecholamine was isolated and perfused with blood free of radioactive material. After stimulation of the splenic nerve, 3H-noradrenaline and its metabolites were measured in the venous outflow. There was a decided increase in the 3H-noradrenalinc in the venous outflow after each stimulation. There was also a smaller increase in thc 3H-normetanephrine which was directly related to the 3H-noradrenaline liberated. These results again demonstrated that noradrenaline can be taken up from the circulation by sympathetic nerves and on stimulation, discharged. When liberated, the noradrenaline interacts with the receptor and part is released into the blood stream, part 0-methylated by catechol-0-methyltransferase, and part returns to the sympathetic nerves to be bound and used again. EFFECT OF D R U G S O N THE U P T A K E , RELEASE A N D METABOLISM OF N O R A D R E N A L I N E

Drugs affecting behavior also alter the uptake, release and metabolism of catecholamines. Antidepressant drugs inhibit monoamine oxidase and raise the concentration of catecholamines in the heart and brain of certain species (Shore et al., 1957);

C A T E C H O L A M I N E S A N D T H E EFFECT O F D R U G S

85

reserpine depletes catecholamines from tissues (Holzbauer and Vogt, 1956). In our laboratory, we have used radioactive catecholamines to study the effect of drugs on uptake, release and metabolism of these hormones. Reserpine and chlorpromazine have been shown to increase the rate of destruction of circulating catecholamines (Axelrod and Tomchick, 1959). These drugs prevented the uptake of the catecholamines, thus interfering with the protective binding. The hormones were then exposed to enzymatic attack and more rapid metabolism. As pointed out above, reserpine also releases the bound noradrenaline within the nerve where it is deaminated by monoamine oxidase. Chlorpromazine blocked the uptake of noradrenaline by the nerve (Axelrod et al., 1961b). Once the neurohumor was bound within the nerve, this drug could not release it (Axelrod et al., 1962b). Chlorpromazine is a drug of many actions: It is an antihistamine, tranquilizer, and an adrenergic blocking agent. The blocking of the uptake of noradrenaline was found to reside only in the antiadrenergic activity of chlorpromazine (Rose11 and Axelrod, 1963). Other antiadrenergic drugs such as Dibenzyline and dichlorisoproterenol affect the uptake and release of noradrenaline (Axelrod et al., 1962b). Like chlorpromazine, the antidepressant drug imipramine increases the metabolism of catecholamines and blocks the uptake of these hormones. Many antidepressant drugs also inhibit monoamine oxidase and elevate the concentration of catecholamines in the brain, heart, and other tissues (Shore et al., 1957). These enzyme inhibitors, however, do not prolong the physiological response to catecholamines and appear to play a minor role in the inactivation of circulating catecholamines. As pointed out above, monoamine oxidase operates mainly within the nerves, inactivating the noradrenaline before it leaves the nerve (Kopin et al., 1962). In the presence of monoamine oxidase inhibitors the released noradrenaline returns to the storage vesicle or escapes into the circulation. Monoamine oxidase inhibitors also blocked the spontaneous release of noradrenaline from stores (Axelrod et al., 1961a), and this also serves to elevate the endogenous catecholamine levels in tissues. Bretylium and ganglionic blocking agents prevent the spontaneous release of 3H-noradrenaline (Hertting et al., 1962a, b). Although these compounds do not inhibit monoamine oxidase, they increase the endogenous catecholamine levels (Hertting et al., 1962b; Ryd, 1962). The releasing action of guanethidine and reserpine was found to be blocked by monoamine oxidase inhibitors, bretylium and ganglionic blocking agents. When noradrenaline is liberated from the easily releasable pool by sympathomimetic amines neither monoamine oxidase inhibitors nor bretylium blocked its release (Potter and Axelrod, 1963). Monoamine oxidase inhibitors, bretylium, guanethidine and reserpine are hypotensive drugs which affect the uptake, release and metabolism of catecholamines. The actions of reserpine and monoamine oxidase inhibitors on noradrenaline have been described above. Bretylium and guanethidine have many actions in common and also differ in their effects on circulating and bound noradrenaline (Hertting et al., 1962a). Both compounds when injected produced a brief rise in blood pressure as well as an immediate release of noradrenaline. These drugs also inhibited the uptake of noradrenaline and potentiated the effects of the hormone. Bretylium and guanethiReferences p . 87-89

86

JULIUS AXELROD

dine blocked the liberation of noradrenaline as well as the physiological effects when sympathetic nerves were stimulated. When the noradrenaline was bound in tissues, bretylium prevented its spontaneous release while guanethidine caused a slow but continuous release of the neurohumor. Many drugs such as cocaine, guanethidine, bretylium, imipramine, chlorpromazine, and Dibenzyline cause supersensitivity to catecholamines. All of these drugs prevented the uptake of circulating catecholamines by tissues (Whitby et al., 1960; Axelrod et al., 1961b; Hertting et al., 1961b). The concentration of catecholamines in the blood was elevated when the animals were pretreated with these drugs. Supersensitivity resulted because these drugs prevented inactivation of the hormone by binding and a higher concentration of the free and active catecholamines was present at the receptor site. Chronic denervation of sympathetically innervated organs also caused supersensitivity to catecholamines. When the nerves degenerate, the vesicles which bind and inactivate noradrenaline are destroyed. Consequently, the amount of unbound and active catecholamines in the vicinity of the receptors in denervated tissues would be expected to persist for longer periods, thus resulting in supersensitivity. Sympathomimetic amines not only liberated catecholamines (Burn and Rand, 1958) but also prevented their uptake and increased the rate of metabolism (Axelrod and Tomchick, 1960; Hertting et al., 1961b). After repeated administration of tyramine

Fig. I . The fate of noradrenaline at the sympathetic nerve endings and the effect of drugs. Bret bretylium; GBO ganglionic blockers; MAOJ = monoamine oxidase inhibitors; COMT -- catechol-0-meth yltransferase.

and other sympathomimetic amines, there was successive decrease in the amount of noradrenaline released from the heart and a concomitant decrease in physiological response (Potter et al., 1962). When there was no further liberation of noradrenaline

CATECHOLAMINES A N D THE EFFECT OF DRUGS

87

by tyramine, tachyphylaxis (loss of responsiveness) resulted. At this time, there is a considerable amount of noradrenaline still present in the heart. The action of sympathomimetic amines could be restored after an infusion of noradrenaline (Cowan et af., 1961). Tachyphylaxis appears to be a result of the depletion of the bound catecholamines easily available for release. Fig. 1 shows a working model of the sympathetic nerve which is consistent with its morphological appearance and would explain differences in storage, metabolism and the action of drugs on noradrenaline. The majority of the storage vesicles are deep within the nerves. Reserpine and guanethidine release noradrenaline from the deep vesicles. The catecholamines are deaminated by monoamine oxidase in the mitochondria and leave the nerve as inactive metabolites. Bretylium appears to block the releasing action of these drugs from the storage vesicles and monoamine oxidase inhibitors prevent the metabolism of noradrenaline liberated by reserpine and guanethidine. A smaller number of vesicles may be close to the synaptic terminals where sympathomimetic amines or nerve impulses release noradrenaline from the nerve cell. Once released, the noradrenaline reacts with the receptor and is metabolized by catechol-0-methyltransferase, discharged into the circulation, or returns to the storage vesicle. Bretylium and guanethidine block the release of noradrenaline by nerve impulses. Noradrenaline is also released from the stores spontaneously and this release is blocked by monoamine oxidase inhibitors, bretylium, and ganglionic blocking agents. The storage vesicles can also take up noradrenaline from the circulation. Cocaine, chlorpromazine, imipramine, bretylium, guanethidine, and reserpine block this uptake thus causing supersensitivity. SUMMARY

Circulating noradrenaline and adrenaline are mainly inactivated by catechol-0methyltransferase or they are taken up and bound in dense core vesicles in sympathetic nerves. When the catecholamines are released they are metabolized by monoamine oxidase within the nerve or by catechol-0-methyltransferase outside the nerve. A part of the catecholamines are also inactivated by being bound again or by diffusing into the circulation. Noradrenaline is stored in an easily releasable or firmly bound form. Many drugs such as cocaine, chlorpromazine, imipramine, reserpine, guanethidine, bretylium and sympathomimetic amines interfere with the uptake, storage and release of catecholamines. REFERENCES AXELROD, J., GORDON, E., HERTTING, G., KOPIN,I. J., AND POTTER, L. T., (1962a); On the mechanism of tachyphylaxis to tyramine in the isolated rat heart. Brit. J. Pharmacol., 19, 56-63. AXELROD, J., HERTTING, G., AND PATRICK,R. W., (1961a); Inhibition of H3-norepinephrine release by monoamine oxidase inhibitors. J . Pharmacol. exp. Ther., 134, 325-328. AXELROD, J., HERTTING, G., AND POTTER,L. T., (1962b); Effect of drugs on the uptake and release of 3H-norepinephrine in the rat heart. Nature (Loncl.), 194, 297.

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JULIUS AXELROD

AXELROD, J., AND LAROCHE, M . J., (1959); Inhibitor of 0-methylation of epinephrine and norepinephrine in vitro and in vivo. Science, 130, 800. AXELROD, J., AND TOMCHICK, R., (1959); Activation and inhibition of adrenaline metabolism. Nature (Lond.), 184, 2027. AXELROD, J., AND TOMCHICK, R., (1960); lncreased rate of metabolism of epinephrine and norepinephrine by sympathomimetic amines. J . Pharmacol. exp. Ther., 130,367-369. H., AND TOMCHICK, R., (1959); The physiological disposition of AXELROD, J., WEIL-MALHERBE, H3-epinephrine and its metabolite metanephrine. J . Pharmacol. exp. Ther., 127, 251-256. AXELROD, J., WHITEY, L. G., AND HERTTING, G., (1961b); Effect of psychotropic drugs on the uptake of H3-norepinephrine by tissues. Science, 133, 383-384. BURN,J. H., AND RAND,M. J., (1958); The action of sympathomimetic amines in animals treated with reserpine, J . Physiol. (Lond.), 144, 314-336. COWAN,F. F., CANNON, C., KOPPANYI, T., AND MAENGWYN-DAVIES, G. D., (1961); Reversal of phenylalkylamine tachyphylaxis by norepinephrine. Science, 134, 1069-1070. CROUT,J. R., CREVELING, C. R., A N D UDENFRIEND, S., (1961); Norepinephrine metabolism in rat brain and heart. J . Pharmacol. exp. Ther., 132, 269-271. GRIESEMER, E. C., BARSKY,J., DRAGSTEDT, C. A., WELLS, J. A., A N D ~ E L L E R E.A.,(1953); , Potentiating effect of iproniazid on the pharmacological action of sympathomimetic amines. Proc. Soc. exp. Biol. ( N . Y . ) , 84, 699-701. HERTTINC, G., AND AXELROD, J., (1961); Fate of tritiated noradrenaline at the sympathetic nerve endings. Nature (Lond.), 192, 172-173. HERTTING, G., AXELROD, J., KOPIN,1. J., AND WHITBY, L. G., (1961a); Lack of uptake of catecholamines after chronic denervation of sympathetic nerves. Nature (Lond.), 189, 66. HERTTING, G., AXELROD, J., AND PATRICK, R. W., (1962a); Actions of bretylium and guanethidine on the uptake and release of (3H)-noradrenaline. Brit. J . Pharmacol., 18, 161-166. HERTTING, G., AXELROD, J., A N D WHITBY, L. G., (1961 b); Effect of drugs on the uptake and metabolism of H%orepinephrine. J . Pharmacol. exp. Ther., 134, 146-1 53. HERTTING, G., POTTER, L. T., AND AXELROD, J., (1962b); Effect of decentralization and ganglionic blocking agents on the spontaneous release of H3-norepinephrine. J . Pharmacol. exp. Ther., 136, 289-292. HOLZBAUER, M., AND VOGT,M . , (1956); Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat. J . Neurochetn., 1, 8-1 1. KOPIN,I. J . , HERTTING, G., AND GORDON, E. K., (1962); Fate of norepinephrine-H3 in the isolated perfused rat heart. J . Pharmacol. exp. Ther., 138, 34-40. MANNARINO, E., KIRSHNER, N . , AND NASHOLD, JR., B. S.,(1962); Themetabolism of noradrenaline-C'd by cat brain in vivo. Fed. Proc., 21, 182. MASAMI, M., HIROSHI, Y.,ANDREIJI, I . , (1962); Effect of pyrogallol on the catecholamine content of the rabbit brain. Biochem. Pharmacol., 11, 1109-11 10. POTTER, L. T., AND AXELROD, J . , (1962); Intracehlar localization of catecholamines in tissues of the rat. Nature (Lond.), 194, 581-582. POTTER, L. T., AND AXELROD, J., (1963); Studies on the storage of norepinephrine and the effect of drugs. J . Pharmacol. exp. Ther., 140, 199-206. POTTER, L. T., AXELROD, J., AND KOPIN,I. J., (1962); Differential binding and release of norepinephrine and tachyphylaxis. Biochem. Pharmacol., 11, 254-256. ROSELL, S., A N D AXELROD, S., (1963); Relation between blockade of 3H-noradrenaline uptake and pharmacological actions produced by phenothiazine derivatives. Experientia (Easel), 19, 3 18. RYD, G., (1962); Protective effect of bretylium on noradrenaline stores in organs. Acra physiol. scand., 56, 90-93. SHORE, P. A., MEAD,J. A. R., KUNTZMAN, R. G., SPECTOR, S., AND BRODIE, B. B., (1957); On the physiologic significance of monoamine oxidase in brain. Science, 126, 1063-1064. VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (Lond.), 123, 451-481. WEIL-MALHERBE, H., AXELROD, J., AND TOMCHICK, R., (1959); Blood-brain barrier for adrenalinc. Science, 129, 1226-1227. WEIL-MALHERBE, H., WHITBY, L. G . , AND AXELROD, J., (1961); The blood-brain barrier for catecholamines in different regions of the brain. Regional Neurochemistry. S. S . Kety and J. Elkes, Editors. Oxford, Pergamon Press (p. 284-292). WHITBY, L. G., AXELROD, J., AND WEIL-MALHERBE, H., (1961); The fate of H3-norepinephrine in animals. J . Pharmacol. exp. Ther., 132, 193-201.

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WHITBY,L. G., HERTTING, G., AND AXELROD, J., (1960); Effect of cocaine on the disposition of noradrenaline labelled with tritium. Nature (Lonil.), 187, 604-605. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine-containing particles from brain. Biochern. J., 72, 694-706. WOLFE,D. E., POTTER,L. T., RICHARDSON, K. C., A N D AXELROD, J. (1962); Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138, 440-442.

90

Investigations on the Storage Sites of Biogenic Amines in the Central Nervous System V. P. WHITTAKER Biochemistry Department, Agricultural Research Council, Institute of Animal Physiology, Babraharn, Cambridge (Great Britain)

The brain is a rich source of biogenic amines, many with pharmacological activity, including an ability to modify the electrical behaviour of central neurones in quite low concentrations. Some of these are listed in Table I, together with an indication of their effect on neurones (for much of this information I am indebted to Dr. K. Krnjevic). Interest in these amines has been greatly stimulated by recent advances in psychopharmacology. Many of the drugs known to produce effects on the psyche are related chemically to brain amines or can be shown to influence their metabolism. Some examples are given in Table 11. Such relationships are held to imply a mode of action of the drugs concerned based on interference with chemical mechanisms regulating neuronal activity. One such mechanism is the transmission of excitation from one nerve cell to another across synapses. In the peripheral nervous system, chemical transmission by acetylcholine (ACh) and noradrenaline (NA) is well established. In the central nervous system, it has been more difficult to prove, but cholinergic transmission has now been fully demonstrated for certain central synapses by all the classical criteria (Mitchell, 1963; Gaddum, 1962).For others, however, the transmitters are unknown and most of the substances in Table I are possible candidates. Can these substances, or other equally or more active on neurones, be shown to be concentrated at nerve endings and released during transmission? If so, by what mechanisms are they stored and released? How do drugs interfere with these processes? The purpose of this review is to give an account of work done in my laboratory during the past few years which, we believe, provides a new experimental approach to some of these problems. Briefly, it has proved possible, using conventional methods of subcellular fractionation, to isolate nerve endings from the central nervous system (Gray and Whittaker, 1960, 1962; Whittaker, 1961). The isolated nerve endings, when prepared from regions containing cholinergic endings, are found to account for as much as 70-80 % of the total ACh present in the tissue sample. They therefore provide an excellent preparation with which to study the mechanism of binding and release of ACh in yitro and the effects of drugs thereon. The preparation also promises to be a useful material for similar studies with other putative transmitters, for the

91

STORAGE SITES OF B I O G E N I C A M I N E S

identification of new transmitters and for the isolation of various components of the nerve ending such as synaptic vesicles, intraneuronal mitochondria and nerve cell membranes (Whittaker, 1962; Whittaker and Gray, 1962). TABLE I PHARMACOLOGICALLY ACTIVE BIOGENIC AMINES PRESENT I N B R A I N A N D T H E I R EFFECTS O N C E N T R A L N E U R O N E S

Biogenic amine*

Bases

ACh HT NA DA Adrenaline Histamine Amino acids Glutamate Aspartate Asparagine Serine, glutamine y- Aminobutyrate u-Alanine Taurine Phosphate ATP

*

Effect on cortical cells (1)

Effect on Effect on transcallosal lateral pathway geniculate body (2) (3)

++

f-

_-_ - 4.) ---

- - (t) -

-t++ + t -t

0 ---

-_

+ -

0

+++

Effect on brain stem (4)

Effect on neurones (5)

+ -(+I (0) + - (0)

+++ 0 0 0 0 0

(+++I (0)

+++ +++ 0 0

-0

0

0

For abbreviations see text.

(I) Results of KrnjeviC and Phillis (1961, 1962); f indicates excitation, - depression of spontaneous activity, synaptic excitation or excitation by glutamate. Effects graded from none (0) through weak (one symbol) to strong (three symbols). With some compounds depression was usually followed by excitation (+). The action of ACh is highly selective and limited to cholinoceptive cells. (2) Results of Marrazzi (1957); - indicates inhibition, number of symbols indicating degree of action on non-linear scale. (3) Results of Curtis and Davis (1962, 1963); -1 indicates excitation, - inhibition. Inhibition was qualitatively different from that seen in the cortex in that the amines blocked synaptic transmission without affecting glutamate firing. Results were assessed by authors on a scale of 1 (one symbol) to 12 (three symbols). ( 4 ) Results of Bradley and Wolstencroft (1962) and Curtis and Koizumi (1961) (latter in brackets). Both excitatory (+) and inhibitory (-) effects on the spontaneous discharge of neurones were found for both ACh and NA by Bradley and Wolstencroft, but only occasional cholinoceptive cells by Curtis and Koizumi. (5) Results of Curtis, Phillis and Watkins (1961), Curtis (1962) and Curtis and Watkins (1960). ACh acts only on Renshaw cells. Excitation (+), inhibition (-) or no action (0) as indicated by

authors. Refeerencesp . 115-117

92

V. P. W H I T T A K E R

T A B L E I1 SOME P S Y C H O T R O P I C D R U G S A N D T H E I R R E L A T I O N S H I P S T O EIOGEN IC AMINES

Brain biogenic amine

Hallucinogen

Tranquillizer

M e r b C H 2 CH2NH2

H O b c H 2 c H 2 N H 2 Dopamine Noradrenaline (2-hydroxydopomine)

Mescaline

%

CH2

\

I: Lysergic acid diethyiamide

'5 - Hydroxytryptamine

Bufotenine ( N N - d i m e t h y i -

5 - hydroxytr;ptamine) Psilocybin ( 4 - phosphoroxy~,~-dimethyltryptomine)

Me0,C OMe

CMe

Reserpine

Me0 Me Hormaiine

CH2-CH-CH,

CH3

I

+NMCCH,CH,.

I

CHzOH

0.CO. CH3 CH,-CH-CH,

CH3

Atropine

Acetylcholine

HOCO

I c?

COOH

I

PNH*

CH2

Glutamic acid

Tholidomide

THE N A T U R E OF BOUND ACETYLCHOLINE

It has long been appreciated that most of the ACh of brain and other tissues is in a form which is pharmacologically inactive unless liberated by suitably vigorous treatment such as extraction with acid ethanol or heating at pH 4 and 100". A considerable literature has grown up round this phenomenon, which is known to apply to other biogenic amines also. Similar effects were found with the enzyme later named choline acetylase (ChA) which synthesizes ACh. Chloroform (Stedman and Stedman, 1939), ether (Mann, Tennenbaum and Quastel, 1939) and snake venom (Braganca and

S T O R A G E S I T E S O F B I O G E N I C AMINES

93

Quastel, 1952) were found greatly to increase the rate of formation of ACh, apparently by breaking down subcellular barriers. The true nature of these phenomena was clarified by subcellular fractionation studies. The now classical work of De Duve and collaborators (reviewed by De Duve, 1959) on the acid hydrolases of liver showed how a group of enzymes could exist

A

B

C

(m)

Fig. 1. Distribution of LDH ( ), bound ACh and SDH (0) in primary (PI, PZ and S Z ) and subfractions (A, B and C ) ofguinea-pig brain (Whittaker, 1959a; Johnson and Whittaker, 1962,1963). PI and PZ are the ’nuclear’ and ‘crude mitochondrial’ fractions prepared at 1000 x g, 11 min and 12,000 x g, 60 min, respectively; S Z is the supernatant from Pz.Fraction A floats on 0.8 M sucrose and fraction B on 1.2 M sucrose.

in a latent or occluded form within a discrete subcellular organelle (in this case the lysosome) to become fully active only when the barrier interposed between the enzymes and their substrates by the walls of the organelle was broken down by disruptive treatments. Blaschko and colleagues (reviewed by Blaschko, 1959) showed that the catecholamines of the adrenal medulla were bound to a specific storage granule which could be isolated by conventional subcellular fractionation techniques. About this time, Hebb and Smallman (1956) showed that most of the ChA of brain tissue was present in an occluded particle-bound form and could be isolated in the large granule (crude mitochondrial) fraction from sucrose homogenates of brain by differential centrifugation. It seemed clear that bound ACh might represent ACh synthesized within the compartment occupied by occluded ChA (Bodian, 1942; Feldberg, 1945), and that further information about the nature of this compartment might throw light on the mode of action of ACh and its mechanism of release. Accordingly Miss Hebb and I (Hebb and Whittaker, 1958) studied bound ACh in sucrose homogenates of brain tissues and fractions derived therefrom. We found that bound ACh survived homogenization in sucrose and that its distribution closely paralleled that of occluded ChA. We further showed that the particles carrying bound ACh and ChA could be separated from mitochondria by a simple density procedure. Fig. 1 (Whittaker, 1959a; Johnson and Whittaker, 1962, 1963) illustrates this. The distribution of bound ACh in three References p. 115-117

94

V. P. W H I T T A K E R

Figs. 2 and 3. For legends see p. 95.

STORAGE SITES OF BIOGENIC AMINES

95

primary fractions (PI, PZ and S2) obtained by centrifuging brain homogenates at increasing speeds closely parallels that of the mitochondrial marker, succinate dehydrogenase (SDH), being mainly recovered in the intermediate, crude mitochondrial fraction (Pz). However, when this fraction is separated into three subfractions by density gradient centrifugation, the bound ACh is recovered mainly in a fraction of intermediate density (B), whereas the SDH migrates with the fraction of greatest density (C). A light fraction (A) contains little of either. A complete morphological survey (first reported in lectures in Philadelphia and Washington in the spring of 1960 and at the lVth Neurochemical Symposium Varenna, June 12-17, 1960; Whittaker, 1961) was carried out by Dr. E. G. Gray and myself (Gray and Whittaker, 1960, 1962) on all the fractions and showed that the particles of the B fraction were, in fact, detached nerve endings which had apparently been pinched off entirely from their nerve terminations during homogenization to form distinct subcellular particles with somewhat similar sedimentation characteristics to mitochondria (Figs. 2-5). We call these particles nerve-ending particles (NEPs). They frequently contain one or more small mitochondria and are packed with synaptic vesicles. Occasionally a length of post-synaptic membrane is seen adhering to the periphery. The existence of NEPs in crude mitochondrial fractions and their isolation by means of sucrose density gradients has been confirmed by De Robertis el al. (1961, 1962a, b) and damaged NEPs can be recognised in the electron micrographs of Petrushka and Giuditta (1959). NEPs are very labile structures and can only be readily identified in electron micrographs of fractions prepared under good conditions. The nature of bound ACh is now simply explained. It is ACh entrapped within discrete nerve endings which survive mincing or homogenization of brain tissue and give rise to NEPs. It becomes a matter of importance to determine how other biogenic amines and the enzymes involved in their metabolism are distributed in the various brain fractions and whether any of these too are localized in the nerve-ending fraction. The presence of a substance in this fraction does not prove that it is involved in chemical transmission; however, localization in this fraction can be regarded as a sine qua non for a candidate for transmitter action. Several groups of workers have now appreciated the possibilities for further work on these lines and rapid progress may be expected. In the following sections our own recent work is reported.

Fig. 2. Thin section of rat cortex showing nerve endings (NE) containing mitochondria (m) and synapticvesicles(sv) within thin external membrane (tm) applied to dendritic process with thickened postsynaptic membrane (psm). Osmium tetroxide-phosphotungstic acid fixing and staining, Araldite embedding. Electron micrograph kindly provided by Dr. E. G. Gray. Fig. 3. Portion of B fraction of guinea-pig brain showing nerve-ending particles (NEP) with same structural features as Fig. 2. S is a shrunken ending. Curved membrane fragments (mem) may be detached psms. Other abbreviations and preparation as in Fig. 2. From Gray and Whittaker (1960,1962). References p . 115-117

V. P. W H I T T A K E R

96

5 Figs. 4 en 5. For legends see p. 97.

S T O R A G E SITES OF B I O G E N I C AMINES

97

THE SUBCELLULAR LOCALIZATION OF ENZYMES IN BRAIN

The existence of NEPs in the B fraction implies the presence of entrapped cytoplasm in this fraction. It is of interest that between 7 and 28 % of the soluble enzymes of the glycolytic pathway are particle bound in brain (Johnson, 1960). More detailed studies with one of these, lactic dehydrogenase (LDH) (Johnson and Whittaker, 1962, 1963), show (Fig. 1) that particulate LDH is mainly recovered in the PZfraction; on density gradient separation, it travels with the NEPs and bound ACh. It cannot be washed out of this fraction but is readily released by disruptive procedures which also liberate bound ACh. The released enzyme is electrophoretically identical with the soluble cytoplasmic enzyme, containing the same four isoenzymes (Johnson and Whittaker, 1963). Interest has also attached to the distribution of three enzymes concerned with the metabolism of ACh in nervous tissue, acetyl-CoA synthetase (acetylthiokinase), ChA and cholinesterase (ChE). A study of the distribution of acetyl-CoA synthetase is complicated by the presence of an inhibitor (Mounter and Whittaker, unpublished observations) which was assayed on a synthetase preparation from acetone-dried pigeon liver. The inhibitor is thermolabile and fluoride-sensitive; its distribution is similar to acid phosphatase (Whittaker, 1959a) and there is relatively little in the high-speed supernatant. It is possibly a phosphatase which destroys coenzyme A. Unlike the synthetase, it is not extracted from acetone-dried powders by saline solutions; this explains why the synthetase activity cannot be detected in untreated particulate fractions but can be assayed in extracts of acetone-dried powders of these fractions. Using this method, it was found that the subcellular distribution of the synthetase resembled that of LDH (Mounter and Whittaker, unpublished observations). The distribution of ChA, as already mentioned, closely resembles that of ACh. By contrast, ChE is not sharply localized in any one fraction but the highest specific activity is in the high-speed particulate (Ps) fraction. It appears to be bound to small membrane fragments (microsomes) which form a very heterogeneous fraction in brain (Aldridge and Johnson, 1959; Toschi, 1959). T H E S U B C E L L U L A R L O C A L I Z A T I O N OF B I O G E N I C AMINES I N BRAIN

The successful isolation of most of the bound ACh of brain in a specific fraction consisting mainly of NEPs prompted us to enquire whether other biogenic amines have a similar distribution. Our results to date are presented in Tables I11 and IV.

Fig. 4.Thin section of PZ fraction showing main types of particles present: NEPs, mitochondria, both free and within NEP cytoplasm, myelin fragments (my) and microsomes (mi). Preparation and acknowledgements as in Fig. 2. Fig. 5. Appearance of similar particles in an unsectioned negatively stained preparation. Abbreviations as in Figs. 2 and 4.The boundary between the dark and light backgrounds shows the limit of spread of phosphotungstate. A mitochondrion and synaptic vesicles are dimly seen within the intact external metnbrane of the NEP. The periodic structure of myelin is visible at arrow. Preparation fixed with neutralized 5 ”/, formaldehyde in 0.32 Msucrose before negative staining. References p . 115-117

T A B L E 111 DISTRIBUTION OF BIOGENIC AMINES I N SUBCELLULAR FRACTIONS OF GUINEA-PIG BRAIN Figures in brackets indicate the % distribution in the primary particulate fractions (PI P2 P3 = 100)

+ +

% Total activify recovered in1

Concentration Amine

in homogenafe

Recovery

% Total activity recovered in

I % of

Recovery (%of

(,ug/g tissue) 2

P1

Pz

pa

S3

hornogenate)

A

B

C

P2)

ACh Whole brain

2.33

11 (14)

55 (77)

6 (9)

28

95

33

67

0

96

HT Whole brain Caudate nucleus 4

0.505 -

5(11)

41 (89) - (49)

O(0) ~ ( 2 3 )

54 -

113 -

12 31

64

- (28)

53

24 13

106 -

DA Caudate nucleus 4

6.61

16 (36)

22 (50)

6 (14)

56

73

42

51

7

74

0.29

28 (44)

25 (39)

11 (17)

36

110

-

-

81 15 (20)

5 39 (54)

-

-

-

-

-

37

53

10

91

Histamine Whole brain Pituitary 4 Hypothalamus

8.45 1.48

a-

14 19 (26) \

27-

95 97

-

Substance P5 Whole brain

72

8 (9)

74 (88)

2 (3)

16

89

34

75

1

72

ATP Whole brain

224

20 (22)

72 (77)

l(1)

7

69

17

71

12

84

Abbreviations as in text. Weights refer to free base or (for ACh) to chloride. Total ACh; ACh in S3 is all in free form. Units/g tissue (fowl rectal caecum). Dog.

99

S T O R A G E SITES O F B I O G E N I C A M I N E S

T A B L E IV D I S T R I B U T I O N O F AMINO ACIDS I N S U B C E L L U L A R F R A C T I O N S OF G U I N E A - P I G B R A I N

For brevity, values for PZhave been omitted and contents of fractions A, B and C have been expressed as % of total aminoacids recovered in all fractions. Concentration in homogenate

% Total amino acid recovered in

Amino acid

(Prnoleslg tissue)

PI

Glutamic Aspartic y- Aminobutyric Glutamine asparagine** Glycine Serine a-Alanine Threonine LDH

7.90 2.54 2.03 1.38 1.13 1.11 0.78 0.28 122***

5 8 5 1 0 7 4 8 5 9 5 8 5 11 3 6 6 5

+-

* ** ***

<;

+ +

recovered in A B Not separated. nEsdo/min/g tissue.

S3;

B

A

1

C

1 7 11

1

2 1 -

1 2 1 0 3 1 6 3 14 4 1 5 0 7 1

P3

S3

3 7 1 3 7 4 82 3 7 2 3 7 0 4 6 4 4 62 0 7 6 3 8 7

Recovery (%of homogenate)

89 102 126* 102 90 89 85 121 86

other fractions not analysed.

Hydroxytryptamine (HT) and dopamine (DA)

These amines have somewhat similar distributions (Whittaker, 1959a, b ; Michaelson and Whittaker, 1962a, 1963; Laverty, Michaelson, Sharman and Whittaker, 1963). Up to 56 % may appear in the high-speed supernatant fraction as against 28 % for ACh. However, in the case of HT, if the high-speed supernatant is prepared directly from the homogenate, the proportion is lower, suggesbing that this amine is not as tightly bound as ACh and leaks out during manipulation. The Pa fraction is the richest in amines of the primary particulate fractions and fraction B (containing NEPs) the richest of the subfractions. Experiments in which the B fraction was subdivided into a number of smaller fractions in semi-continuous density gradients showed some separation between HT and ACh (Michaelson and Whittaker, 1962a, 1963),ACh being preferentially concentrated in the fractions containing lighter and smaller NEPs. Substantial overlap occurred, however, and the densest fraction of NEPs and the mitochondria1 fraction were both low in these amines. The localization of DA and HT in the particulate fractions from caudate nucleus was not as sharp as those of H T and ACh in whole brain; electron microscopy showed that large NEPs were present in both the PI and A fractions, so that a location in NEPs is not excluded. 5-Hydroxyindole acetic acid

In contrast to HT, 5-hydroxyindole acetic acid was localized mainly in the high-speed supernatant fraction (Sharman and Whittaker, unpublished observations). Histamine

Michaelson and Whittaker (1962b) and Michaelson and Dowe (1963) showed a bimodal distribution of the bound fraction in whole guinea-pig brain, slightly more References p . 115-11 7

100

V. P. W H I T T A K E R

being recovered in the PI than in the PZfraction. The histamine content of the Pi fraction was shown to be of mast-cell origin by tagging experiments with rat peritoneal mast cells which increased only the PI moiety and by fractionation experiments with dog pituitary, rich in mast cells, in which almost all the histamine was recovered in the P1 fraction. The PZfraction appears to represent non-mast cell histamine, since with dog hypothalamus, an area containing appreciable amounts of histamine but no mast cells (Adam, 1961), particulate histamine was recovered mainlyin the Pa fraction. On subfractionation, the most active particulate fraction was B and the general pattern of distribution was similar to that of the other amines. Substance P This has proved difficult to study owing to the presence of interfering substances including other polypeptides and UDP. The recent availability of pure substance P and the development of a new and very sensitive preparation, the gold fish intestine suspended in a 0.05 ml capacity organ bath, have made possible new and very stringent tests. Parallel assays have revealed the previously unsuspected presence of interfering substances even in highly purified standard preparations of substance P (Gaddum, 1961; Cleugh, Gaddum, Mitchell and Smith, 1963). Preliminary results (reported by Whittaker, 1962 and Gaddum, 1961), using the fowl rectal caecum (Cleugh, Gaddum, Holton and Leach, 1961) (Table III), indicated a localization of substance P in the PZand B fractions even sharper than that of ACh. However, parallel assays using the guinea-pig ileum and fowl rectal caecum, the latter regarded as the most reliable and specific assay method, often failed to agree. It seems clear that distribution studies based on one assay procedure alone are unreliable. A new assay procedure (Cleugh et al., 1963) based on a separation of substance P from interfering substances on an ion-exchange column followed by parallel assays on the gold fish intestine, fowl rectal caecum and guinea-pig ileum is proving promising. Adenosine triphosphate ( A T P ) This compound has been proposed as a transmitter substance in sensory pathways (Holton, 1959) and has been implicated in the binding of pressor amines to nonnervous storage granules (Blaschko, 1959). NEPs were found to contain considerable quantities of bound ATP. The bound nucleotide was rather uniformly distributed throughout the brain, being present in both white matter and grey, and there was no correlation with the distribution of pressor amines (Nyman and Whittaker, 1963; Whittaker, 1962). It seems unlikely, therefore, that all the ATP measured in these experiments was concerned with the two functions mentioned above, though it is not excluded that some of it was. Amino acids" The general excitant action of glutamate and certain other amino acids on central neurones when applied iontophoretically and the depressant effect under the same conditions of y-aminobutyrate and related compounds (Table I) has suggested that

* Mangan and Whittaker, unpublished work.

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certain amino acids may act as chemical moderators or regulators of neuronal activity and perhaps also as transmitters. It was therefore of interest to determine the free amino acid content of the various brain fractions. The analyses were carried out on an automatic amino acid analyser. Eight main amino acid peaks were obtained. These corresponded (in order of increasing retention volume) to aspartic acid, threonine, glutamine and/or asparagine (not resolved by the procedure used), glutamic acid, glycine, a-alanine and finally y-aminobutyric acid. In addition, there were small peaks corresponding to taurine, urea and three unidentified compounds and larger peaks corresponding to glycerophosphoethanolamine, phosphoethanolamine and phosphoserine. The chromatograms were qualitatively similar for all fractions with minor differences. Preliminary results for the distribution among the various fractions of the 8 main amino acids are given in Table IV together with that of LDH for the same fractions. It is interesting that the 3 amino acids most active on neurones, the excitatory compounds glutamate and aspartate and the inhibitory y-aminobutyrate, are the most abundant. The distribution pattern of all the amino acids is remarkably similar. Of the total amino acid recovered 62-76 % of each (82 % in the case of y-aminobutyrate for which only three fractions were analysed) were found in the high-speed soluble cytoplasmic fraction, SS.The particulate fractions richest in amino acids were the PZ fraction (usually over 20 %) and the B fraction (10-1 6 % of the total material recovered). This pattern of distribution is in marked contrast to that of the pressor amines and ACh but closely resembles that of the soluble cytoplasmic marker, LDH. It would thus appear that the free amino acids of brain either leak out from their binding sites very rapidly, or, more likely, are constituents of the soluble cytoplasm and that the content of free amino acids in the nerve-ending fraction can be accounted for by the presence of entrapped cytoplasm in this fraction. In summary, a number of biogenic amines known to exert stimulatory or inhibitory effects on neurones are found to be preferentially localized in the nerve-ending fraction. In almost all cases, a considerable proportion is also recovered in the nonparticulate supernatant fraction where they are presumably in the free form; however, the proportion in this fraction is not as high as would be expected of compounds possessing simply a soluble cytoplasmic localization. By contrast, free amino acids, including those with marked excitatory or inhibitory effects on neurones, do not appear to be specifically localized in nerve endings although the nerve-ending cytoplasm contains them in fairly high concentrations. This may be regarded as primafacie evidence against a transmitter role for these amino acids, though it does not preclude some other kind of regulatory role in nervous function. Other substances and enzymes which are not likely to be involved directly in synaptic function (e.g. the metabolite of HT, 5-hydroxyindole acetic acid) are also not specifically localized in the nerve-ending fraction. FORMATION A N D PROPERTIES OF

NEPS

At first sight it may seem remarkable that nerve endings survive homogenization and References p . 115-117

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can be isolated in particulate form. There is, however, good evidence that nerve endings possess a greater mechanical strength than the structures which surround them and further that sucrose assists in the pulling away and pinching off of the nerve ending. Electron micrographs of the torn edges of thin sections of whole cortical tissue show the club-like nerve endings pulling away from disintegrating dendrites (Gray, 1959). The nerve endings also survive as organized structures in teased or squashed brain and can be seen clearly by negative staining methods (vide infru) (Gray, personal communication). When brain slices are soaked in sucrose for 5-30 min characteristic changes occur which aid the separation of nerve endings (Gray, observations reported by Whittaker and Gray, 1962; Whittaker, 1963). These include shrinking and pulling away of the nerve endings from surrounding structures. There seems little doubt, therefore, that the mechanical properties of nerve endings and their response to exposure to sucrose assist the formation of NEPs during homogenization. NEPs can be formed from endings of greatly varying size. Thus homogenates of cerebellar cortex contain giant NEPs several ,u in diameter derived from the mossy fibre endings. These sediment in the nuclear fraction and have postsynaptic granulecell processes still adhering to them. Smaller NEPs apparently derived from the granule-cell endings in the molecular layer were identified in the supernatant from this fraction; these could be seen still enveloping torn off dendrite spines derived from Purkinje cells (Whittaker, 1963). Large NEPs are also seen in preparations from the caudate nucleus (Laverty, Michaelson, Sharman and Whittaker, 1963). The isolation of NEPs of distinctive morphology from localized areas of the CNS opens up the possibility of identifying the chemical transmitter substances operating at such synapses. This approach may help to obviate the difficulties which arise when working with a tissue as heterogeneous as whole brain. Although the structure of nerve endings is robust enough to ensure their survival during homogenization, it must be stressed that they are extremely labile structures (Gray and Whittaker, 1962), much less stable than mitochondria, for example, and osmotically and thermally sensitive. Brief exposure to temperatures above O", to acid or alkaline pH, organic solvents, lytic enzymes or detergents leads to extensive breakdown and release of ACh. Hypotonic dilution, freezing and thawing and mechanical disintegration have less dramatic effects and release about 50% of bound ACh (Whittaker, 1959a). In partially disrupted preparations, particularly those subjected to treatments such as brief autolysis, mechanical disintegration and hypotonic dilution, which affect the external membrane first, the synaptic vesicles are often seen hanging together in clumps as though embedded in a sticky cytoplasm (Whittaker, 1963). In hypertonic solutions, shrinkage occurs without release of ACh; NEPs become bizarre in shape and the cytoplasmic organelles within tend to fuse into an intensely osmiophilic mass. This process is reversible if it has not gone too far (Gray and Whittaker, 1962). NEPs treated with fixatives such as formaldehyde or osmium tetroxide are particularly fragile and very easily disrupted by mechanical means. Thus the morphological appearance of fixed preparations is readily affected by the method of prepa-

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ration. Any mechanical handling tends to cause disruption of NEPs and may give a false idea of the degree of organization of the preparation. T h s explains the differences between the first electron micrographs of the B fraction (Whittaker, 1959a), in which isolated vesicles were seen, and subsequent ones (Gray and Whittaker, 1962), in which the vesicles are seen to be present within the external membrane of the nerve ending. A combined morphological and chemical investigation is in progress in order to establish conditions which will permit the isolation of synaptic vesicles. This work is described more fully in a later section. N E G A T I V E S T A I N I N G METHODS

Conventional methods of preparing specimens for electron microscopy - positive staining, embedding and thin sectioning -have considerable limitations when applied to subcellular fractions. They are extremely time consuming, so that in one day a biochemist may produce enough fractions to keep an electron microscopist busy for several weeks. They lack resolution at the macromolecular level and do not do justice to the electron-optical performance of the best commercially available electron microscopes. Recently an alternative technique has been developed known as the negative staining or negative contrast method. In positive staining the contrast between biological membranes and their background, usually too low for effective image-formation, is artificially enhanced by attaching heavy electron-scattering atoms to them. In negative staining the membranes are not chemically modified but are instead surrounded by an electron-dense embedding medium. This is usually potassium phosphotungstate which has the property of drying out of thin films of solution as an electron dense glass, structureless at the limit of resolution of the electron microscope. At pH 7.4, combination of phosphotungstate with proteins or lipids does not take place so that structures containing these substances are clearly seen against the electron dense background of the phosphotungstate. In collaboration with Dr. R. W. Horne (Horne and Whittaker, 1962; Whittaker, 1963) negative staining, originally developed for the study of viruses and bacteria, has been found to be an excellent method for examining subcellular particles from mammalian tissues. Suspensions are simply applied directly to specimen grids, with or without prior fixation, and are allowed to dry in air in the presence of phosphotungstate. Conditions for negative staining are fairly critical and must be found by trial and error for each type of preparation. One difficulty is that in concentrations suitable for negative staining (1-2 %) the phosphotungstate solution is hypotonic, but the presence of sucrose or other solutes in concentrations sufficient to maintain isotonicity disrupts the even pattern of phosphotungstate deposition, and may produce artefacts, difficult to distinguish from certain kinds of subcellular structures on casual examination. The study of osmotically sensitive structures such as NEPs and mitochondria thus References p. 115-117

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presents difficulties which have not been entirely overcome. We have usually diluted out solutes with phosphotungstate after light fixation of suspended material with neutralized aqueous isotonic formaldehyde (0.5-5 %) or osmium tetroxide (0.01-0.5 %) ; however, this fixation does not render biological membranes completely permeable ; it tends to reduce contrast and may modify macromolecular structures. On the other hand, if no fixative is used, osmotically sensitive particles are badly disrupted and the resultant stretching and distortion of membranes may create other types of artefact. Provided the period for which the fixed material is exposed to hypotonic conditions is kept very short, appreciable distortion does not occur and for simple identification of subcellular particles negative staining provides a rapid and extremely effective procedure. Application of material to the grid is usually made by micropipette rather than by spraying to avoid mechanical damage to labile structures. The suspension may be diluted with phosphotungstate before application and the droplet removed from the grid with filter paper to leave a thin film of suspension which rapidly dries, or the suspension may be applied without prior addition of phosphotungstate and be followed by a drop of the latter. Critical factors are: particle concentration, final concentration of solute (for sucrose this should be 0.04 M or less) and the formation of thin films (wetting of the grid, assisted by soluble protein, is essential for this). It will be appreciated from the foregoing description that negatively stained preparations are not sectioned; the collapsed and dried particles are examined as a whole (cp. Figs. 4 and 5). Initially, problems of identification arise but when these have been solved by the examination of more or less homogeneous fractions by both positive and negative staining methods, the latter may give valuable additional morphological information, as for example in the case of mitochondria1 structure (Whittaker, 1963; Horne and Whittaker, 1962). To sum up, the advantages of the negative staining method are: rapidity, high resolution down to macromolecular levels, and provision of morphological information complementary to that obtained from thin sections. The main disadvantage of the method is that it cannot be applied to intact tissues, though valuable results are being obtained with teased or squashed tissue preparations (Gray, personal communication). DISRUPTION OF

NEPS

A N D ISOLATION OF S Y N A P T I C VESICLES

As already mentioned, the isolation of nerve endings as distinct subcellular particles in which all the morphological features of the nerve ending have been preserved, opens up the possibility of isolating synaptic vesicles, intraneuronal mitochondria, outer membranes, postsynaptic membranes and nerve-ending cytoplasm in relatively pure form for biochemical analysis. In this section an account will be given of work which has been carried out in my laboratory along these lines. Preliminary results with positive staining and thin sectioning showed that disruptive techniques such as hypotonic dilution and incubation at 37" which liberated acetylcholine in part or completely led to extensive morphological changes in the nerveending fraction (Whittaker, 1962). NEPs lost their synaptic vesicles and mitochondria

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and appeared as empty synaptic bags (NEP ghosts) ;clumps of disintegrating vesicles with no outer membrane were sometimes seen especially in the incubated preparations. When, however, attempts were made to define more accurately the changes induced by various kinds of treatment, the inherent limitations of positive staining and thin sectioning became apparent. The transformation of the material accompanying

8oL

d

60

P

20 10 0 Sucrose concentration (cM)

30

Fig. 6. The effect of suspension in hypotonic media (continuous lines) and freezing and thawing (dotted lines) on the release of LDH (triangles) and bound ACh (circles) from the crude mitochondria1 (Pz)fraction of guinea-pig brain (Johnson and Whittaker, 1962, 1963). The open symbols relate to measurements made on hypotonic suspensions at 40 min, the filled symbols to observations at 70 min (LDH) or 80 min (ACh). Their coincidence shows that an end-point had been reached with each solution. The half-filled symbols refer to freezing and thawing.

fixation and staining introduced the possibility of changes greater than those brought about by the treatments themselves, especially the gentler ones. It was not clear that structures as small and delicate as synaptic vesicles, not always very clearly defined when packed together in nerve endings, would be visible at all when liberated. The time taken to process the material was so great that electron microscopy could not be used effectively to help design the techniques required for systematic disruption of the NEPs. The development of negative staining, as described in the previous section, transformed this situation. We also required biochemical markers for the various components of the NEP. In SDH and LDH we had markers for mitochondria and soluble cytoplasm. The components of the acetylcholine system, ACh, ChE and ChA and another putative transmitter, HT, were also measured. We first screened a number of disruptive procedures comparing the release of ACh and LDH. It was reasoned that if ACh was sequestered inside the NEP within a structure such as the synaptic vesicle which survived disruption of the outer membrane, ACh would remain at least partly bound under conditions causing the extensive liberation of LDH. A search was therefore made for conditions which caused a relatively greater release of LDH than of ACh. Fig.6 shows that these conditions are attained when NEPs are suspended in hypotonic media, but not by freezing and thawing. Electron-microscopic investigation of such preparations, using negative staining, revealed an interesting difference in morphology. In hypotonic preparations, there were few intact NEPs, but many in which the outer References p . 115-11 7

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Figs. 7 and 8. For legends seep. 107.

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membrane had ruptured leaving the intraneuronal mitochondria and synaptic vesicles intact (Fig. 7). Empty external membranes (NEP ghosts) could also be seen. The synaptic vesicles were usually present in clumps, but occasionally isolated vesicles were seen, sometimes in drifts relatively free from other membranes (Fig. 8). Isolated vesicles were more easily seen if water-treated NEPs were re-extracted with sucrose (Figs. 9 and 10) or if the larger membrane masses were removed by centrifuging in fields of moderate intensity (e.g. 10,000 x g, 20 min). By contrast, electron micrographs of frozen and thawed preparations from which LDH and ACh are released pari passu, showed few, if any, isolated synaptic vesicles or synaptic vesicle clumps but many NEPs with intact external membranes whose contents showed varying degrees of disruption. It would seem that during hypotonic disruptioc, the external membrane is ruptured first, but during freezing and thawing disruption occurs from within. Our next step (Whittaker, Michaelson and Kirkland, 1963a, b) was to fractionate hypotonic suspensions of NEPs in order to obtain pure monodisperse synaptic vesicles free from partially disrupted NEPs and other membrane fragments. Various procedures have been used, but the most successful so far has been separation on a density gradient built up from sucrose solutions of concentrations ranging from 0.4 to 1.2 M in 0.2 M steps (Fig. 11). Usually NEPs are isolated as rapidly as possible by centrifuging 10 % (w/v) homogenates of guinea-pig brain at 10,000 x g for 20 min after removal of nuclei, large myelin fragments and tissue debris at 1000 x g, 10 min. This preparation, like our usual Pz fraction, contains myelin fragments, microsomes, and free mitochondria as well as NEPs but the microsomal contamination is not as great, Similar results have also been obtained with the B fraction, consisting mainly of NEPs. The pellet is suspended in water and recentrifuged at 10,000 x g for 20 min to remove the larger mitochondria and myelin fragments together with some intact or partially disrupted NEPs. The supernatant is transferred to the density gradient and centrifuged at 53,500 x g for 2 h. The appearance of the tube after spinning is shown in Fig. 11. The density gradient is sliced into seven fractions. The first (0) consists of an optically clear water layer which had originally contained the disrupted NEPs. The second (D) is the now slightly hazy layer of 0.4 M sucrose immediately below 0. The third, fourth, fifth and sixth fractions (E-H) correspond to particulate layers lying between sucrose solutions of increasing density. Fraction I is the pellet in the bottom of the tube.

Fig. 7. Portion of supernatant from water-treated PZpreparations (Pz Ws in text Fig. 11). Note: damaged NEP (DN) with ruptured external membrane, internal mitochondrion and synaptic vesicles which remain clumped together as though embedded in sticky cytoplasm; NEP ghosts (NG) to left containing afew vesicles; microsomes (mi); isolated dispersed particles of about 500 8, in diameter, assumed to be isolated synaptic vesicles (sv); portion of free mitochondrion (rn); non-vesicular membrane fragments (some possibly postsynaptic membranes). This and subsequent Figs. are all of preparations negatively stained after formaldehyde fixation as in Fig. 5. Fig. 8. Area immediately adjacent to that shown in Fig. 7 showing drift of isolated synaptic vesicles relatively uncontaminated with other membrane structures. References p. 115-117

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Figs. 9 and 10. For legends see p. 109.

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1

Nuclri Mytiin

Mitochondria Myriin

bcbris

Mictotorncr

(dtrcardtd)

(discord ad)

N€Ps

Fig. 1 1. Preparation and density gradient fractionation of hypotonically disrupted NEPs. Left : SW 25 lustroid tube of Spinco ultracentrifuge showing water-treated preparation and sucrose density gradient (numbers give molarity of sucrose layers). Right: same after centrifuging showing fractions taken (Whittaker, Michaelson and Kirkland, 1963a, b).

Morphology of fractions

All fractions were subjected to electron-microscopic examination by negative staining. The results are presentedinTable V and Figs. 12-1 7. The most important finding was that the D band consisted almost entirely of monodisperse vesicles identical in size and appearance to synaptic vesicles (Fig. 12). The E layer, though consisting mainly of larger vesicular elements (microsomes) about 0.1-0.2 p in diameter (Fig. 13) also contained considerable numbers of synaptic vesicles, often in clumps. Noteworthy also is the high concentration of non-vesicular membrane fragments in F and G Fig. 9. Sucrose extract of water-treated PZfraction showing isolated synaptic vesicles mixed with microsomes and disintegrated NEPs. Fig. 10. Same as Fig. 9 a t higher magnification showing isolated synaptic vesicles (sv) mixed with larger vesicular structures (microsomes) 0.1-0.4 /c in diameter. From Johnson and Whittaker (1963). References p. 115-1 17

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Figs. 12, 13 and 14. For legends see p. 111.

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TABLE V MORPHOLOGICAL APPEARANCE OF FRACTIONS OBTAINED B Y DENSITY GRADIENT S E P A R A T I O N O F W A T E R - T R E A T E D C R U D E M I T O C H O N D R I A L F R A C T I O N OF G U I N E A - P I G B R A I N

*

Fraction

Density*

0 D E F G

0 0.4 0.4-0.6 0.64.8 0.8-1 .O

H I

1.O-1.2

I .2

Morphology

No organized structures Synaptic vesicles, occasional microsomes Microsomes, clumps of synaptic vesicles NEP ghosts, myelin fragments, postsynaptic membranes (?) NEP ghosts, membrane fragments, postsynaptic membranes (?) Damaged NEPs, NEP ghosts Small mitochondria, some shrunken NEPs

Expressed as the sucrose concentrations in molar units in which the particles float.

resembling postsynaptic membranes (psms) (Figs. 14, 15). H consisted mainly of partly damaged NEPs (Fig. 16) inside which synaptic vesicles could often be clearly seen. Fraction I (Fig. 17) had a high concentration of small mitochondria (presumably released from disrupted NEPs) together with some shrunken NEPs. Fractions F, G and H all contained particles identified as NEP ghosts - damaged NEPs consisting of empty synaptic bags with few or no synaptic vesicles within. All the structures mentioned above were seen in the parent fraction P2Ws (Fig. 7). This corresponds to the combined M2 and Ms fraction of De Robertis eta]. (1962a, b). In electron micrographs of this fraction, we sometimes found areas containing free synaptic vesicles relatively uncontaminated by disrupted NEPs and other components (Fig. 8) but all the particles in Table V could be readily identified in adjacent areas (Fig. 7).

Assays The results of the assays are given in Fig. 18. LDH, representing soluble cytoplasm released from disrupted NEPs, was recovered mainly in the optically clear fraction 0, containing no organized membrane structures in the electron microscope. A dummy run with bovine serum albumin showed that 76% of this soluble protein likewise

Fig. 12. Fraction D derived from a water-treated PZ fraction by density gradient separation (see Fig. 1I). Note abundance of small vesicles (presumably synaptic vesicles) about 500 A in diameter. The contamination with microsomes is much less than in Fig. 10. The mottled background is due to the supporting film. Fig. 13. Fraction E showing drift of closely packed microsomes. In more spread out portions of the grid synaptic vesicles either in isolation or (more usually) in clumps could be seen between the microsomes. Figs. 14 and 15. Fractions F and G showing non-vesicular membranes, some of which could be postsynaptic membranes (psm?), also NEP ghosts (NG), (see p. 112). Fig. 16. Fraction H showing damaged NEPs (DN) and membrane fragments, (see p. 112). Fig. 17. Fraction I showing swollen mitochondria (m). Occasional particles in this fraction could be identified as shrunken or damaged NEPs, (see p. 112). References p . 115-117

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V. P. WHITTAKER

Figs. 15, 16 and 17. For legends see p. 111.

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remained in the top fraction during centrifuging. The lack of appreciable amounts of LDH in lower fractions (H, containing incompletely disrupted NEPs and I, containing a few shrunken NEPs) suggests that few NEPs in P2Ws escaped damage. SDH, in contrast to LDH, was concentrated, as expected, mainly in fraction I, consisting mainly of mitochondria. Appreciable amounts also occurred in fraction H which contained some free mitochondria and others within NEPs. The remaining activity may represent solubilized SDH, the subsidiary peak of relative specific activity (RSA) in fraction E simply resulting from the low nitrogen content in this fraction. ChA had a distribution similar to LDH suggesting that it too is located in the soluble cytoplasm of the nerve ending. By contrast, ChE had a distribution different from both LDH and SDH, peaking in fraction F which is rich in isolated psms. Some activity was present in all fractions. This might have been due to pseudoChE, known to be a readily diffusible enzyme. The most interesting distribution was obtained with ACh. This was bimodal: the greatest activity and specific activity was associated with the synaptic vesicle fraction (D), while a second peak occurred in H in which the incompletely disrupted NEPs were concentrated. These preparations contained no eserine and all had some ChE activity. The ACh measured was therefore bound ACh; it was released before assay by heating at pH 4 and 100" for 10 min according to our usual procedure (Whittaker, 19.59a). The water treatment in fact released over half the bound ACh originally present in PZand this was destroyed. Further losses occurred when PzWl was removed due to the presence of intact or slightly damaged NEPs in this fraction, leaving about one-third for fractionation on the density gradient. The assay of ACh in the extremely small amounts present in the final fractions was achieved by the microassay procedure of Szerb (1961) using a small (8 x 0.2.5 mm) slip of the dorsal muscle of the leech in an organ bath of 0.05 ml capacity. The ACh activity of fraction D was rapidly destroyed after release by blood cholinesterase. The isolation of almost pure synaptic vesicles in fractions containing ACh is the first positive evidence in favour of the view originally advanced by del Castillo and Katz (19.55), De Robertis and Bennett (1955) and others (Fernandez-Morin, 19.57; Palade and Palay, personal communication by Dr. S. L. Palay) that the synaptic vesicles are storage sites for ACh and other transmitters. The separation of the three functional components of the ACh system is of interest; it suggests that ACh is synthesized in the nerve-ending cytoplasm and taken up by the synaptic vesicles. A small uptake of added ACh by intact NEPs has already been observed (Whittaker,'l959a). ChE appears to be localized, at a n y rate in part, postsynaptically. The distribution of bound HT differed from ACh; it was found in all fractions with a diffuse peak. Dummy runs with free H T showed that this small molecule, unlike bovine serum albumin, diffused throughout the density gradient in 2 h, though it did not reach equilibrium in this time. H T is known to be less firmly bound than ACh (Michaelson and Whittaker, 1962~)and the presence of H T throughout the gradient probably represents diffusion of free H T from a binding site in H. There is some evidence of bimodal distribution but it is much less marked than that of ACh. References p. 115-117

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3t

.:LsA 0

D E F G H I

DEFGH I

ChE

Ih

1. 2 1 , 0 ,

0 D E F

H

H I

HT

ACh

0 DEfG

G

I

,

0

00-0

N (% recoveredhl)

50

100

N@recovered N)

Fig. 18. Histograms showing distribution of marker enzymes, components of ACh system and HT in density gradient prepared as in Fig. 11 (Whittaker, Michaelson and Kirkland, 1963a,b). Ordinates give % of total activity recovered in each fraction divided by % nitrogen recovered in each fraction. Each block represents one fraction, the width representing the % of total recovered nitrogen found in that fraction. This varied slightly from one experiment to another.

The yield of synaptic vesicles is difficult to estimate. The protein content of fraction D is not a good guide, because some probably represents soluble protein which has diffused from the nitrogen-rich fraction 0 immediately above. About 10% of the ACh originally present in P2 is recovered in fraction D, so this may be taken to represent the yield of synaptic vesicles in this fraction. The low yield is accounted for by the fact mentioned in an earlier section that when NEPs are disrupted, the vesicles behave as though they are stuck together in a sticky cytoplasm. The properties of this cytoplasm merit further investigation. Possibly, it restrains the vesicles from spontaneous fusion with the outer nerve-ending membrane and so from spontaneous discharge of transmitter. A change in ionic environment (e.g. rise of intracellular Ca") accompanying the arrival of a nerve impulse might liquefy this cytoplasm so speeding up the rate of spontaneous release of transmitter. However, we have not so far found that the addition of ions including Ca2f ion increases the yield of monodispersed synaptic vesicles. Now that the feasibility of subfractionating isolated nerve endings has been demonstrated, it should be possible to study the metabolism and binding of putative

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transmitters within the nerve ending. In preliminary experiments we have found interesting changes in H T distribution in response to drugs which imply changes in intracellular pools. It should also be possible to study in detail the chemical and enzymic content of the various structures involved. Further work along these lines may be expected to yield significant results for our understanding of the role of functionally important biogenic amines in brain. SUMMARY

When brain tissue is homogenized in sucrose, nerve endings are torn away from their pre- and postsynaptic attachments to form subcellular particles which can be isolated in relatively pure form by a combination of differential and density gradient centrifugation. The isolated nerve-ending fraction is rich in biogenic amines: acetylcholine, hydroxytryptamine, dopamine, noradrenaline and substance P. Histamine is bimodally distributed in subcellular brain fractions, mast cell histamine being recovered in the nuclear fraction, while non-mast cell histamine has a distribution resembling the other amines. The nerve-ending fraction is also rich in aminoacids, but this is probably due to the presence of entrapped cytoplasm within the endings. Using lactic dehydrogenase as a marker for the soluble cytoplasm of the nerveending particle, it has been found possible to rupture the external membrane and release most of the soluble cytoplasm of the nerve-ending particles under conditions which permit the survival of about half of their bound acetylcholine. Such preparations contain many intact synaptic vesicles, often in clumps. Density gradient fractionation of the disrupted material resolved it into several subfractions: soluble cytoplasm, isolated synaptic vesicles, microsomes and other membrane fragments, partially disrupted nerve-ending particles and mitochondria. Bound acetylcholine was bimodally distributed, being present in the isolated synaptic vesicle fraction and in the fraction containing partially disrupted nerve endings. Choline acetylase was mainly recovered in the soluble fraction. Cholinesterase was mainly recovered in the microsome fraction. Hydroxytryptamine showed slight evidence of bimodal distribution but much of it appeared to be released during disruption. Lactic dehydrogenase and succinic dehydrogenase had markedly contrasting distributions, the former being recovered in the soluble fraction, the latter in the mitochondria1 fraction. ACKNOWLEDGEMENTS

This work was supported by a grant, No. B-3928, from the National Institute of Neurological Diseases and Blindness, USPHS. Electron microscopy was carried out on a Siemens Elmiskop I electron microscope provided by the Wellcome Trust. REFERENCES ADAM,H. N., (1961); Histamine in the central nervous system and hypophysis of the dog. Regional Neurochemistry : the Regional Chemistry, Physiology and Pharmacology of the Nervous System. S. S. Kety and J. Elkes, Editors. Oxford, Pergamon Press (p. 293).

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ALDRIDCE, W. N., AND JOHNSON, M. K., (1959); Cholinesterase, succinic dehydrogenase, nucleic acids, esterase and glutathione reductase in sub-cellular fractions from rat brain. Biochem. J., 73, 270-216. H., (1959); The development of current concepts of catecholamine formation. Pharmacol. BLASCHKO, Rev., 11, 307-316. BODIAN,D., (1942); Cytological aspects of synaptic function. Physiol. Rev., 22, 146-169. P. B., AND WOLSTENCROFT, J. H., (1962); Excitation and inhibition of brain stem neurones BRADLEY, by nor-adrenaline and acctylcholine. Nature (Lond.), 196, 840 and 873. BRAGANCA, B. M., AND QUASTEL, J. H., (1952); Action of snake vemm on acetylcholine synthesis in brain, Nature (Lond.), 169, 695-697. J., GADDUM, J. H., HOLTON,P., AND LEACH,E., (1961); Assay of substance P on the fowl CLEUGH, rectal caecum. Brit. J. Pharmacol., 17, 144-158. J., GADDUM, J. H., MITCHELL, A . A,, SMITH,M. J., A N D WHITTAKER, V. P., (1963); J . CLEUGH, Physiol. ( L mI.). CURTIS,D. R., (1962); Action of 3-hydroxytyramine and some tryptamine derivatives on spinal neurones. Nature (Lond.), 194, 292. CURTIS,D. R., AND DAVIS,R., (1962); Pharmacological studies upon neurones of the lateralgeniculate nucleus of the cat. Brit. J. Pharmacol., 18, 217-246. CURTIS,D. R., AND DAVIS,R.. (1963); The excitation of lateral geniculate neurones by quaternary ammonium derivatives. J . Physiol. (Lond.), 165, 62-82. CURTIS,D. R., AND KOIZUMI,K., (1961); Chemical transmitter substances in the brain stem of the cat. J. Neurophysiol., 24, 80-90. J. C., (1961); Cholinergic and non-cholinergic transCURTIS,D. R., PHILLIS,J. W., AND WATKINS, mission in the mammalian spinal cord. J . Physiol. (Lond.), 158, 296-323. CURTIS,D. R., AND WATKINS, J. C., (1960); The excitation and depression of spinal neurones by structurally related amino acids. J . Neiirochetn., 6, 117-141. C . , (1959); Subcellular Particles. T. Hayashi, Editor. New York, Ronald Press (p. 128). DE DUVE, DELCASTILLO,J., AND KATZ,B., (1955); Local activity at a depolarized nerve muscle junction. J. Physiol. (Lond.), 128, 396-41 I . DE ROBERTIS, E., AND BENNETT, H. S., (1955); Some features of the submicroscopic morphology of synapses in frog and earthworm. J. biophys. bioclietn. Cytol., 1, 47-56. E., DE TRALDI,A. P., DEL ARNAIZ,G. R., AND SALGANICOFF, L., (1962a); Cholinergic DE ROBERTIS, and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J. Neurochem., 9, 23-35. DE ROBERTIS, E., DEL ARNAIZ,G. R., AND DE IRALDI,A. P., (1962b); Isolation of synaptic vesicles from nerve endings of the rat brain. Nature (Loncl.), 194, 794-795. DEROBERTIS, E., DE IRALDI,A. P., RODRIGUEZ, G., AND GOMEZ,J., (1961); On the isolation of nerve endings and synaptic vesicles. J. biophys. biochem. Cytol., 9, 229-235. FELDBERG, W., (1945); Present views on the mode of action of acetylcholine in the central nervous system. Physiol. Rev., 25, 596-642. H., (1957); Metabolism of the Nervous System. D. Richter, Editor. London, FERNANDEZ-MORAN, Pergamon Press (p. 1). GADDUM, J. H., (1961); Symposium on Substance P, June 9-10, Sarajevo. Proc. Sci. Soc., Bosnia and Herzegovina, Yugoslavia, 1 (Dept. med. Sci. Issue l), 7. GADDUM, J. H., (1962); Substances Released in Nervous Activity. Proc. First intern. Pharmacol. Meeting. 8. ModeofActionofDrugs. W.D. M. PatonandP.Lindgren,Editors.London,PergamonPress(p. I). GRAY,E. G., (1959); Axo-somatic and axo-dendritic synapses of the cerebral cortex; an electron microscope study. J . Anat. (Lond.), 93, 420-433. V. P., (1960); The isolation of synaptic vesicles from the central GRAY,E. G., A N D WHITTAKER, nervous system. J. Physiol. (Lond.)? 153, 3 5 P . GRAY,E. G., AND WHITTAKER, V. P., (1962); The isolation of nerve endings from brain: an electronmicroscopic study of cell fragments derived by homogenization and centrifugation. J . Anat. (Lond.), 96, 79-88. HEBB,C . O., AND SMALLMAN, B. N., (1956); Intracellular distribution of choline acetylase. J. Physiol. (Lond.), 134, 385-392. V. P., (1958); Intracellular distributions of acetylcholine and choline HEBB,C . O., AND WHITTAKER, acetylase. J . Physiol. (Lond.), 142, 187-196. HOLTON,P., ( I 959); The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves. J . Physiol. (Loncl.), 145, 494-504.

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HORNE,R. W., AND WHITTAKER, V. P., (1962); The use of the negativeIstaining method for the electron-microscopic study of subcellular particles from animal tissues. Z. Zellforsch., 58, 1-16. JOHNSON, M. K., (1960); The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondria1 preparations. Biocliem. J., 77, 610-618. JOHNSON, M. K., AND WHITTAKER, V. P., (1962); Abstracts of’the International Neurochemical Symposium. Cotlienburg, June 17-21 (p. 60). Reprinted in Acta neurol. Scand., Suppl. I , 38, 60. JOHNSON, M . K., AND WHITTAKER, V. P., (1963); Lactic dehydrogenase as a cytoplasmic marker in brain. Biocliem. J., 88, 404-409. KRNJEVIC, K., AND PHILLIS, J. W., (1961); The actions of certain aminoacids on cortical neurones. J . Physiol. (Lontt.), 159, 62-63P. KRNJEVIC, K . , AND PHILLIS, J. (1962); Actions of certain amines on cerebral cortical neurones. Brit. J . Pliarmacol., 20, 471-490. LAVERTY, R.,MICHAELSON, 1. A., SHARMAN, D. F., AND WHITTAKER, V. P., (1963); The subcellular localization of dopamine and acetylcholine in the dog caudate nucleus. Brit. J . Pliarmacol., in the press. MANN, P. J. G . , TENNENBAUM, M., A N D QUASTEL, J . H., (1939); Acetylcholine metabolism in the central nervous system. Biochem. J., 33, 1506-1518. MARRAZZI, A. S., (1957); The effects of certain drugs on cerebral synapses. Ann. N . Y. Acad. Sci., 66, 496-507. MICHAELSON, 1. A., A N D DOWE, G., (1963); The subcellular distribution of histamine in brain tissue. Biochem. Pharmacol., 12, 949-956. MICHAELSON, I. A., AND WHITTAKER, V. P., (1962a); The distribution of hydroxytryptamine in brain fractions. Biochem. Pharmacol., 11, 505. MICHAELSON, 1. A,, A N D WHITTAKER, V. P., (1962b); The subcellular distribution of histamine in guinea pig brain. Biochem. J., 84, 3 1 ~ . MICHAELSON, T. A., AND WHITTAKER,V. P., (1962~);The subcellular localization of S-hydroxytryptamine in guinea-pig brain. Biochem. Pharmacol., 12, 203-21 1. MITCHELL, J. F., (1963); The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol. (Lond.), 165, 98-116. NYMAN,M . , A N D WHITTAKER, V. P., (1963); The distribution of adenosine triphosphate in subcellular fractions of brain tissue. Biochem. J . , 87, 248-255. PETRUSHKA, E., AND GIUDITTA, A,, (1959); Electron microscopy of two subcellular fractions isolated from cerebral cortex homogenate. J . biophys. biochem. Cytol., 6, 129-1 32. STEDMAN, E., A N D STEDMAN, E., (1939); The mechanism of the biological synthesis of acetylcholine. Biochem. J., 33, 811-821. SZERB,J. C., (1961); The estimation of acetylcholine, using leech muscle in a microbath. J . Pliysiol. (Lontl.), 158, 8P. TOSCHI,G., (1959); A biochcrnical study of brain microsomes. Exp. Cell Res., 16, 232-255. WHITTAKER, V. P., (195Ya); The isolation and characterization of acetylcholine containing particles from brain. Biochem. J., 72, 694-706. WHITTAKER, V. P., (195Yb); A comparison of the distribution of lysosome enzymes and S-hydroxytryptamine with that of acetylcholine in subcellular fractions of guinea pig brain. Biochem. Pharniacol., 1, 351. WHITTAKER, V. P., (1961); The binding of neurohormones by subcellular particles of brain tissue. Regional Neurocheniistry : the Regional Chemistry, Physiology and Pharmacology of the Nervous System. S . S . Kety and J. Elkes, Editors. Oxford, Pergamon Press (p. 479). WHITTAKER, V. P., (1962); Pharmacological studies with isolated cell components. 5. Methods for the study of pharmacological effects at the cellular and subcellular levels. Proc. First intern. Pharmacol. Meeting. 0. H. Lowry and P. Lindgren, Editors. London, Pergamon Press (p. 61). WHITTAKER, V. P., (1963); The separation of subcellular structures from brain tissue. Methods of Separation of Subcellular Structural Components (Biocliemical Society Symposium No. 23). Cambridge, University Press. WHITTAKER, V. P., A N D GRAY,E. G., (1962); The synapse: biology and morphology. Brit. med. Bull., 18, 223-228. WHITTAKER, V. P., MICHAELSON, I. A,, AND KIRKLAND, R. J., (1963a); The separation of synaptic vesicles from disrupted nerve ending particles. Biochem. Pharmacol., 12, 300-302. WHITTAKER, V. P., MICHAELSON, I. A., AND KIRKLAND, R. J . A,, (1963b); The separation of synaptic vesicles from nerve-ending particles (synaptosomes). Biochem. J., in the press.

w.,

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Electron Microscope and Chemical Study of Binding Sites of Brain Biogenic Amines * E D U A R D O D E ROBERTIS Instituto de Anatomia General y Embriologia, Facultad de Ciencias Midicas, Buenos Aires (Argentina)

Modern neurobiology has recognized the importance of an integrated study of the chemical and structural organization of the CNS. The concept has emerged, among other facts, from the high degree of functional complexity of the nervous tissue, the discovery of numerous metabolic pools and the extraordinary cellular and subcellular compartmentalization demonstrated by high-resolution morphology. The need for such a correlation becomes more pronounced when specific neuronal functions such as the energy-dependent ion shifts occurring in nerve conduction, the release of transmitters at the nerve endings or their reaction with special chemical receptors are taken into account. Such an integrated analysis can be made by combining histochemical methods, or biochemical or pharmacological techniques of assay with morphological observations at all levels of organization. Thus the classical work of Vogt (1954) on the neuroanatomical localization of catecholamines has been recently continued with fluorescence microscopy to a cellular level by Carlsson et al. (1962), and to the resolution provided by the electron microscope into special synaptic vesicles, within axons and nerve endings of the hypothalamus, in our laboratory (Pellegrino de lraldi et al., 1963). Cell fractionation methods which have produced such important data on the biochemical architecture and metabolism of subcellular particles has yielded up to recent times rather meager results when applied to the central nervous system. This is due to the finely subdivided structure of this tissue and to the fact that a greater part of it is composed of submicroscopic components that escape analysis with light optical instruments. The so-called neuropils that constitute the larger proportion of the brain gray matter appear under the electron microscope as formed by tightly packed axons, dendrites, glial processes and synaptic endings, many of which are of submicroscopic dimcnsions (Fig. 1). The effect of sheer force used in homogenization on this profusely branched tissue is difficult to predict and certainly can not be inferred from what is known for the cell fractionation of liver, kidney and other parenchymal tissues. In the case of the brain it is indispensable to have on hand the

*

This work was supported by a grant of the National Institutes of Health.

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Fig. 1. Electron micrograph of a neuropil region of the molecular layer of the cerebellar cortex. Numerous fine axons, dendrites (d) and nerve endings (e) are observed. mi = mitochondria; my = myelin fibre; v = vacuoles in dendrites, x 30,000. References p . 135l136

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E. D E R O B E R T I S

electron microscope identification of the structures present in the subcellular fractions. In recent years the work of our laboratory has beenconcentrated on the development of methods for the isolation of brain components such as myelin, neuronal and glial mitochondria, nerve endings and synaptic vesicles (De Robertis et a/., 1961 ; De Robertis et al.,1962a; Rodriguez de Lores Arnaiz and De Robertis, 1962; De Robertis et al., 1962b). These methods permit a morpho-biochemical correlation in order to determine the localization and metabolism of components that may be involved in important nerve functions. Thus to consider a substance as a transmitter or involved in some other synaptic function, it is prerequisite that it should be localized in nerve endings and preferably in synaptic vesicles. Furthermore, the enzymes directly involved in its synthesis or inactivation should be localized within the synaptic complex or in their immediate proximity. Due to their possible role in synaptic transmission or in other neuronal functions, this work was started with the localization of some biogenic amines and related enzymes (De Robertis et a/., 1962a; Rodriguez de Lores Arnaiz and De Robertis, 1962; De Robertis et a/., 1963). At present, it is being continued with an investigation of substance P and of the enzymes involved in the metabolism of y-aminobutyric acid (Salganicoff and De Robertis, 1963). We will devote the greater part of this presentation to a short description of the isolation methods and a brief account of the pertinent results. ISOLATION OF NERVE ENDINGS FROM THE

CNS

The discovery of synaptic vesicles as the most characteristic morphological component of nerve endings (De Robertis and Bennett, 1955) paved the way to the identification TABLE I TECHNIQUES USED IN PREPARATION O F PRIMARY FRACTIONS AND 'SUBMITOCHONDRIAL' FRACTIONS FROM RAT BRAIN

Fractions

Conditions

N

900 x g, 10 min 2 washings 11,500 x g, 30 min 1 washing

Mit

:/ C

.I

lsorlensity of fraction (d)

0.32

Ultrastrrtcture

Nuclei, capillaries, myelin Myelin, mitochondria, nerve endings

0.32

0.8

1.090 1.120

50,000 x g, 2 h

1.o 1.2 1.4

1. I60 I . 175 1.190

100,000 x g, 30 min Supernatant

0.32 0.32

E

M ic SUP

Sucrose gradietrt ( M )

Myelin Synaptic debris, membranes Nerve endings Nerve endings Free mitochondria Microsomes Soluble

Primary fractions: N=nuclear, Mit =mitochondrial, Mic=microsomal and Sup-supernatant, and submitochondrial fractions A, B, C, D, E (see De Robertis et a/. 1962a).

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of these structures in mammalian brain homogenates. This was done independently by Gray and Whittaker (1960) and De Robertis et al. (1960 and 1961) after their demonstration that the so-called mitochondrial fraction of the brain (Mit) contains numerous nerve endings in addition to free mitochondria and myelin. Recently a technique was worked out for the isolation of intact nerve endings or of large fragments of them, which also avoids considerable breakage (De Robertis et al., 1962a) (Table I). The presence of a certain level of concentration of Ca2+ ions was found to be important to improve the results and to obtain more purified subfractions. The excellent preservations of these isolated nerve endings permitted the demonstration of the intersynaptic $laments and subsynaptic web whch together with the synaptic membranes, are parts of the synaptic complex (De Robertis, 1962). The subfractionation of the crude mitochondrial fraction in a gradient of density varying between 0.8 and 1.4 A4 sucrose gave the subfractions shown in Table I. With this method two fractions (C and D) of nerve endings and one fraction (B) containing membranes and broken nerve endings can be separated from myelin fragments(A) and free neuronal and glial mitochondria (E) (De Robertis et al., 1962a).

Fig. 2. (A) Isolated nerve ending showing mitochondria (mi) and synaptic vesicles (sv). ssw = subsynaptic web. Arrows indicate the synaptic cleft, x 100,000.(B) Nerve ending ghost after hyposmotic shock found in fraction MI. See the disappearance of the synaptic vesicles and mitochondria from the ending. ssw = subsynaptic web, Arrows point to the synaptic cleft, x 72,000. References p . 135/136

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E. D E R O B E R T I S

Fig. 3. (A) Isolated nerve ending showing one mitochondrion (mi) and synaptic vesicles (sv). ssw -~ subsynaptic web. Arrows point to the synaptic cleft, x 80,000. (B) Isolated synaptic vesicles (sv) found in fraction Ma, x 80,000.

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I S O L A T I O N O F S Y N A P T I C VESICLES

More recently a technique was developed in our laboratory which permits a mechanical dissection of the nerve endings and the separation of synaptic vesicles (De Robertis et al., 1962b). If freshly prepared nerve endings present in the primary fraction Mit (Figs. 2 and 3) are hyposmotically shocked, the synaptic vesicles are released from the ending and can be isolated by differential centrifugation. Under the electron microscope one may study: a subfraction MI, containing myelin fragments, swollen mitochondria and nerve ending ghosts, which have lost most of the synaptic vesicles, a subfraction Mz mainly containing synaptic vesicles and curved membranes of probable synaptic nature and a soluble supernatant M3 (Table 11). By studying the vesicles with a TABLE 11 T E C H N I Q U E FOR T H E P R E P A R A T I O N OF S U B F R A C T I O N S O F T H E M I T O C H O N D R I A L

FRACTION

MIT A F T E R

HYPOSMOTIC SHOCK

(see De Robertis et al., 1962b, 1963)

Fractions Mi Mz M3

Conditions

Ultrasrructure

1 1,500 x g, 30 min Myelin, mitochondria, nerve ending ghosts 100,000 x g, 30 min Synaptic vesicles, membranes Supernatant Soluble

negative staining technique using phosphotungstate one may observe fine details of structure such as the presence of small pores, which may be due to the osmotic shock, and fine particles on the vesicular membrane (Figs. 4 and 5). In addition to the most common or simple type of synaptic vesicles there are others that have a ring of particulated or microvesicular material around them and which we have described as annular vesicles (De Robertis et al., 1963). There are also some dense vesicles containing a dark granule of reduced osmium. These granular vesicles are similar to those described by De Robertis and Pellegrino de Iraldi (1961) in adrenergic nerves and endings and probably contain reducing catecholamines (Fig. 6). Similar granules containing vesicles have now been observed in synapses of the anterior hypothalamus of the rat (Pellegrino de Iraldi et al., 1963) (Fig. 7). ACETYLCHOLINE SYSTEM

Acetylcholine (ACh) and other transmitter substances are contained in the nervous tissue mainly in a “bound” form, suggesting an association with some subcellular particle. Indirect support for this concept may be found in the early experiments of Loewi and Hellauer (Loewi, 1956) who showed that, in an extract of nerve tissue in Ringer, ACh is found mainly in the insoluble residue. Feldberg (1957) gave some of the first pieces of evidence that ACh is bound with a protein particulated component, which was generally identified as mitochondria; other authors maintained a similar References p. 1351136

I24

E. D E R O B E R T I S

Fig. 4. Isolated synaptic vesicles (sv) found in fraction Mz after negative staining of the pellet, x 60,000.

viewpoint and actually measured ACh in the mitochondria1 fraction (Bellamy, 1959). It is generally admitted that the presence of ChAc in any neuron identifies it as cholinergic (see Hebb, 1957). Feldberg and Vogt (1948) observed the presence of cholinergic neurons in the CNS, but only in a fraction of its total population. They concluded that the concentration of ChAc in the different parts of the CNS is directly related to the concentration of cholinergic neurons. This contention has also been

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125

neurotubule; sv = synaptic vesicles. Arrows Fig. 5. The same as Fig. 4 at higher magnification. nt point to vesicles in which fine structure is better seen, x 200,000.

Rrferences p . 1351136

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E. D E R O B E R T I S

Fig. 6. The same as Fig. 5, showing different types of synaptic vesicles found in fraction Mz. asv annular synaptic vesicle; fsv = filled synaptic vesicle without penetration of the phosphotungstate; esv = empty synaptic vesicle in which the staining penetrates. With arrows are shown some granulated synaptic vesicles containing a dense granule, x 100,000. ~

supported by the more detailed work of Hebb and Silver (1956). Burgen and Chipinan (1951) postulated that between the three components of the ACh system, i.e. ACh content, ChAc, and AChE activity, “there is a reasonable degree of correlation in

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127

Fig. 7. Synaptic endings in the anterior hypothalamus of the rat showing typical synaptic vesicles (sv) and granular vesicles (gv) with a dense granule inside. x 45,000.

most areas of the brain”. However there are some definite exceptions to this rule. In fact there are some pieces of evidence that ChAc and AChE may be spatially separated (Hebb, 1957). References p . 1351136

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E. D E R O B E R T I S

Regarding the intracellular distribution of ChAc, Hebb and Smallman (1956) showed that between 50 and 70 % exists in the particulate fraction of the brain identified by Brody and Bain (1952) as being mitochondrial in nature. In a later paper based on subfractionation Hebb and Whittaker (1958) could separate on a gradient the particles containing ChAc from those, presumably true mitochondria, containing SDH. In these papers and in that of Bellamy ( I 959) the close relationship of ChAc with ACh was emphasized. It was also found that while a small part of the enzyme was in the microsomes, about 20-28 % was found in the supernatant in a rather soluble form. Whittaker (1959) observed that ACh and 5-hydroxytryptamine were localized in a vesicular layer which could be separated on a gradient from mitochondria and first interpreted it as being composed of synaptic vesicles. This interpretation was difficult to reconcile with the fact that synaptic vesicles have a mean diameter of 400-500 A, which corresponds to small microsomes. This contradictory point was clarified independently by De Robertis ct a/. (1960 and 1961) and Gray and Whittaker (1960) with the demonstration that in the mitochondrial fraction there were numerous intact nerve endings. The studies of subcellular localization of ACh have also been extended to AChE, another enzyme related to the physiological inactivation of this transmitter. Richter and Hullin (1951) observed a considerable concentration of AChE in nuclei isolated from the human cortex by the technique of Dounce. This unusual result was most certainly due to contamination. Nathan and Aprison (1955) found that the large cytoplasmic particles and mitochondria of the caudate nucleus of the rabbit had 70 of the AChE. Toschi (1959) and Aldridge and Johnson (1959) reported that the largest concentration of AChE was in the cytoplasmic particles (mitochondria microsomes) with the highest specific activity in the microsomes. Toschi (1959) postulated that the AChE activity is bound to membranes lacking ribosomes, which probably originate from the endoplasmic reticulum (see also Hanzon and Toschi, 1959). Smallman and Wolfe (1957) observed that in homogenates of insect nervous tissue, the greater concentration of this enzyme is in the large cellular fragments with a lesser amount in the microsomes. The cholinesterase activity found in the CNS is, i n its greater part, the true or AChE type. According to Davison (1 953), in the whole homogenate of the rat brain, there is a low percentage of pseudo-ChE. With histochemical methods this enzyme has been found to occur only in the fibrous astrocytes i n the spinal cord of the cat and rooster and in the endothelium of capillaries in the spinal cord of the rat and goldfish. In the CNS of other vertebrate species, including man, there is no histologically demonstrable pseudo-ChE (Brightman and Albers, 1959).

+

ISOLATION OF NERVE E N D I N G S C O N T A I N I N G THE

A C H SYSTEM

De Robertis et a]. (1962a, 1963) have studied the ACh, AChE and ChAc content of the primary N, Mit, Mic and Sup and of the submitochondrial fractions A, B, C, D and E (see Table I). The results shown in Table 111 demonstrate that there is a close parallelism in the distribution of the three components of the ACh system.

129

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TABLE 111 PROTEIN,

SI)H

AND

ACn-smmM

I N PRIMARY A N D SUBFRACTIONS OF

MIT

IN THE

CNS

O F T H E R A T (SEE T A B L E I)

Data from De Robertis

et al.,

RSA

(1962a, 1963) recalculated as percent recovery of total homogenate. = recovery activity/% recovery protein ~

Fraction

P (%)

ACh ( R S A )

ChAc ( R S A )

AChE 1RSA)

N Mit

15.8 39.2 11.0 3.9 4.9 9.4 10.0 20.0 25.3

0.35 I .20 0.27 2.35 4.70 0.79 0.32 1.17 0.98

0.59 1.39 0.51 2.35 2.80 0.92 0.76 0.92 0.51

0.54 1.34 0.2 I 2.95 3.90 I .26 0.77 2.00 0.00

A

B C D E Mic

SUP

SDH (RSA) 0.65 2.02 0.00 0.00 0.65 2.90 5.65 0.12 0.00

All of them are specially concentrated in the nerve endings of subfraction C and to a lesser extent in subfraction B containing membranes and fragmented nerve endings. In the large population of nerve endings represented by subfraction D there is no concentration of the ACh system and for this reason they were early defined as non-cholinergic (De Robertis et a/., 1962a). In the present stage of our work this denomination may be misleading since other non-cholinergic substances thought to be central transmitters such as NA, 5-HT and substance P are also concentrated in subfractions C and B (see below). This still leaves the nerve endings of D with an unknown composition although some positive evidence is now becoming available (Salganicoff and De Robertis, 1963). The difference between the nerve endings of C and D is the density at which they equilibrate in the gradient (Table I). This may be related to the larger content of mitochondria evidenced by the SDH concentration (Table 11). Unpublished work of Gomez (personal communication) demonstrates that the protein/lipid ratio is higher in D than in C, a fact that may also explain the difference in specific gravity between these two nerve ending fractions. In the primary fractions there is a difference between ACh and ChAc as compared to AChE. The latter enzyme is also concentrated in microsomes (Table Ill). L O C A L I Z A T I O N OF THE

ACH SYSTEM

WITHIN THE SYNAPTIC COMPLEX

From the time when the electron microscope revealed the complex organization of the synaptic region it was suggested that within it there would be a fine compartmentalization of enzymes and transmitter substances. From the very beginning, after the discovery of the synaptic vesicles, it was suggested that they could be associated with the storage and probably with the synthesis of transmitter substances. The technique for the isolation of synaptic vesicles described above has now allowed the References p . 135/ 136

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E. DE R O B E R T I S

verification of this hypothesis for the ACh system. As shown in Table IV after the osmotic shock of the mitochondrial fraction, both ACh and ChAc, in spite of some solubilization into Ms, are concentrated into subfraction Mz containing the synaptic vesicles. The ratio RSA M2/RSA MI which expresses the concentration of the different components between the two structural compartments, is very high for ACh and specially for ChAc. These two findings are considered as definite proof that the synaptic vesicles are the carriers of ACh and that they also contain the enzyme directly involved in its synthesis. TABLE IV PROTEIN,

SDH

AND

ACH-SYSTEM IN

MITOCHONDRIAL S U B F R A C T I O N S OBTAINED A F T ~ R

H Y P O S M O T I C SHOCK

(SEE

T A B L E 11)

Data from De Robertis et a/. (1963) recalculated as percent recovery of total homogenate. RSA recovery activity/ % recovery protein

‘7,

~~

M2

Mi

M3 ~

Fraction

Protein SDH AChE ACh ChAc

u/ /o

RSA

31.5

7;

RSA

%

RSA

RSA M2 RSA M i

11.2

6.3 2.7 1.2 0.7 0.7

--__

~~

0.3 2.3 3.6 5.6

0.0 0.0 1.5 0.9

0.1 1.9 5.8 7.8

AChE, which was previously shown to be related to ACh and ChAc in the total nerve ending (Table III), now appears dissociated from the rest of the ACh system (Table IV). Most of it (64% taking Mit as 100%) remains in M I and is concentrated in M2 to a very small degree. Further experiments have shown that the AChE in Mz is attached to the membranes present in this fraction and not to the synaptic vesicles (De Robertis et al., 1963). It is concluded from this experiment that the synaptic vesicles are the morphological units of ACh and ChAc while AChE is probably localized in the membranes of the nerve ending. 5-HYDROXYTRYPTAMINE

Knowledge of the subcellular distribution of 5-HT may be important to ascertain the exact role of this monoamine in the function of the CNS. Walaszek and Abood (1959) found 5-HT concentrated in the crude mitochondrial fraction which, as was mentioned above, is highly heterogeneous. Whittaker (1 959) found 5-HT in the so-called vesicular fraction later shown to be composed of nerve endings. The high content of 5-HT found by this author was interpreted by Inouye et al. (1962) as being due to the extraction technique used, which apparently did not eliminate substance P or other active polypeptides. More recently Michaelson and Whittaker ( I 962) found 5-HT in 3 subfractions of the crude mitochondrial fraction which contained nerve endings.

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131

TABLE V 5-HT ( Z I E H E R A N D D E R O B E R T I S , 1963), 5-HTPD ( R O D R I G U E Z D E L O R E S A R N A I Z A N D D E R O B E R T I S , 1963) NA ( Z Y L B E R G E R , U N P U B L I S H E D ) , M A 0 A N D SDH ( R O D R I G U E Z D E L O R E S A R N A I Z A N D D E R O B E R T I S , 1962) C O N T E N T I N P R I M A R Y A N D S U B F R A C T I O N S O F M I T O F T H E CNS O F T H E R A T (SEE T A B L E I) PROTEIN,

Data recalculated as percent recovery of total homogenate. RSA protein

N Mit A B C D E Mic SUP

14.0 47.1 8.5 3.7 11.5 15.6 7.7 13.4 25.5

0.3 I 0.71 0.36 0.45 I .38 1.20 0.28 0.68 2.05

0.49 1.oo 0.61 0.78 2.35 0.66 0.53 3.33 0.16

=

% recovery activity/% recovery

1.10 1.80 0.00 0.00 0.35 2.70 4.20 0.35 0.00

0.26 0.83 0.22 2.60 1.80 0.35 0.22 0.98 1.66

1.36 1 .80 0.00 0.00 0.80 2.90 4.00 0.37 0.00

Zieher and De Robertis (1963) have applied the techniques for the isolation of nerve endings and synaptic vesicles described above to the study of the subcellular localization of 5-HT. Table V shows that most of 5-HT is bound, only 4 % existing in the soluble supernatant. About half of bound 5-HT is in the crude mitochondria1 fraction Mit while 40 ”/, is in the microsomes. Because of the low protein content ofthe latter fraction there is a high concentration of 5-HT with an RSA of 3.33. With further TABLE VI N A ( Z Y L B E R G EURN, P U B L I S H E D ) , 5-HT(ZlEHER A N D D E R O B E R T I S , 1963), 5-HTPD 1963), M A 0 A N D SDH ( R O D R I G U E Z D E A R N A I Z A N D D E R O B E R T I S , 1962) I N M I T O C H O N D R I A L S U B F R A C T I O N S O B T A I N E D

PROTEIN,

(RODRIGUEZ DE LORES ARNAIZ A N D D E ROBERTIS, LORES

A F T E R H Y P O S M O T I C S H O C K (SEE T A B L E 11)

Data recalculated as percent recovery of total homogenate. RSA protein

Fractions

Protein NA 5-HT 5-HTPD MA0 SDH References p . 135/136

%

% recovery activity/ % recovery

Mz

MI

-

=

RSA

32.5

%

M3 RSA

RSA

RSA Mz RSA Mi

9.1

5.5 0.33 0.90 0.26 3.07 2.70

%

1.52 1.10 0.30 0.00 0.30

2.25 1.10 2.00 0.00 0.00

4.6 1.o 1.o 0.1

132

E. D E R O B E R T I S

fractionation of Mit, most of the 5-HT appears in fraction C of nerve endings; that is, in the same in which the ACh system is present. Several interpretations can be proposed to explain this finding. It may be that both ACh and 5-HT are in the same nerve terminal, but it is also possible that they are contained in two structural entities that sediment at the same level. In favour of this last hypothesis are the published reports indicating a different distribution of ACh and 5-HT in certain regions of the CNS. Furthermore, the distribution of the ACh system and 5-HT in subfraction B is not strictly parallel (see Table V). The resdts of the osmotic shock are more difficult to interpret. The fact that most of 5-HT remains in M1 (60.4%, using Mit as 100%) may be interpreted as an indication that it is not in the synaptic vesicles and a n alternative explanation could be a different sensitivity of ACh and 5-HT containing nerve endings towards the osmotic shock. This different behaviour of 5-HT is even more striking when compared with that of adrenalin (see below). 5-HYDROXYTRYPTAMINE DECARBOXYLASE

Some preliminary results from our laboratory (Rodriguez de Lores Arnaiz and De Robertis, 1963) show that 5-hydroxytryptamine decarboxylase (5-HTPD), the enzyme directly concerned with the decarboxylation of 5-hydroxytryptophan to form 5-HT, has to some extent a similar subcellular distribution as 5-HT (Zieher and De Robertis, 1963). This enzyme, which is considered by others (Giarman, 1956; Hagen et at., 1960) to be essentially soluble, appears with our fractionation method to be half bound to the structure, the rest being in the soluble supernatant. The total bound enzyme is about 60% in the crude mitochondria1 fraction and, upon further subfractionation, most of it goes to the subfractions C and D of nerve endings. The RSA shows a definite concentration of 5-HTPD in the subfraction C of nerve endings in which 5-HT is also concentrated (Table V). Under the influence of the osmotic shock of Mit most of the enzyme becomes solubilized and concentrated i n the soluble supernatant M3 (Table Vl) indicating that it was only loosely bound or occluded within the membranes of the nerve ending. NORADRENALIN

(NA)

The presence of catecholamines in the nervous system has been known for many years. The highest concentrations of NA occur in the hypothalamus, central gray matter of the mesencephalon and area postrema (Vogt, 1954) and dopamine is mainly localized in the corpus striatum and associated with basal ganglia (Carlsson, 1959). The subcellular distribution of catecholamines has been mainly studied in the adrenomedullary cells. Von Euler and Hillarp (1956) isolated a granular fraction rich in NA from homogenates of the spleen and splenic nerves and Weil-Malherbe and Bone (1957) from brain homogenates. More recently Chrusciel(1960) has found NA to be concentrated in the vesicular fraction of Whittaker (l959), which contains nerve endings.

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133

In our laboratory, Zylberger (unpublished) carried out some preliminary work using the fluorimetric method of Bertler et al. (1958) on fractions of rat brain. In the primary fractions, it was found that about 40% of NA is in the soluble Sup. Bound NA remains mainly in the crude mitochondrial fraction Mit, which on the gradient shows the maximum distribution in subfractions C and B. Due to the low protein coiltent of B, the highest RSA is found in this subfraction containing synaptic debris and membranes (Table V). After the hyposmotic shock of Mit most of the NA is liberated into the soluble supernatant M3 (Table Vl). In spite of this, some NA remains with the vesicular fraction MZ and there is a slight concentration (RSA 1.52). The most interesting result is shown by the ratio RSA Mz/RSA M1 which shows a 4.6 concentration of NA. This is particularly interesting if compared with ACh (Table IV) and specially with 5-HT, which shows practically no concentration in Mz (Table VI). From all these results it can be concluded that NA is contained in nerve endings which sediment at the same density as those containing 5-HT and ACh; for NA, there is a higher concentration in the synaptic fragments of subfraction B. The results of the osmotic disruption support the concept that NA is localized in synaptic vesicles although these are more sensitive to the shock than those containing ACh. N A is probably present in the granular vesicles found by De Robertis et a/. (1963) in MZand in sections of the anterior hypothalamus (Pellegrino de Iraldi et a/., 1963) (Fig. 7). M 0N O A M I N E 0 X I D AS E

(M AO)

Knowledge of the subcellular localization of M A 0 in brain is of considerable interest in view of its possible role in the physiological inactivation of indolamines (Sjoerdsma et a/., 1955) and catecholamines (Shore et a/., 1957; Spector et al., 1960). I n this respect M A 0 could have a similar role for these amines as AChE for ACh. M A 0 was found in the particulated fraction of severzl tissues including brain (Davison, 1958). Weiner (1960) found M A 0 in the mitochondrial fraction of the brain. The use of similar methods of fractionation as reported above has shown that M A 0 is strictly a mitochondrial enzyme having exactly the same distribution as SDH in true primary fractions and in the subfractions of Mit (Table V) (Rodriguez de Lores Arnaiz and De Robertis 1962). This is further corroborated by the results of the osmotic shock, which demonstrates that all the M A 0 activity remains in subfraction M1 (Table VI). This finding indicates that M A 0 and AChE can not be equal in their action on the respective amines. While AChE is localized in the membranes of the cholinergic nerve endings and probably acts at the synaptic membrane, M A 0 is present in neuronal and synaptic mitochondria. This fine topochemical difference suggests a different physiological function of these two enzymes. The enzyme catechol-0-methyltransferase involved in the methylation of catecholamines (Axelrod et a/., 1959) has apparently a different topography than MAO, but its exact localization with respect to the synaptic region has not yet been determined. References p . 135!136

134

E. DE R O B E R T I S

SUMMARY

Due to the extraordinary morphological complexity and presence of numerous submicroscopic components, any neurochemical study of brain fractions should be checked with the electron microscope. Methods have been developed in our laboratory for the isolation of two populations of nerve endings in addition to myelin and free mitochondria from the crude mitochondrial fraction. In addition, a technique based on the hyposmotic shock of the crude mitochondrial fraction permits the disruption of nerve endings and the isolation of synaptic vesicles. Several types of vesicles, some having a ring of fine material around them (annular vesicles), others containing a dense granule (granular vesicles), in addition to the most frequent and simple type, have been recognized. The ACh system represented by the acetylcholine content and the cholineacetylase (ChAc) and acetylcholinesterase (AChE) activities were determined in all the above fractions. ACh and ChAc have a parallel distribution in the primary fractions, in the nerve endings and in the fraction obtained after hyposmotic shock. AChE follows the same distribution only in the nerve endings. In the primary fraction it is also present in the microsomes. All three components of the ACh system were concentrated in a single fraction C of nerve endings and in fraction B of synaptic debris. The larger population of nerve endings of the brain represented by fractionDlacks the ACh system. After the hyposmotic shock and in spite of some solubilization, ACh and ChAc became highly concentrated in fraction M2 containing the synaptic vesicles. This result is interpreted as definite evidence that synaptic vesicles are the carriers of ACh and that they also contain the enzyme directly involved in its synthesis. Within the synaptic complex, AChE appears dissociated from ACh and ChAc, as most of it remains in fraction MI containing the membranes of the nerve endings. 5-Hydroxytryptamine (5-HT) is bound mainly to the structure and is divided between the crude mitochondrial fraction and microsomes in which it is considerably concentrated. Most of the mitochondrial 5-HT exists in the nerve endings of fraction C, which also contains the ACh system. After hyposmotic shock, most 5-HT remains in fraction MI. Preliminary studies on 5-hydroxytryptamine decarboxylase (5-HTPD) show that despite the fact that this enzyme is about half soluble, the bound portion is present in the crude mitochondria and in nerve endings of type C . With hyposmotic shock practically all the enzyme is released showing that it was loosely occluded within the membranes of the ending. Noradrenalin (NA) becomes solubilized up to 40 in the primary fractions. The bound portion is concentrated in the crude mitochondrial fraction from which, upon further fractionation, it appears in fractions C of nerve endings and B of synaptic debris. After the hyposmotic shock and despite considerable solubilization, there is concentration in the synaptic vesicles. It is concluded that NA exists in nerve endings of type C and probably in some granular type of synaptic vesicles. The enzyme monoamine oxidase, which is involved in physiological inactivation of indolamines

BINDING SITES OF BIOGENIC AMINES

135

and probably of catecholamines in the CNS, was found to be strictly a mitochondria1 enzyme exactly following the distribution of succinodehydrogenase (SDH). All these findings demonstrate that there is a fine compartmentalization in the CNS of the binding sites for biogenic amines and enzymes related to their metabolism.

REFERENCES

w.

M. K., (1959); Cholinesterase, succinic dehydrogenase, nucleic ALDRIDGE, N., AND JOHNSON, acids, esterase and glutathione reductase. Biochem. J., 73, 270. AXELROD, J., ALBERS,W., AND CLEMENTE, c. D., (1959); Distribution of catechol-0-methyl transferase in the nervous system and other tissues. J . Neurochem., 5, 68. BELLAMY, D., (1959); The distribution of bound acetylcholine and choline acetylase in rat and pigeon brain. Biochem. J . , 72, 165-168. BERTLER, A,, CARLSSON, A., AND ROSENGREN, E., (1958); A method for the fluorometric determination of adrenalin and noradrenalin in tissues. Acta physiol. scand., 44, 273-292. M. W., AND ALBERS,R. W., (1959); Species differences in the distribution of extraBRIGHTMAN, ~ieuronalcholinesterases within the vertebrate central nervous system. J . Neurochem., 4, 244-250. BRODY,T. M., AND BAIN,S. A., (1952); A mitochondrial preparation from mammalian brain. J . biol. Chem., 195, 685-696. L. M., (1951); Cholinesterase and succinic dehydrogenase in the BURGEN, A. S. V., AND CHIPMAN, central nervous system. J . Physiol. (Lond.), 114, 296-305. CARLSSON, A., (1959); The occurrence, distribution and physiological role of catecholamines in the nervous system. Pharmacol. Rev., 11, 490-493. CARLSSON, A., FALCK,B., AND HILLARP,N. A., (1962); Cellular localization of brain monoamines. Acta physiol. scand., 56, 196. CHRUSCIEL, T. L., (1960); Observations on the localization of noradrenalin in homogenates of dog’s hypothalamus. Adrenergic Mechanisms. Ciba Foundation Symposium. Churchill, London (p. 539-543). DAVISON, A. N., (1953); Return of cholinesterase activity in the rat after inhibition by organophosphorus compounds. Biochem. J., 54, 583-590. DAVISON, A. N., (1958); Physiological role of monoamine oxidase. Physiol. Rev., 38, 729-747. E., (1962); Fine structure of synapses in the CNS. Int. Congress Neuroparh. 2 , 35-38. DE ROBERTIS, E., AND BENNETT, H. S., (1955); Some features of the submicroscopic morphology of DE ROBERTIS, synapses in frog and earth-worm. J . biophys. biochem. Cyrol., 1, 47-58. E., AND PELLEGRINO DE IRALDI, A., (1961); A plurivesicular component in adrenergic DE ROBERTIS, nerve endings. Anat. Rec., 139, 299. E., PELLEGRINO DE IRALDI, A., RODRIGUEZ DE LORES ARNAIZ,G., AND GOMEZ,C. J., DE ROBERTIS, (1960) ; Aislamiento de terminaciones nerviosas y vesiculas sinapticas. Sesiones de la Sociedad Argentina de Biologia, Mendoza (p. 24-25). DE ROBERTIS, E., PELLEGRINO DE IRALDI, A., RODRIGUEZ DE LORES ARNAIZ,G., A N D GOMEZ,G. J., (1961); The isolation of nerve endings and synaptic vesicles. J . biophys. biochem. Cytol., 9,229-235. E., PELLEGRINO DE IRALDI, A,, RODRIGUEZ DE LORES ARNAIZ,G., AND SALGANICOFF, L., DE ROBERTIS, (1962a); Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J. Neurochem., 9, 23-35. E., RODRIGUEZ DE LORESARNAIZ, G., AND PELLEGRINO DE IRALDI, A., (1962b); Isolation DE ROBERTIS, of synaptic vesicles from nerve endings of the rat brain. Nature (Lond.), 194, 794-795. E., RODRIGUEZ DE LORESARNAIZ,G., SALGANICOFF, L., PELLEGRINO DE IRALDI,A., DE ROBERTIS, AND ZIEHER, L. M., (1963); Isolation of synaptic vesicles and structure organization of the acetylcholine system within brain nerve endings. J . Neurochem., 10, 225-235. FELDBERG, W., (1957); Acetylcholine in Metabolism of the Nervous System. Pergamon, London (p. 493). FELDBERG, W., AND VOGT, M., (1948); Acethylcholine synthesis in different regions of the central nervous system. J. Physiol. (Lond.), 107, 372-381. GIARMAN, N. J., (1956); Biosynthesis of 5-hydroxytryptamine (serotonin, enteramine). Fed. Proc., 15, 428. GOMEZ,C . J., Personal communication.

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E. D E R O B E R T I S

GRAY,E. G., AND WHITTAKER, V. P., (1960); The isolation of synaptic vesicles from the central nervous system. J . Physiol. (Lond.), 153, 35P-39P. HAGEN,P., WEINER,N., ONO,S., AND LEE, F. L., (1960); Amino acid decarboxylases of mouse mastocytoma tissue. J . Pharmacol. exp. Ther., 130,9-12. HANZON, V., AND TOSCHI,G., (1959); Electron microscopy on microsomal fractions from rat brain. Exp. Cell Res., 16, 256-271. HEBB,C. O., (1957); Biochemical evidence for the neural function of acetylcholine. Physiol. Rev., 37, 196-220. HEBB,C. O., AND SILVER, J., (1956); Choline acetylase in the central nervous system of man and some other mammals. J. Physiol. (Lond.), 134, 718-728. HEBB,C. O., AND SMALLMAN, B. N., (1956); lntracellular distribution of the choline acetylase. J. Physiol. (Lond.), 134, 385-392. HEBB,C . O., AND WHITTAKER, V. P., (1958); Intracellular distributions of acetylcholine acetylase. J. Physiol. (Lond.), 142, 187-196. INOUYE, A., KATAOKA, K., AND SHINAGAWA, J., (1962); 5-Hydroxytryptamine in the subcellular particles of rabbit brain. Nature (Lond.), 194, 286-287. LOEWI,O., (1956); On the intraneuronal state of acetylcholine. Experientia (Easel), 12, 33 1-333. MICHAELSON, J. A,, AND WHITTAKER, V. P., (1962); The distribution of hydroxytryptamine in brain fractions. Biochem. Pharmacol., 11, 505-506. NATHAN, P., AND APRISON, M. H., (1955); Cholinesterase activity in cytoplasmic particles from rabbit brain. Fed. Proc., 14, 106-107. PELLEGRINO DE IRALDI, A., FARINI DUGGAN, H. J.,ANDD E ROBERTIS, E., (1963); Adrenergic synaptic vesicles in the anterior hypothalamus of the rat. Anat. Rec., 145, 521-531. RICHTER, D., AND HULLIN, R. P., (1951); Isolated nuclei from cells of the cerebral cortex preparation and enzyme content. Biochem. J., 48, 406410. RODRIGUEZ DE LORES ARNAIZ, G., AND DE ROBERTIS, E., (1964) ; 5-Hydroxytryptophane-decarboxylase activity in nerve endings of the rat brain. J . Neuvochem., in the przss. RODRIGUEZ DE LORES ARNAIZ,G., AND DE ROBERTIS, E., (1962); Cholinergic and non-cholinergic nerve endings in the rat brain. 11. Subcellular localization of monoamine oxidase and succinate dehydrogenase. J . Neurochem., 9, 503-508. SALGANICOFF, L., AND DE ROBERTIS, E., (1963); Subcellular distribution of glutamic decarboxylase and gamma-aminobutyric a-ketoglutaric transaminase. Life Sci., 2, 85-9 I. SHORE, P. A., MEAD,J. A. R., KUNTZMAN, R. G., SPECTOR, S., A N D BRODIE, B. B., (1957); On the physiologic significance of monoamine oxidase in brain. Science, 126, 1063-1064. SJOERDSMA, A., SMITH,T. E., STEVENSON, T. D., A N D UDENFRIEND, S., (1955); Metabolism of 5hydroxytryptamine (serotonin) by monoamineoxidase. Proc. SOC.exp. Biol. ( N . Y.), 89, 36-38. SMALLMAN, B. N., AND WOLFE,L. S., (1957); (Quoted by HEBB,C.O.) in Biochemical evidence for the neural function of acetylcholine. Physiol. Rev., 37, 196-220. SPECTOR, S., KUNTZMAN, R., SHORE, P. A., AND BRODIE, B. B., (1960); Evidence for release of brain amines by reserpine in presence of monoamine oxidase inhibitors; implication of monoamine oxidase in norepinephrine metabolism in brain. J . Pharmacol. exp. Ther., 130, 256-261. TOSCHI,G., (1959); A biochemical study of brain microsomes. Exp. Cell Res., 16, 232-255. VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after administration of drugs. J. Physiol. (Loncl.), 123, 451481. VON EULER, U. s., AND HILLARP, N. A., (1956); Evidence for the presence of noradrenalin in submicroscopic structures of adrenergic axons. Nature (Lond.), 177, 44-45. WALASZEK, E., AND ABOOD,L., (1959); Fixation of 5-hydroxytryptamine by brain mitochondria. Proc. SOC.exp. Biol. ( N . Y.).,101, 3740. WEIL-MALHERBE, H., AND BONE,A. D., (1957); Intracelhlar distribution of catecholamines in the brain. Nature (Lond.), 180, 1050-1051. WEINER,N., (1960); The distribution of monoamineoxidase and succinic oxidase in brain. J . Neurochem., 6, 70-86. WHITTAKER, V. P., (1959); The isolation of acetylcholine-containing particles from brain. Biocheni. J., 72, 694-706. ZIEHER, L. M., AND DE ROBERTIS, E., (1963); Subcellular localization of 5-hydroxytryptamine in rat brain. Biochem. Pharmacol., 12, 596-598. ZYLBERGER, R., Unpublished.

137

Acidic Glycolipoprotein Granules (Lysosomes) as Probable Binding Sites of Biogenic Amines HAROLD KOENIG Neurology Service, V. A . Research Hospital; and Departnient of Neurology and Psychiatry, Northwestern University Medical School, Chicago, IN. ( U S .A . )

In view of the prevailing inclination to localize the biogenic amines to nerve endings, I approach my rather heretical title with some trepidation. I would point out to you, however, that morphologically homogeneous subcellular fractions of brain have yet to be produced, making it somewhat precarious to ascribe any particular compound to a given organelle in such fractions. Furthermore, uninnervated tissues also possess

Fig. 1. Spinal motoneuron of cat stained in frozen section for carbohydrate by the periodic acid-Schiff method. Note numerous glycolipoprotein granules. ZOO0 X

.

References p . I41

138

H. K O E N I G

biogenic amines, e.g. mast cells grown in vitro have considerable 5-OH tryptamine and non-innervated heart of hagfish (Augustinsson et al., 1956) and chick embryo prior to innervation (Lee e t a / . , 1960) contain adrenalin. The purpose of this brief communication is to invite attention to a special class of cytoplasmic granules as a potential binding site for biogenic amines. These granules,

Fig. 2. Spinal motoneuron of rat stained intravitally by the basic dye, neutral red (80 mg, injected intraperitoneally 3 h before removal). Glycolipoprotein granules are basophilic in vivo. 1600 x .

which I have been studying for the past several years, are <0.2-0.6 ,u in diameter. They occur in the cytoplasm of neurons, glia and other non-neuronal cells (and in a wide assortment of other tissues), give reactions for non-glycogenic carbohydrate, such as the periodic acid-Schiff method, and are extracted from fresh, but not fixed, tissues, by chloroform-methanol (2 : 1) (Koenig, 1962). These histochemical features denote the presence of protein-bound glycolipids. Histochemical and biochemical data at hand suggest that gangliosides, a heterogeneous group of acidic glycosphingolipids containing N-acetylneuraminic acid, are localized to these inclusions within the central nervous system (Koenig, 1962; Koenig and Jibril, 1962). These granules furthermore display a highly selective affinity for intravitally administered basic dyes and metalic ions, suggesting that they are negatively charged in vivo (Koenig, 1963a). See Figs. 1-3.

LYSOSOMES A N D BIOGENIC AMINES

139

These acidic glycolipoprotein granules seem to correspond to the lysosomes of De Duve, a class of cytoplasmic particles containing a number of acid hydrolases (see De Duve, 1959). Thus they give staining reactions for acid phosphatase (Koenig, I962), a thiolacetic acid esterase resembling cathepsin C and acid deoxyribonuclease

Fig. 3. Spinal motoneuron of cat stained in frozen section for acid phosphatase activity by Gomori’s lead glycerophosphate method. Glycolipoprotein granules give strong reaction for this enzyme.2000 x

.

(Koenig, Gaines et al., 1964). They also have the same fine structure as lysosomes in liver and other organs in electron micrographs where they appear as dense bodies which give reactions for acid phosphatase (Novikoff and Essner, 1962; Koenig and McDonald, unpublished) and thiolacetic acid esterase (Torack and Barrnett, 1962). We have presented evidence to show that ionic bonds are involved in the binding of several acid hydrolases to lysosomal matrix in brain, liver and kidney. Ionic bonds also seem to be responsible for the structure-linked latency of lysosomal enzymes (Koenig and Jibril, 1962). Lysosomes are autofluorescent, but the fluorescent constituents, which are extractable by chloroform-methanol, have not been chemically identified (Koenig, 1963b). Although not previously correlated, biogenic amines and acid hydrolases share a number of properties in common. (1) They are bound to particles which can be References p. 141

140

H. K O E N I G

separated from mitochondria by centrifugation in a sucrose density gradient. (2) They are metabolically inert while in particulate state but metabolically active in solution. (3) They are released from particles by a number of disruptive treatments, such as hyposmotic, thermal and mechanical shock, detergents and organic solvents. ( 4 ) Ionic bonds appear to be involved in their binding to particles. These considerations, together with the demonstrated ability of lysosomes to accumulate exogenous cationic molecules in vivo, lead me to propose that biogenic amines, like acid hydrolases, are stored in the same acidic glycolipoprotein granules. This proposal receives a measure of support from an observation of Whittaker (1 959) that a large portion of the total acid phosphatase activity in brain was associated with acetylcholine and 5-OH tryptamine in this nerve ending fraction. Preliminary experiments in my laboratory also have revealed considerable acid phosphatase activity and numerous acid phosphatase granules, i.e. lysosomes, in a similarly prepared fraction. Studies are in progress to shed additional light on this possibility.

ADDENDUM

Since presenting this paper, lysosome-rich fractions of rat brain have been prepared in this laboratory by centrifugation of mitochondria1 and microsomal fractions over a complex sucrose density gradient. These studies indicate that, contrary to expectation (Koenig, 1962), gangliosides do not occur in brain lysosomes (Koenig, Gaines et al., 1964). The biochemical composition of these fractions, including the biogenic amines and lipid components, are under investigation.

SUMMARY

It is proposed that lysosomes may be the binding sites of biogenic amines on the following grounds : 1. Lysosomes are polyanionic lipoprotein granules with demonstrated ability to segregate cationic substances, such as basic dyes and metallic cations. 2. Lysosomal enzymes and biogenic amines share the following features: (a) they are particulate; (b) they show structural latency, disruptive procedures releasing them into solution in active state; and (c) ionic bonds have been implicated in their binding to particles. This proposal is currently under investigation in my laboratory.

ACKNOWLEDGEMENTS

This investigation was assisted by Grant B-1456 (CI-C3), U.S. Public Health Service, Contract No. AT (11-1)-1180, U.S. Atomic Energy Commission, and No. 304, National Multiple Sclerosis Society.

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141

REFERENCES

AUGUSTINSSON, K.-B., FANGE,R., JOHNELS,A., AND ~ S T L U N D E., , (1956); Histological, physiological and biochemical studies on the heart of two cyclostomes, hagfish (Myxine) and Lamprey (Lampetra). J. Physiol. (Lond.), 131, 257-276. DE DUVE,C., (1959); Lysosomes, a new group of cytoplasmic particles. Subcellular Particles. T. Hayashi, Editor. New York, Ronald Press Co. (p. 128). KOENIG,H., (1962); Histological distribution of brain gangliosides; lysosomes as glycolipoprotein granules. Nature (Lond.), 195, 782-784. KOENIG, H., (1963a); Intravital staining of lysomes by basic dyes and metalic ions. J. Histochem. Cytochem., 11, 120-121. KOENIG, H., (1963b); The autofluorescence of lysosomes. Its value for the identification of lysosomal constituents. J . Histochem. Cytochem., 11, 556-557. KOENIG,H., GAINES, SCOTT,D., AND GRAY,E. G., (1964); Studies of brain lysosomes. Second International Symposiuni of Response of Central Nervous System to Ionizing Irradiation. T. Haley and R. Snider, Editors. New York, Academic Press, in the press. KOENIG, H., A ND JIBRIL, A. (1962); Acidic glycolipids and the role of ionic bonds in the structurelinked latency of lysosomal hydrolases. Biochim. biophys. Acta (Amst.), 65, 543-545. LEE,W. C., MCCARTY, L. P., ZODROW, W. W., AND SHIDEMAN, F. E., (1960); The cardiostimulant action of certain ganglionic stimulants on the embryonic chick heart. J. Pharmacol. exp. Ther., 130, 30-36. NOVIKOFF, A. B., AND ESSNER, E., (1962) ; Pathological changes in cytoplasmic organelles. Fed. Proc., 21, 1130-1 142. TORACK, R. M., AND BARRNETT, R. J . , (1962); Fine structural localization of cholinesterase activity in the rat brain stem. Exp. Neurol., 6, 224-244. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine-containing particles from brain. Biochem. J., 72, 694-706.

142

General Discussion *

GOOD:I would like briefly to discuss the problem of the origin of synaptic vesicles, and to ask Dr. Whittaker and Dr. De Robertis for their viewpoints on this subject. As has been demonstrated here today most of the acetylcholine and choline acetylase appear to be stored in the purified synaptic vesicle subfraction of crude mitochondrial fraction of brain homogenate. The fact that there is still considerable neurohumoral and associated enzyme activity present in the purified mitochondrial fraction, however (Hebb and Whittaker, J . Physiol. (Lond.), 142 (1958) 187; Whittaker, Biochem. J., 72 (1959) 694), falls in line with observations we have been making in the giant mossy fibre synapses of the cerebellar glomerulus which suggests the possibility of a mitochondrial origin of synaptic vesicles. I have a number of micrographs with me from a paper we are preparing, the more pertinent observations of which I would like to schematize for you in a composite drawing. In Fig. l a presynaptic mitochondria may be found which contain circular profiles of such regularity and number as to suggest that they may be vesicles. These circular profiles compare in size to the synaptic vesicles just outside these mitochondria. In Fig 1b similar mitochondria may be seen with poorly defined borders, and others in Fig. l c may be seen to be in continuity with a train of vesicles. These Figs. are not seen in every field and they may represent capricious sections

Fig. 1. In this Fig. a, b, and c are variant forms of mitochondria taken from electron micrographs of presynaptic regions of cerebellar glomeruli. Circular profiles of the same size as synaptic vesicles are demonstrated (a) within intact mitochondria, (b) within imperfectly bound mitochondria, (c) within and trailing off from the open ends of imperfect mitochondrion, and (d) from dense reticulated body in bouton of anterior horn thought to be a mitochondrion undergoing reorganization of structure.

* This discussion refers to the papers of Dr. N. Giarman, Dr. J. Axelrod, Dr. V. P. Whittaker and Dr. E. De Robertis.

DISCUSSION

143

and uneven fixation. The circles, for example, may be peduncles, tubules, or hollow appendages of cristae, but the possibility that these circular profiles may represent the outlines of vesicles containing acetylcholine is suggested by the followingconsiderations.

Fig. 2. Cerebellar glomerulus showing: (a) Dense accumulations of mitochondria (arrow) shown at higher magnification in Fig. 3 to be associated with numerous synaptic vesicles. Pre = presynaptic; Post = postsynaptic, X 9605. (b) Mitochondrion containing circular profiles of the same size as synaptic vesicles, x 63,945.

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DISCUSSION

First, there is the possibility of local autonomy for the replacement of chemical synaptic mediator - viz. synaptic vesicles - ( a ) in the mere remoteness of giant presynaptic terminals from their perikarya of origin, and (b) in the persistence of spontaneous electrical activity in the granular layer of the cerebellum isolated by undercutting (Snider, personal communication). Second, as we and others (Palade and Palay, Anat. Rec., 118 (1954) 335; Palay, J . hiophys. biochem. Cytol., 2 (1956) No. 4, Suppl.) have demonstrated (Fig. 2 and 3) there is a predominance of mitochondria

Fig. 3. Presynaptic mossy fibre intumescence. Neurotubules (nt) in center are nearly surrounded and separated by a wall of many mitochondria. While not conclusive, direct observation so far seems to implicate the mitochondria in synaptic vesicle formation. Many of the vesicles are ringed with miniature satellite vesicles (mv) -structures which may have significance in terms of the quantum electrogenic observations of Fatt and Katz (J. Physiol. (Lond.), 117 (1952) 109) and Del Castillo and Katz (J. Physiol. (Lond.), 124 (1954) 560; J . Physiol. (Lond.), 124 (1954) 574). sp = ~ -thickenings or plaques on apposing synaptic membranes; Pre = presynaptic; Post = postsynaptic, x 56,600.

DISCUSSION

145

on the presynaptic side of the synapse, and often we see a great massing of mitochondria in the presynaptic regions of the cerebellar glomerulus. Since energy underlying the recovery processes for synaptic electrogenesis is almost certainly supplied by postsynaptic mitochondria (Eccles, The Physiohgy of Nerve Cells, Johns Hopkins Press, 1957), it might be asked if the meaning of the preponderance and abundance of mitochondria on the presynaptic side of the synapse might not be that these mitochondria are engaged in an extra-respiratory role related to the function of their likewise abundant companions, the acetylcholine-containing synaptic vesicles. Third, the fact that acetyl coenzyme A, choline acetylase, and the required ATP are present in brain mitochondria leads us to speculate that this role of the presynaptic mitochondria may be that of a synthesis of acetylcholine, and that such synthesis may take expression in the reorganization of part of the mitochondrial structure into microvesicles, which we see here with the mitochondria in the terminal axon swellings. Finally, although we hesitate to argue from the standpoint of negative evidence, it must be said that we have not yet been able to find alternative morphological evidence for vesiculation in synaptic regions, nor of a migration or formation of vesicles within axons. DEROBERTIS: There are very few observations on the probable origin of the synaptic vesicles. One interesting point is observed when the nerve is sectioned. In this case, just at the site of the cut, the axon develops vesicles very similar to the synaptic vesicles in great quantities. This is a very good material to study the origin of vesicles because the content of the choline and the choline acetylase increases considerably. We believe there is no good explanation but we think that the neurotubules which are all along the axon may bring some of the essential material (enzymes) to the ending for the formation of the synaptic vesicles. : Dr. Good has raised some interesting points regarding the metabolism WHITTAKER of the synaptic vesicles, that is to say, their formation and ultimate fate. We really know very little indeed about this, and I have no results of my own to offer. Regarding the cerebellar endings also described by Gray ( J . Anat. (Lond.), 95 (1961) 345) we have shown (Whittaker, 1963) that homogenates of cerebellar cortex contain giant nerve-ending particles apparently formed from these giant nerve endings. I think there is a very good possibility of scaling down our methods of fractionation and of isolating them as a preliminary to studying the transmitters contained within them. They come down in the nuclear fraction instead of in the mitochondrial fraction. I do not find it puzzling that there are large numbers of mitochondria in these nerve endings. There must be a tremendous metabolism connected with the synthesis and storage of the transmitters and possibly the replacement sf the vesicles themselves after they have been discharged. Many of our negatively stained preparations, which show the whole mitochondrial structure and not just a section, reveal that the series of small oval profiles seen in thin section are probably, in most cases, sections of a single, very long coiled mitochondrion. I would now like to comment, if I may, on some of the points of disagreement between Dr. De Robertis and myself. At the time of the Varenna Symposium (Regional Neurochemistry, S. S. Kety and J. Elkes, Editors, New York, Pergamon Press, 1961)

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DISCUSSION

when I presented my work with Dr. Gray on the nerve-ending particles, Dr. De Robertis was mainly looking for the synaptic vesicles in the microsomal fraction and that would seem to be a very reasonable thing to if one had not appreciated that homogenization in sucrose does not rupture the nerve endings. I think that now as a result of further work by Dr. De Robertis, there is basically no disagreement between us regarding the primary fractions, the presence of nerve-ending particles, and the distribution of the various components. However, there does seem to be disagreement on the composition of the isolated vesicles. He referred to his Nature article (De Robertis, De Traldi, Arnaiz and Salganicoff, Nature (Lond.), 194 (1962) 794) which was published while we were engaged in our work but this paper does not contain very much in the way of experimental details and certainly no pharmacological or density gradient work. As far as we can tell from this paper, our PzW, fraction which we submit to density gradient separation is equivalent to his combined Mz and MS fractions. Now, T think the presence of hydroxytryptamine and choline acetylase in his M2 fractions which Dr. De Robertis showed in one of his slides and which we do not find localized in our D fraction of isolated vesicles (Fig. 4, De Robertis, this Symposium) simply means that his Mz preparation is heterogenous and contains unbroken nerve endings. The question we then have to account for is the picture in his Nature article (1962) which does not, in fact, show unbroken nerve-ending particles. This reminds me of a sad experience which beset when I first started this work. When we first prepared nerve endings for electron microscopy (Whittaker, Biochem. J., 92 (1959) 694) we were unfamiliar with handling central nervous system material, and we got what subsequently turned out to be very extensive disruption of our material after fixation (Gray and Whittaker, J . Physiol. (Lond.), 153 (1960) 33; Whittaker and Gray, Brit. med. Bull., 18 (1962) 223). So we reported that the B fraction consisted mainly of isolated synaptic vesicles. We had every reason to believe that we were doing perfectly sound work because our mitochondria1 ( C ) fraction, treated in exactly the same way, gave quite decent pictures of mitochondria. It was only later on that we appreciated that there is an immense difference in stability between these two types of particles. The nerve-ending particles are wery much more fragile than the mitochondria and particularly after fixation they become extremely labile. They are also much more sensitive to autolysis. Now I am wondering whether the failure to observe the intact nerve-ending particles in the MZ fraction may not be due to some difference between our two procedures for preparation for the electron microscope. We are extremely careful about fragility and we do not spray our material; we put it on very carefully with a micropipette. We have further shown that even after fixation in formaldehyde the particles are still osmotically active and can still be disrupted by suspension in hypotonic PTA giving a false idea of the degree of organization of the material. Now T just want to reiterate that in fact our PzW, fraction contains all the fractions with all the structures which we subsequently see in the density gradient and if you would agree, Dr. Page, T could just show two slides which illustrate this point. This first slide (reproduced as Fig. 6, Whittaker, this Symposium) shows part of the field of our original PzW, fraction. As you can see it is almost indistinguishable from the D fraction (reproduced in Fig. 3, Whittaker,

DISCUSSION

147

this Symposium). In fact I was rather tempted to swap them around! But it does happen that sometimes one sees these drifts of isolated synaptic vesicles in this fraction. But now the next slide (reproduced as Fig. 5, Whittaker, this Symposium) shows that within adjacent areas one can readily see organized material including, right in the middle of the field, a very nice nerve-ending particle, mitochondria, the non-vesicular membranes which we subsequently find in the F fraction and of course many of the microsomes which we find in the E fraction. They are all there. DE ROBERTS:I would like to tell Dr. Whittaker that we study all our fractions after osmotic shock, not only by the negative staining technique but also by section of the fixed pellets. We first described the isolation of the synaptic vesicles in the Nature article (1962). In September 1962 we gave at the Philadelphia meeting of Electron Microscopy the first information about the acetylcholine and choline acetylase content of these fractions showing the high concentration of ACh and ChAc in the synaptic vesicles and Dr. Whittaker was present at that meeting. I think that our technique has given a far more rewarding picture of the vesicles and also a much higher concentration of the active components as shown by the relatively specific activities. BLASCHKO: I am interested in the centrifugation technique, because it had led to many interesting results. We must remember, however, that the terms used for the fractions obtained by centrifugation do not necessarily refer to a defined structural element; they are defined in operational terms. This has been repeatedly pointed out, e.g., by Dr. H. Holter of Copenhagen. Let me illustrate this by three points. Dr. Koenig compares the amine-carrying granules with lysosornes. But can one find the typical lysosomal enzymes in these granules? They are very highly specialized elements, and maybe they have sacrificed enzyme content as they become fully mature. Then I was interested in Dr. De Robertis’ study of the distribution of acetylcholinesterase. It was Dr. Paul Hagen who showed that in the adrenal medulla this enzyme was specifically located in the microsomal fraction; he studied both the specific activity and the percentage distribution (Hagen, J . Physiol. (Lond.), 129 (1955) 50). This does not tell us where enzyme is situated in the intact cell; it may well be located in or near the cell membrane. One has the feeling that in the centrifugation of nervous tissue a complication is brought about by the fact that the ‘NEP’s’ enclose so many different things. This brings me to the last point I wish to make; this refers to the interesting difference in the distribution of acetylcholine and 5-hydroxytryptamine that Dr. Whittaker showed in his last slide (Fig. 4, Whittaker, this Symposium). The distribution of acetylcholine was bimodal, but that of the amine was not. One possible explanation that I would like to propose is a little different from his: we do know from work in other tissues that the amine-carrying granules are in fact very dense. The surprising thing in the work on the CNS was that here the amine-carrying elements were always found in regions of density lower than that where the mitochondria were recovered. This is of course explained by the discovery that the elementary structures are enclosed in what you call the ‘NEP’s’. What you do in the centrifugation is to determine their overall density. If you ‘unpack’ the different elements I would not be surprised if

148

DISCUSSION

they showed up their true density. The amine-carrying granules might then well all go lower than the acetylcholine-carrying granules (or vesicles) and you would then expect to recover the amine activity at much lower levels in the density gradient. DE ROBERTIS: Regarding Dr. Blaschko’s question on the distribution of acetylcholinesterase, the work of our laboratory on the isolation of nerve endings has shown that in addition to a microsomal fraction there is another related to the cholinergic nerve endings. By disruption of this unit it has been possible to show that this enzyme is located in the membranes of the ending and not in the synaptic vesicle. COSTA:We have also studied uptake of 3H-NE in thyrotoxic animals (Life Sciences, 1962, No. 4, 241). We have found that the difference in uptake between thyrotoxic and control animals is only about 15%, at most, when the 3H-NE level is expressed at c.p.m./heart. Most important, however, was the indication that the N E turnover rate is not changed in thyrotoxic mice. We have concluded, therefore, that the symptoms of hyperthyroidism must be based on some other factor than a more rapid formation of the amine. AXELROD: We have found that the uptake of 3H-epinephrine in the hyperthyroid heart was reduced when expressed as uptake per gram of heart or in total heart despite the fact that the hyperthyroid heart is larger (Wurtman, Kopin and Axelrod, Endocrinology, in press). Furthermore, the amount of 3H-epinephrine delivered to the whole hyperthyroid heart is greater than to the normal heart. Consequently i n hyperthyroid hearts more catecholamine is delivered and less is inactivated by binding and this may be the mechanism for super-sensitivity in a hyperthyroid animal. I did not have an opportunity to talk very much about little dense core vesicles because I had so much ground to cover. PAGE:Would you please explain what you mean by little dense core vesicles? AXELROD: Electron microscopic studies (Milofsky, Thesis, Yale Univ., 1958; De Robertis eta/., J. biophys. biochem. Cytol., 10 (1961) 361) have revealed that autonomic axons contain granulated vesicles. These granulated vesicles are 40 to 50 mp wide, contain a 20 to 30 mp electron dense core and seem to be concentrated in preterminal axoplasm. Dr. Wolfe et a/. (Science, 138 (1962) 440) have already clearly shown that the dense core vesicles in the adrenergic axons of the pineal body are always associated with labelled norepinephrine. The labelled compound was shown to be almost exclusively norepinephrine by chemical estimation. I would like to ask Dr. De Robertis what evidence he has showing that the dense core vesicles he described are in the axon and contain norepinephrine. According to Dr. De Robertis, the depletion of granulated vesicles after reserpine and the correlated reduction of norepinephrine indicate the presence of norepinephrine in the granule. It is well known that the effects of reserpine are nonspecific and there is just as much serotonin depleted as norepinephrine. As I understand, your evidence indicated a different subcellular distribution of serotonin and norepinephrine. DE ROBERTIS: In addition to the action of reserpine on the granulated vesicles we have several other data in favor of their catecholamine contents, i.e., the injection of DOPA and dopamine increases the number of granulated vesicles. The injection of 5-hydroxytryptophan, which increases to 400 % the 5-HT content of the pineal,

DISCUSSION

149

does not change the granulated vesicles. Gangliectomy of superior sympathetic cervical ganglia produces degeneration of nerve endings and disappearance of granulated vesicles. These and other data are soon to be published in Int. J. Neuropharmacol. CARLSSON: I think we have to be a little bit careful with the terminology here. Dr. Axelrod said that he had demonstrated that in these pineal nerves norepinephrine was present. Well, 1 do not think he has demonstrated that norepinephrine is present normally in these nerves but what he has demonstrated is that these nerves are capable of taking up norepinephrine and this may be an entirely different study. As a matter of fact, Dr. Falck showed a slide yesterday disclosing these particular pineal nerves which are a type of sympathetic nerve ending, and according to the histochemical evidence these nerves contain the 5-HT and not norepinephrine. But, he also has evidence that they are able to take up norepinephrine because after injection of norepinephrine, the fluorescence color changes from yellow, which is the color of 5-HT, to green, which is the color of catecholamines. AXELROD : Giarman has demonstrated that endogenous norepinephrine is present in pineal gland in relatively large amounts. Furthermore, Dr. Potter and I (1962) found that exogenous and endogenous norepinephrine are associated in the same subcellular fraction. If Dr. Carlsson’s interpretation is correct, then endogenous norepinephrine in pineal glands is outside the nerve while exogenous norepinephrine is taken up in the axon. It is possible that both serotonin and endogenous norepinephrine are in the nerve but the fluorescence of serotonin is more intense so that it masks the fluorescence of norepinephrine. KOENIC: Dr. Blaschko has questioned my hypothesis on the ground that the catecholamine-rich granular fraction from adrenal medulla contains little acid phosphatase activity. Although this would at first glance appear to be a serious obstacle to my proposal, it can be explained. The fundamental feature of lysosomes, in my view, is that they are solid granules consisting of a polyanionic glycolipoprotein matrix. This enables them to exert an electrostatic attraction on diverse endogenous soluble substances whch carry basic groups. Salt linkages, possibly aided by H bonds and other type bonds, would retain cationic molecules within these granules and simultaneously would confer upon them a metabolic latency or inertness, restricting their accessibility to cell sap constituents. I n this view, hydrolytic enzymes such as acid phosphatase are only one class of soluble molecules that would be stored in these granules. Other types of endogenous substances, for example, non-enzyme protein such as ferritin, and constituents of small molecular weight, such as amino acids and biogenic amines, could also be sequestered in these granules. In the case of the adrenal medulla, the principal product of cell metabolism is presumably exportable catecholamines. If the storage granules are acidic glycolipoprotein granules, as I have suggested, the accumulation of catecholamines would tend to displace by mass action through a cation-exchange reaction acid phosphatase and other hydrolytic enzymes from these granules. The paucity of granule-bound acid hydrolases also might reflect low levels of synthesis of these enzymes in adrenal medullary cells.

150

Biochemical-Pharmacological Studies With 5-Hydroxytryptophan, Precursor of Serotonin HARRY GREEN

AND

JOHN L. S A W Y E R

Research and Development Division, Smith Kline and French Laboratories, Philadelphia I , Pa. (U.S.A.)

Previous publications (Green et al., 1960, 1962) from this laboratory have described the effects of the monoamine oxidase (MAO) inhibitors, tranylcypromine (tvans-2phenylcyclopropylamine) and iproniazid (~-isonicotinyl-2-isopropylhydrazine) and of reserpine upon endogenous rat brain levels and intracellular distribution of norepinephrine (3,4-dihydroxyphenylethanolamine) (NE) and serotonin (5-hydroxytryptamine) (5-HT). Although pronounced changes in the endogenous brain concentrations of these amines have been produced, the presence or absence of accompanying observable pharmacologic responses could not be correlated in terms of a general biochemical-pharmacological relationship applicable under all of the experimental conditions studied. Another approach to the study of such a relationship is based upon the demonstration by Udenfriend et al. (1957) that the intraperitoneal administration of 5hydroxytryptophan (5-HTP) was rapidly converted to 5-HT in the brain. However, the results of such investigations (Udenfriend et al., 1957; Bogdanski et al., 1958; Tedeschi et al., 1959; Costa and Rinaldi, 1958; Costa et al., 1960; Hess and Doepfner, 1961) have not unequivocally established that the pharmacologic responses elicited following the administration of 5-HTP were dependent upon the resulting changes in brain 5-HT concentration. Furthermore, no attempt was made to determine whether the 5-HTP-induced increases in brain 5-HT concentration represented changes in endogenous levels of the amine. The present report describes the results of a systematic kinetic study of the biochemical and pharmacological effects of exogenousIy administered serotonin precursor (5-HTP) in normal rats and rats pretreated with M A 0 inhibitors or reserpine. The results clearly indicate that the pharmacologic responses and changes in brain 5-HT concentration elicited following the administration of 5-HTP did not reflect changes in the concentration of endogenous brain 5-HT. Indeed, the biochemical and pharmacologic properties of the 5-HTP-induced increases in brain 5-HT concentration were distinctly different from those of increases in the endogenous content of the amine.

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MATERIALS

Fasted, male albino rats, Dierolf, Wistar strain, weighing between 100-200 g were used in all experiments. The HC1 salt of tranylcypromine and the phosphate salt of iproniazid were dissolved in HzO and administered orally by stomach tube in volumes of 1.5-3.0 ml. Reserpine was prepared for intraperitoneal injection (0.2 m1/100 g) according to the procedure of Pletscher et al. (1956) by dissolving 100 mg of the free base in a few drops of glacial acetic acid, adding 2.5 ml of propylene glycol and 2.5 ml of ethyl alcohol, and diluting to a final volume of 40 ml with distilled HzO. DL -~ - HT P (California Corporation for Biochemical Research) was dissolved in 0.9 % NaCl for intraperitoneal injection. a-Methyldopa (3,4-dihydroxyphenyl-u-methylalanine) (Merck, Sharp and Dohme) was used as an aqueous solution. Rats were sacrificed by decapitation, the brains immediately removed, rinsed with HzO to remove adsorbed blood, lightly blotted and weighed. The brains were either immediately processed for analysis or were frozen overnight and processed the following day. METHODS

Brain amine concentration The individual amine concentrations were measured photofluorometrically by means of an Aminco-Bowman Spectrophotofluorometer: 5-HT by the method of Bogdanski et al. (1957), N E by the method of Shore and Olin (1958) as modified by Green and Erickson (1960) and dopamine IDA) by the method of Drujan et al. (1959) after extraction by the procedure of Shore and Olin (1958) for NE. Since the presence of 5-HTP in brain homogenates interferes with the analysis of 5-HT, it was necessary to remove the amino acid. This was done by washing the butanol extracts 3 times with borate buffer, pH 10.0. Recovery experiments indicated that such a procedure did not result in any apparent loss of 5 H T , but completely removed at least up to 25 pg of added 5-HTP. These findings were in agreement with those of Clark et al. (1954). Since freezing tends to destroy the structural integrity of the brain amine storage granules, as well as of other particulate material, in the intracellular distribution studies the brains were chilled without freezing and homogenized in 0.30 M sucrose soon thereafter, as previously described (Green and Sawyer, 1962). Also, in order to prevent the breakdown of 5-HTP in the brain to 5-HT during homogenization in the sucrose solution, as suggested by the findings of Gey and Pletscher (1960), it was found necessary to have a-methyldopa (1 lop3 M ) (Smith, 1960) present during the homogenization. The presence of the decarboxylase inhibitor did not interfere with the analysis of 5-HT. As a matter of fact, in the alkaline medium used for the extraction of 5-HT, a-methyldopa was converted into a pink compound which was not extracted by butanol. In the other experiments in which brains were homogenized in 0.1 N HCI at 0", no measurable conversion of 5-HTP to 5-HT was found. Also, 5-HTP was stable in brains frozen overnight. References p . 1661167

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H. GREEN A N D J. L. S A W Y E R

5-HTP concentration Measurement of the brain 5-HTP concentration was made according to the procedure of Udenfriend et al. (1957). Brains were homogenized in 0.1 N HC1 and proteins removed with trichloroacetic acid. The protein-free filtrate was extracted with ether to remove the trichloroacetic acid and any 5-hydroxyindoleacetic acid present. The 5-HT was then removed from an alkaline solution with butanol and the 5-HTP remaining was measured photofluorometrically. Internal standards were run simultaneously. Recoveries were about 83 %. 5-HTP decarboxylase activity The enzyme assay was conducted according to the method of Clark et a/. (1954) using a supernatant of rat brain homogenate as described by Brodie et al. (1957). Substrate-activity studies showed enzyme saturation at a concentration of about 7 10-5 M 5-HTP (55 pg/3.5 ml). Monoamine oxiduse activity The enzyme assay was performed according to the procedure of Sjoerdsma et al. (1955) and Bogdanski et al. (1957). Identijication of possible 5-HT metabolites The general procedure of McIsaac and Page (1959) was followed. Three rats were pretreated with tranylcyproniine (5 mg/kg, p.0.) followed 2 h later with an i.p. injection of 5-HTP (25 mg/kg). The rats were sacrificed by decapitation 2 h after the injection of 5-HTP. The 3 brains were pooled and homogenized in 2 volumes of 0.1 N HCI. Six ml of the high-speed supernatant (100,000 x g), equivalent to about 2 rat brains, were adjusted to pH 5 with 0.04 ml of 20% N a ~ C 0 3and shaken with 20 ml of ether for 20 min. The ether layer was removed (acid ether extract) and the aqueous layer adjusted to pH 8 with 0.09 ml of 20% Na2COs and shaken again with 20 mi of ether for 20 min. The ether layer was removed and represented the alkaline ether extract. Both ether extracts were evaporated to dryness in a stream of N Z and the residues dissolved in a few drops of methanol and spotted on Whatman Nr. 1 filter paper. Descending chromatography in n-butanol, acetic acid and HzO (4 : 1 : 5) was conducted at room temperature overnight in a cylindrical jar lined with filter paper saturated with the solvent mixture. The developed chromatogram was air-dried and sprayed with Ehrlich's reagent. The absence of any detectable blue spot indicated the absence of any measurable indole compound (about 0.2 pg). The following possible metabolites of 5-HT were, therefore, presumed to be absent : 5-hydroxyindoleacetic acid, 5-methoxytryptamine, N-acetyltryptamine and N-acetyl-5-methoxytryptamine*. The aqueous residue remaining after the 2 ether extractions was filtered and the filtrate evaporated to dryness under reduced pressure. The residue was dissolved in a small volume of methanol, spotted on filter paper and developed as above. Spraying

* Although not detected in brains of normal rats; about 1 /fg/g was found I h after injection of 200 mg/kg of 5-HTP.

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of the developed, dried chromatogram revealed 2 blue spots with RF values of 0.27 and 0.45. The latter spot was identical with 5-HTP and was present in the brains of rats treated with tranylcypromine and 5-HTP, but not in the normal brains. The blue spot corresponding to an RF value of 0.27 was present in the brain preparations from both groups of rats and was not further identified. The material did not correspond to 5-HT. RESULTS

EfSect of' intraperitoneal.5-HTP upon rat brain 5-HT Concentration All values of 5-HT concentration following the administration of 5-HTPare averages of 3 to 6 individual brain analyses, unless otherwise indicated. Control values are

'1/ H AFTER 5HTP

Fig. 1 . Effect of 5-HTP upon rat brain serotonin concentration. Each point is an average of 3 separate experiments, and is compared to average value from a pool of 6 normal brains. Normal brain concentration (pglg) from 9 experiments (average & S.E.M.) was 0.534 I 0.012.

averages of a pool of 6 brains. In each experiment all experimental values have been compared to values derived from a pool of 6 brains from normal untreated rats. Analyses of replicates of a brain homogenate agreed withm 10%. ( A ) Normal rats (Fig. I ) . The intraperitoneal injection of 5-HTP resulted in a rapid increase in brain 5-HT concentration which reached a peak after 30-60 min. The concentration then returned to normal levels after 2-8 h, depending upon the dosage of 5-HTP. The magnitude of the increase varied with the quantity of 5-HTP administered. The smallest significant increase in brain 5-HT concentration was produced by the administration of 25 mg/kg of 5-HTP. Pharmacology: No overt responses were apparent. ( B ) Pretreatment with iproniazid (Fig. 2). Pretreatment of rats with iproniazid (100 mg/kg, p.0.) for 3 and 6 h resulted in marked increases of brain 5-HT concentrations following the administration of 5-HTP (25 mg/kg). The 5-HT concentration reached a peak 1-2 h after 5-HTP administration, then returned to the same level as References p . 1661167

H. G R E E N A N D J. L. S A W Y E R

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3

0

6

9

12

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21

H A F T E R IPRONIAZID

Fig. 2. Effect of 5-HTP upon brain serotonin concentration of rats pretreated with iproniazid. Each point is an average of 3-6 separate experiments and is compared to average value from a pool of 6 normal brains. Normal brain concentration (pg/g) from 12 experiments (average f S.E.M.) was 0.506 & 0.01 1 . The arrows indicate time after iproniazid that 5-HTP was injected.

that produced by treatment with iproniazid alone 8 h after 5-HTP. Nineteen h after iproniazid the brain M A 0 activity of the rats, both treated and non-treated with 5-HTP, was inhibited more than 90 %. Pharmacology: Fore-paw clonus and slight body tremors with increased motor activity were observed in some animals within 40 min after administration of 5-HTP to rats pretreated with iproniazid 3 h earlier. These responses lasted from 2 to 4 h. Within any group the presence of observable responses could be related to a higher brain concentration of 5-HT. When 5-HTP was administered to rats 6 h after iproniazid treatment, 9 out of 12 exhibited fore-paw clonus within 10 min and body tremors after about 35 min, with ataxia, Straub tail and increased salivation. The brain 5-HT concentration of the 3 rats that did not show the obvious pharmacological responses was significantly lower than the others, ( C ) Pretreatment with tranylcypromine (Fig. 3 ) . Pretreatment of rats with tranyl-

-

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+-t

Tronylcypromine ( 5 mg/kg P O )

5 HTP ( 2 5 m q l k g

t

p )

'5HTP

li)

Y 0

8

2

4

6

8

H AFTER

10

I2

14

16

1

10

TRANYLCYPROMINE

Fig. 3. Effect of 5-HTP upon brain serotonin concentration of rats pretreated with tranylcypromine. Each point is an average of 3-6 separate experiments and is compared to average value from a pool of 6 normal brains. Normal brain concentration (pg/g) from 9 experiments (average 5 S.E.M.) was 0.516 0.012. The arrow indicates time after tranylcypromine that 5-HTP was injected.

EFFECTS O F

5-HYDROXYTRYPTOPHAN

155

350-

a 3000

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._

2 5 0 -

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c 200-

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2

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p )

Fig. 4. Effect of varying doses of 5-HTP upon brain serotonin concentration of rats pretreated with tranylcypromine. 5-HTP was injected 2 h after administration of tranylcypromine, and the rats were sacrificed 1 h later. Each point is an average of 3 separate experiments and is compared to average value from a pool of 6 normal brains. Normal brain concentration from 1 experiment was 0.513 pg/g. Pharmacology: 6.25 mg/kg, intermittent fore-paw clonus and increased motor activity; 12.5 mg/kg, sustained fore-paw clonus, mild body tremors, backward locomotion, salivation, Straub tail ;15.0-20.0 mg/kg, more severe reactions.

cypromine (5 mg/kg, p.0.) for 2 h resulted in a very marked increase in 5-HT concentration following the administration of 5-HTP (25 mg/kg). The 5-HT concentration reached a maximum (590 % above normal) about 2 h after the administration of 5-HTP, then fell steadily during the next 14 h, at the end of which time it reached the same level as that which resulted after the administration of tranylcypromine alone". Pharmacology: All rats showed well-defined fore-paw clonus within 15 min followed by severe body tremors within the next 15 min, which lasted for about 3-4 h. About 20 % of the rats died during the first 3 h after 5-HTP. All rats appeared normal and free of overt pharmacologic responses 16 h after 5-HTP administration. It can be seen from the results in Fig. 4 that the magnitude of the increase in brain 5-HT concentration was directly related to the dose of 5-HTP. In addition, the appearance and intensity of the pharmacologic responses were directly related to the magnitude of the increase in brain 5-HT concentration. (D)Pretreatment with reserpine (Fig. 5 ) . The administration of 5-HTP to rats whose brain level of apparent 5-HT had been depleted by 85 % by the previous administration of reserpine resulted in a marked increase in the 5-HT concentration, the magnitude of which was dependent upon the dose of 5-HTP. The peak increase occurred in 30 min, after which the 5-HT concentration fell to the reserpine-depleted level. With doses of 5-HTP from 25 mg/kg to 100 mg/kg the depleted level was reached about 5 h later, while a somewhat longer period was required for rats treated with 200 mg/kg of 5-HTP. Comparison of the effects of 5-HTP upon the brain 5-HT concentration of normal

* The administration of 5-HTP had no significant effect upon the tranylcypromine-induced increase in brain NE or DA concentration. References p. 1661167

I56

H . G R E E N A N D J. L. S A W Y E R

f-+

150

Reserpine ( 5 r ns /hg.l P I 5 H T P DOSE

I25

(8

p1

- 25 r n ~ l k o - eo rnp,*s c - 100 m s l " 0 A

6l.O

e

:

C 0

L

D -200 mQlW

ec .

s

2 5

2e s o 75

100 0

,-----

1

2

--

-

3

4

5

9

H A F T E R RESERPINE

Fig. 5 . Effect of 5-HTP upon brain serotonin concentration of rats pretreated with reserpine. Each point is an average of 3 separate experiments and is compared to average value from a pool of 6 normal brains. Normal brain concentration (,ug/g) from 14 experiments (average f S.E.M.) was 0.494 t 0.022. Arrow indicates time after reserpine that 5-HTP was injected.

(,PI

5HTP

DOSE

W C -

25mp/kg 50rng/hg

A

100rng/kp D -200rng/kg

-Normal

-----

n

iiiTER

rat*

Resermne pretreated rot

5HTP

Fig. 6. Comparison of effect of 5-HTP upon brain serotonin concentration of normal and reserpinepretreated rats. The: ordinate represents the absolute increase in terms of the normal brain serotonin concentration, calculated from the data of Figs. 1 and 5.

and reserpine-pretreated rats is shown in Fig. 6, in which the absolute increases, as % of normal endogenous content, are plotted. It can be seen that the maximum increase in 5-HT concentration was greater in the normal rats than in the reserpinepretreated rats for all doses of 5-HTP. By calculation, however, it can be shown that the differences between the two groups for each dose of 5-HTP were: 58% for 25 mg/kg, 47% for 50 mg/kg, 10% for 100 mg/kg and 20% for 200 mg/kg. Thus, with the higher doses of 5-HTP the absolute increases in brain 5-HT concentration of reserpine-pretreated rats were not significantly different from those in normal rats. In both groups of rats the rates of decreases in brain 5-HT concentration were

157

EFFECTS O F 5 - H Y D R O X Y T R Y P T O P H A N

TABLE I INTRACELLULAR DISTRIBUTION OF BRAIN RATS PRETREATED W I T H

MA0

5-HT

CONCENTRATION O F

INHIBITOR A N D F O L L O W E D BY

5-HTP

Serotonin concentration Treatment *

Total homogenate

Supernatant

(X

(Pg/gJ

of totnf) ~~

Iproniazid (3.5 h) Iproniazid (3 h), 5-HTP (0.5 h) Tranylcypromine (4 h) Tranylcypromine (2 h), 5-HTP (2 h)

0.577 1.16 1.05 2.88

0.680 1.13 0.869 2.80

0.942 2.88

24.0 25.0 27.3 34.7

28.2 28.8 28.0 33.3

23.1 35.1 * Doses of drugs administered were: iproniazid, 100 mg/kg. p.0.; tranylcypromine, 5 mg/kg, p.0.; 5-HTP, 25 mg/kg, i.p. Pretreatment time, before injection of 5-HTP, is indicated by the time shown in parentheses after the M A 0 inhibitor; rats were sacrificed after 5-HTP at the time indicated.

comparable. Pharmacology: The administration of 5-HTP in doses up to 200 mg/kg had no apparent effect upon the reserpine-induced depression or ptosis. ( E ) Intracellular distribution of brain 5-HT (Table I). Attempts to determine the intracellular distribution of brain 5-HT of normal or reserpine-pretreated rats following the injection of 5-HTP failed because of poor recovery of the total 5-HT from the sucrose homogenate as measured by the amount of 5-HT recovered from an acid homogenate. Recovery was not improved when tranylcypromine was added to the brain suspension in sucrose prior to homogenization. Since it was previously shown (Green and Sawyer, 1962) that normal brain 5-HT concentration was quantitatively recovered from a sucrose homogenate, it is apparent that destruction of the 5-HTPinduced increase of brain 5-HT occurred during the homogenization, presumably by the action of the uninhibited M A 0 activity. Indeed, the formation of 5-hydroxyindoleacetic acid during the homogenization was established. Moreover, in the experiments in which the rats were pretreated with iproniazid or tranylcypromine prior to the injection of 5-HTP, quantitative recovery of the total brain 5-HT was obtained. The results of the intracellular studies in the experiments in which the rats were pretreated with a n M A 0 inhibitor are shown in Table I. It can be seen that the intracellular distribution of brain 5-HT concentration of rats pretreated with iproniazid and injected with 5-HTP was the same as that of rats given iproniazid alone. In the experiments in which rats were pretreated with tranylcypromine, the subsequent injection of 5-HTP resulted in an increase in the % of total 5-HT concentration present in the supernatant from 26 % to 34 %. However, it can be calculated that about 64 % of the 5-HTP-induced increase in brain 5-HT concentration was associated with the particulate material, while 36 % was present in the supernatant. Thus, although the % of total 5-HT concentration in the supernatant increased, a greater percentage of the total was associated with the particulate material. ( F ) Brain concentration of 5-HTP (Fig. 7). The results of previous experiments (Figs. l , 4 and 5) indicated that the increase in brain 5-HT concentration was directly References p. 1661167

158

H . GREEN A N D J . L. S A W Y E R

related to the dose of administered 5-HTP. The marked difference between the 5-HTP-induced increase in brain 5-HT concentration in rats pretreated with tranylcypromine (2 h) and in normal rats suggested the possibility that the availability of 5-HTP in the brain under the 2 cxperimental conditions may have been different. The results shown in Fig. 7 indicate that the measured brain levels of 5-HTP during the

::L

* 00

105

I

2

0,s

I

H AFTER

2

5 HTP ( 2 5 mg/kg. 1.p )

Fig. 7. Effect of pretreatment upon brain 5-HTP concentration of rats following injection of 5-HTP. Pretreatment time and dose of M A 0 inhibitor are indicated at top of column. Each point is an individual value from a sing!e brain. The effect of the indicated experimental condition upon brain serotonin concentration is shown in the previous figures.

first 2 h after the administration of the amino acid were considerably higher in rats pretreated with tranylcypromine or iproniazid than in untreated rats. Indeed, since the conversion of 5-HTP to 5-HT was very marked in the brains of rats pretreated with the M A 0 inhibitors (Figs. 2 and 3) compared to that in untreated control rats, the actual concentration of 5-HTP present in those brains was obviously much higher than indicated by the measured values. Distribution of 5-HT in rat brain (Table I I ) The data in Table IT compare the distribution of 5-HT in various sections of normal rat brain with that following injection of rats with 5-HTP, with and without prior treatment with tranylcypromine. In all cases the increase in 5-HT concentration occurrcd in both the cerebrum and brain stem, and the ratio of the increase in these 2 sections did not appear to be significantly different from the ratio of the 5-HT concentrations found in 9 normal brains (1.76& 0.26). The cerebellum of the normal rat contains a relatively small concentration of 5-HT (0.1 10 ,ug/g) which was not apparently increased 4 h after the oral administration of tranylcypromine. However, following the administration of 5-HTP, in all cases, very marked increases in the 5-HT concentration of the cerebellum resulted. Indeed, the percentage increase in this tissue was greater than either in the cerebrum or brain stem. DISCUSSION

EfSect of 5-HTP upon untreated rats and rats pretreated Mtth M A 0 inhibitor ( A ) Brain 5-HT concentration. The results of the present investigation confirm the

w2 m

T A B L E I1

P

. 4

h

DISTRIBUTION OF SEROTONIN I N RAT BRAIN

c

Average values in parentheses

u rn

Serotonin concentration ( % above normal) * m

Treatment**

Brain stern* * *

Cerebrum

n .

Brain stern/ cerebrum

Cerebellum 5

m 0

* v1

5-HTP (50 mg/kg, 0.5 h)

53

46

5-HTP (100 mg/kg, 2 h)

128

T (4 h)

53

(51)

58

98

132

121 (127)

135

127

136

118

126 (127)

188

T (2 h), 5-HTP (6.25 mg/kg, 0.5 h)

233

208

200 (214)

T (2 h), 5-HTP (6.25 mg/kg, 2 h)

323

245

T (2 h), 5-HTP (25 mg/kg, 0.5 h)

416

T (2 h), 5-HTP (25 mg/kg, 2 h)

342

63

(73)

(85)

1.76

2.32

1.82 (1.95)

106 (123)

(228)

1.76

1.66

1.59 (1.67)

193

130 (170)

(0)

2.04

2.29

1.74 (2.02)

253

267

253 (258)

(343)

1.78

2.01

1.99 (1.92)

257 (275)

373

375

312 (353)

(345)

1.88

2.33

1.95 (2.05)

360

519 (432)

434

410

452 (433)

(1013)

1.77

1.89

1.52 (1.70)

494

652 (496)

414

480

594 (496)

(846)

1.99

1.67

1.57 (1.71) ~

*

Normal distribution of 9 brains @gig i S.E.M.): cerebrum, 0.347 i0.021 ; brain stem, 0.590 & 0.24; brain stem/cerebrum, 1.76 f 0.26; cerebellum, 0.1 10 (average of 3 pools of 3 tissues). * * T = tranylcypromine (5 mg/kg, p.0.). *** Brain stem represents brain section remaining after removal of cerebrum and cerebellum. Average weight of sections, g & S.E.M. (number of samples): cerebrum, 0.913 0.009 (30); brain stem, 0.532 & 0.008 (30); cerebellum, 0.232 0.011 (average of 10 pools of 3 tissues). 8 Average of a pool of 3 tissues.

0

a

160

H. GREEN A N D J. L. S A W Y E R

observations of Udenfriend et al. (1957) that 5-HTP readily penetrated the central nervous system of the rat and was rapidly converted to 5-HT in the brain. Tt is quite apparent, however, that the time-course of changes in the brain 5-HT concentration, following the administration of either tranylcypromine or iproniazid, w:.s quite different from that following the administration of 5-HTP to rats pretreated with either M A 0 inhibitor (Figs. 2-3). Thus, in the latter circumstances, after a marked increase in brain 5-HT concentration 1-2 h following the administration of 5-HTP, the 5-HT concentration fell rather rapidly and after 8- I6 h reached the same elevated level as that which resulted from the continuous action of the M A 0 inhibitor itself upon endogenous brain 5-HT. This contrasted sharply with the continuous rise in brain 5-HT concentration for 12-16 h after the administration of tranylcypromine or iproniazid to normal rats. Since the tranylcypromine or iproniazid inhibition of brain M A 0 activity was not reversed for at least 16 h after the administration of 5-HTP, it is apparent that the rapid decrease of the elevated brain 5-HT concentration was probably not due to M A 0 activity. It logically follows, therefore, that since the increase in 5-HT concentration induced by 5-HTP in the brains of rats pretreated with the M A 0 inhibitor disappeared during the time that the enzyme activity remained inhibited, the disposition of this 5-HT was different from that of the increase in endogenous 5-HT concentration following the M A 0 inhibitor itself. The question now arises, why was the increase in brain 5-HT concentration, following the administration of 5-HTP to rats pretreated with M A 0 inhibitor, markedly higher than in untreated rats, for comparable doses of the amino acid (25 mg/kg). Tt is neither reasonable nor consistent to presume that the much greater increase in brain 5-HT concentration in rats pretreated with the M A 0 inhibitor reflected the inhibiton of the metabolism of the rapidly formed 5-HT, since a similar mechanism is unable to explain the disappearance of the accumulated 5-HT during the subsequent time while the M A 0 activity remained inhibited. The discussion of the biochemical results thus far involves at least 2 fundamental considerations, namely, the factors responsible for ( I ) the rapid and marked increase in brain 5-HT concentration of rats pretreated with M A 0 inhibitor and following the administration of 5-HTP, and (2) the subsequent complete disappearance of the increase while the brain M A 0 activity remained inhibited. A clue to possible factors responsible for the first consideration arose out of the observation that the administration of 5-HTP to rats treated with tranylcypromine 2 h earlier resulted in a brain 5-HT concentration 600 % above normal, while in rats treated with iproniazid 3 h earlier the resulting brain 5-HT concentration was only 218% above normal. Since in earlier experiments (Green and Erickson, 1960) it had been found that rat brain M A 0 activity was virtually completely inhibited 3 h after the oral administration of 100 mg/kg of iproniazid or 1 h after the oral administration of 5 mg/kg of tranylcypromine, this difference in the effect of 5-HTP suggested that the rapid and marked increase in brain 5-HT concentration may not be a reflection of the inhibition of the breakdown of the rapidly formed amine by M A 0 activity. It may be suggested, of course, that iproniazid, being a hydrazide, may have partially inhibited 5-HTP decarboxylase activity and thereby decreased the rate of formation

EFFECTS OF 5 - H Y D R O X Y T R Y P T O P H A N

I61

of 5-HT. This is unlikely, however, particularly in view of the well-documented ability of a single dose, as well as repeated daily oral administrations, of iproniazid to produce marked incrcases in rat brain 5-HT concentration (Green et a!., 1962). Furthermore, i n the present experiments, prolonging thc pretrcatment time of iproniazid to 6 h, prior to the administration of 5-HTP, resulted in a much greeter increase than aftcr a pretreatment time of 3 h. Also, there is no evidence that iproniazid inhibits 5-HTP decarboxylase activity of rat brain in vivo. It is possible, however, that the extent of in vivo inhibition of brain M A 0 activity, as assayed in a homogenate of the tissue, may not represent the effect of the inhibitor in situ in all parts of the brain where the enzyme may be present. Therefore, iproniazid may enter various areas of the brain at different rates and 6 h, rather than 3 h, may be required for it to inhibit M A 0 activity in all areas of brain where exogenously administered 5-HTP may penetrate. Tranylcypromine, on the other hand, has been shown to act more quicldy upon brain M A 0 activity after a single administration (Green and Erickson, 1960), and, therefore, may be considercd to penetrate more rapidly into the various areas of the brain. However reasonable this explanation may appear, the existence of a direct relationship between the dose of 5-HTP and the resulting increase in brain 5-HT concentration in normal untreated rats, as well as in rats pretreated with tranylcypromine, suggested that the increase in brain 5-HT concentration was more directly related to the relative availability of 5-HTP in the brain, and that inhibition of brain M A 0 activity was not a necessary or singular prerequisite for such an increase, Therefore, the possibility that pretreatment of rats with the M A 0 inhibitor may have increased the availability or penetration of 5-HTP in the brain was tested. The results shown in Fig. 7 lent support to this concept, since the increase in brain 5-HTP content after administration of the amino acid was significantly higher in rats pretreated with the M A 0 inhibitor than in untreated rats. The difference in brain 5-HTP content was even more pronounced when the measured value was corrected for the amount converted to 5-HT. Although the factors responsible for the complete disappearance of the 5-HTPinduced increase of brain 5-HT concentration, while the M A 0 activity was inhibited, remain to be elucidated, the following considerations are pertinent: (I) It is conceivable that the very rapid and marked increase in brain 5-HT concentration exceeded the capacity of the cells to retain or to incorporate the amine into their storage depots. Under these conditions the newly formed 5-HT may have diffused out of the brain cells into the blood or the cerebrospinal fluid. Not in support of this concept, however, is an earlier demonstration of the existence of a dynamic steady-state relationship between the brain 5-HT concentration in the soluble cytoplasm and that associated with particulate material (Green and Sawyer, 1962). Marked increases in endogenous brain 5-HT concentration were shown to persist for several days in rats which received repeated daily administrations of tranylcypromine or iproniazid. Furthermore, in the present investigation the results shown in Table 1 indicate also that 60% of the 5-HTP-induced increase in brain 5-HT was associated with the particulate material. Admittedly the results obtained from the high-speed centrifugation studies with sucrose-brain homogenates may not reflect the true in situ iiitracellular picture. References p . 1661167

162

H. G R E E N A N D I. L. S A W Y E R

Nevertheless, even assuming the possibility that the rapid formation of brain 5-HT may have exceeded the storage capacity of the brain, then it is reasonable to expect that not all of the 5-HTP-induced increase in brain 5-HT would have disappeared from the brain, but that the 5-HT concentration should have reached a level significantly elevated above that produced by the effect of the M A 0 inhibitor alone. This expectation, of course, does not accord with the results of the present investigation. (2) The rapid formation of a large amount of brain 5-HT in rats pretreated with an M A 0 inhibitor may have called forth the operation of an alternate metabolic pathway by which the 5-HT is metabolized. Although it has been well established that endogenous brain 5-HT is metabolized preponderantly via the M A 0 pathway, recently N-acetyl-5-methoxytryptamine (melatonin) was shown to be present in the pineal gland of man, monkey and cow (Lerner et a/., 1958, 1959a,b). Also Axelrod (1960) demonstrated the presence of an enzyme system in cows’ brains which was capable of methylating N-acetyl-5-hydroxytryptamine (N-acetylserotonin) to the corresponding methylated derivative (melatonin). However, in the present study no measurable amounts of the N-acetyl, the 5-methyl and the N-acetyl-5-methyl derivatives of serotonin could be detected in brains of rats pretreated with tranylcypromine and after the administration of 5-HTP. In view of these clear differences between the kinetics of disposition by the rat brain of the 5-HTP-induced increase of 5-HT and that resulting from the action of the M A 0 inhibitor alone, it may be inferred that different pools of brain 5-HT are involved. As to the comparative anatomic localizations of the two pools of 5-HT, the results of Table I1 indicate that the major bulk of the 5-HTP-induced increase in 5-HT occurred in the cerebrum and brain stem sections and that the distribution of this 5-HT in these sections was not significantly different from the distribution of endogenous 5-HT in normal brain or in brains of rats administered tranylcypromine alone. Although the 5-HT concentration in the cerebellum increased most markedly after injection of 5-HTP, the actual increase represented only a relatively small proportion (about 20%) of the increase in the whole brain. The fact that no apparent increase could be detected in the cerebellar 5-HT concentration after administration of tranylcypromine suggests that the metabolism of endogenous 5-HT in this section may not proceed via the M A 0 pathway. Thus, while the results of the present investigation suggest that the 5-HT formed from exogenously administered 5-HTP may constitute a pool different from endogenously formed 5-HT, they do not indicate different anatomic localizations of the two pools. The present results are in agreement with those of Costa et a/. (1961), Bogdanski et al. (1958) and Himwich and Costa (1960) who reported marked increases in 5-HT concentration in most of the component parts of the rabbit and dog brains after the administration of 5-HTP, with or without prior treatment with tranylcypromine. Also, the increase in 5-HT occurred even in those areas of the brain where the normal concentration of 5-HT is relatively small. Returning now to a consideration of the mechanism whereby the elevated 5-HT concentration disappeared, in the absence of any measurable M A 0 activity or any

EFFECTS O F 5 - H Y D R O X Y T R Y P T O P H A N

163

evidence for the presence of other possible 5-HT metabolites, one cannot ignore the possibility of diffusion into the blood and/or into the cerebrospinal fluid. Admittedly while there is no evidence of 5-HT penetrating the brain from the general blood circulation (Udenfriend et al., 1957), Bogdanski et a]. (1958) have reported a small increase in 5-HT concentration in the cerebrospinal fluid of dogs 2 h after the i.v. administration of 60 mg/kg of 5-HTP. Further work along these lines is necessary. The fundamental differences between the changes in endogenous brain 5-HT concentration and those in the brain 5-HT concentration derived from exogenously administered 5-HTP raise the question as to the source of the immediate precursor of the endogenous amine in the brain. In this connection it is interesting to note that the temporal kinetics of brain 5-HT concentration following the intraperitoneal injection of L-tryptophan into rats pretreated with tranylcypromine were similar to those following the injection of 5-HTP (Green and Sawyer, unpublished results). Thus, the available evidence suggests that extracranial tryptophan or 5-HTP may not be the immediate biological precursor of brain 5-HT, but that the brain proteins themselves may uniquely provide the amino acid necessary. This phenomenon, however, implies that the brain is capable of hydroxylating tryptophan to 5-HTP, a reaction so far not demonstrated to take place in that tissue (Cooper and Melcer, 1961). ( B ) Correlation of pharmacologic activity with biochemical changes. Only in rats pretreated with an M A 0 inhibitor could observable pharmacologic responses (forepaw clonus, severe body tremors, convulsions) be elicited by the injection of 5-HTP. The appearance and intensity of the responses seemed to be dependent upon the magnitude of the increase in brain 5-HT concentration (Fig. 4), with responses clearly visible in rats whose brain 5-HT concentration was raised about 177 % above normal, or only 52% higher than the concentration that resulted from the administration of tranylcypromine alone. However, the absence of any observable responses in rats administered 200 mg/kg 5-HTP, without pretreatment with M A 0 inhibitor, despite an increase of brain 5-HT concentration to levels of 250-300 % (average 280 %) above normal (Fig. l), and in rats whose brain 5-HT concentration was elevated 300-400 % above normal by repeated daily administration of tranylcypromine or iproniazid (Green et al., 1962), indicated that such elevated brain levels of 5-HT are not a sufficient condition for the elicitation of observable pharmacologic responses, as described in the present report. Moreover, it is reasonable to conclude, in accord with the suggestion made previously, that the 5-HTP-induced increase in brain 5-HT concentration represented an amine pool that was different from that of the increase resulting from the action of the M A 0 inhibitor alone. While the available evidence argues against the concept that the pharmacologic responses were dependent upon a critical threshold level of brain 5-HT concentration, it is conceivable that the 5-HTP-induced responses may have been related to a smaller concentration of 5-HT at some effector site made accessible and/or sensitive to 5-HT by pretreatment with tranylcypromine or iproniazid. In any event, it does not seem justified to ascribe the 5-HTP-induced pharmacologic responses to increases in endogenous brain 5-HT concentration. References p . 1661167

164

H . G R E E N A N D J. L. S A W Y E R

Efect o j ' 5 -H T P upon rats pretreated with reserpine Previous investigators (Carlsson et al., 1957; Bogdanski et a ] . , 1958) reported that large doses of 5-HTP did not reverse reserpine-induced stupor or sedation in mice, rats, dogs and cats. These observations were confirmed in the present study and extended to show that the overt pharmacologic effects of reserpine were not reversed by 5-HTP in doses up to 200 mg/kg, despite the restoration of the brain 5-HT concentration to normal and 150 % above normal values. One might infer from these results that the relative level of brain 5-HT concentration may not be the fundamental criterion for the elicitation of sedation by reserpine. However, the results of attempts to determine the intracellular distribution of the 5-HT derived from exogenous 5-HTP between the particulate material and the soluble cytoplasm again revealed different biochemical properties of this pool of 5-HT compared to endogenous 5-HT. Thus, while sucrose homogenates of brains from untreated rats gave the same total 5-HT concentration as brains homogenized in 0.1 N HCI, the 5-HTP-induced increase in brain 5-HT concentration was only partially recovered from sucrose homogeiiates. This was true whether or not reserpine was administered to the rats prior to the injection of 5-HTP. The failure to achieve complete or quantitative recovery was probably due to the action of MAO, since the loss of 5-HT was accounted for by the formstion of 5-hydroxyindoleacetic acid. Also, pretreatment of rats with tranylcypromine or iproniazid permitted the 5-HTP-induced increase in brain 5-HT to be quantitatively recovered from sucrose homogenates. However, the addition of tranylcypromine to the sucrose solution prior to homogenization of brains of rats not administered the M A 0 inhibitor did not enhance the recovery of the 5-HT derived from the administered 5-HTP. This disparity in relative stability between endogenous 5-HT and 5-HT derived from administered 5-HTP lends support to the concept that 2 pools of amines, with perhaps different biochemical and physiological properties, are herein represented. Thus, although 5-HTP is a useful experimental pharinacologic agent, the results of such studies should not be uncritically interpreted in terms of the physiologic properties of endogenous brain 5-HT. In view of the foregoing discussion it seems appropriate to call attention to a report by Brodie et al. (1957) that reserpine impaired the ability of rabbit brain to store 5-HT following the administration of 5-HTP. This conclusion was based upon the finding that 1 h after intraperitoneal administration of 50 mg/kg of 5-HTP, the brains of normal rabbits took up 4 times as much 5-HT from 5-HTP as did the brains of rabbits treated 16 h earlier with reserpine (5 mg/kg, i.v.). The results of the present kinetic studies (Fig. 5), however, showed that the extent of the increase in brain 5-HT concentration of both normal and reserpine-pretreated rats was dependent upon the dosage of 5-HTP and the time after administration of the amino acid. At the higher doses of 5-HTP 110 marked difference was found in the maximum increase in brain 5-HT concentration of normal and reserpine-treated rats. Moreover, in both groups of animals, after attainment of the peak increase, the 5-HT concentrations fell at approximately the same rates to the levels existent prior to the administration of 5-HTP. Obviously, the effect of reserpine upon the conversion of exogenously ad-

EFFECTS OF 5 - H Y D R O X Y T R Y P T O P H A N

165

ministered 5-HTP to 5-HT in the brain and/or upon the uptake of 5-HT by the brain was more apparent at the lower concentrations of 5-HTP, while at the higher concentrations the rate of conversion and/or uptake was the same as in normal rats. These observations lend support to our previous suggestion (Green and Erickson, 1962) that, although brain 5-HTP decarboxylase activity was apparently not inhibited by reserpine, the drug may have disrupted the functional in vivo activity of the enzyme. Consequently, when exogenous 5-HTP was administered, the availability of excess substrate may have reversed the interference due to reserpine and restored the functional activity of the enzyme. This sequcnce of events, of course, would be expected to be dependent upon the dosage of 5-HTP administered as, indeed, was the case. It is particularly pertinent to the present discussion that recently Drain et a/. (1962) reported that the dose of N-(3-hydroxybenzyl) N-methylhydrazine (NSD 1034) which inhibited mouse brain 5-HTP decarboxylase activity by 75 %, without any effect on the rate of formation of endogenous 5-HT (Brodie et al., 1962; Hirsch et a/., 1962), did block the formation of 5-HT from exogenous 5-HTP. These findings suggest that 2 pools of decarboxylase enzymes may exist in the mouse brain and lend support to the concept that 5-HT derived from exogenously administered 5-HTP is biochemically different from 5-HT formed from endogenous precursors. SUMMARY

The kinetics of brain serotonin (5-HT) concentration and the observable pharmacological responses induced by the intraperitoneal administration of DL-5-hydroxytryptophan (5-HTP) were studied in untreated normal rats and in rats pretreated with the monoamine (MAO) inhibitors tranylcypromine or iproniazid and reserpine. The ability of 5-HTP to raise the brain 5-HT concentration increased with increasing dosages of 5-HTP, at least up to 200 mg/kg. Except at the lower dosages, the absolute increase in brain 5-HT concentration of reserpine-pretreated rats was not significantly different from that of normal untreated rats. In rats pretreated with either M A 0 inhibitor the 5-HTP induced increase in brain 5-HT concentration was markedly higher than in untreated rats. It was found that pretreatment with the M A 0 inhibitor increased the penetration of 5-HTP into the rat brain, resulting in a much greater increase in the amount of 5-HT formed per unit of time. In sharp contrast to the continued increase of brain 5-HT concentration for 12-16 h in rats given the M A 0 inhibitor alone, the HTP-induced increase in rats pretreated with the M A 0 inhibitor reached a maximum within 2 h and then disappeared within the next 6-12 h. This observation, together with others, suggested that the 5-HTP-induced increase in brain 5-HT Concentration constituted a pool different from that of the increase in endogenous brain 5-HT concentration. Studies of the distribution of 5-HT in the cerebral and brain stem sections, under the experimental conditions, did not indicate different anatomic localizations of the 2 pools. The appearance and intensity of the pharmacologic responses (forepaw clonus and body tremors) induced by 5-HTP in rats pretreated with the M A 0 inhibitor were References p . 166/167

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dependent upon the dosage of 5-HTP administered, and could be correlated with increased levels of brain 5-HT concentration. However, in normal rats administered 5-HTP or given repeated daily oral administrations of either M A 0 inhibitor, observable pharmacologic responses were not elicited, despite equal or even larger increases in brain 5-HT concentration. Consequently, it was concluded that elevated brain levels of 5-HT are not a sufficient condition for the elicitation of overt pharmacologic responses in rats. The results of the present study clearly emphasize the biochemical and pharmacological differences between brain 5-HT formed from endogenous precursors and that formed from exogenously administered 5-HTP. The data also argue against the concept that the biological precursor (5-HTP) of rat brain 5-HT is formed outside of the brain, and suggest that the brain proteins themsclves may provide the amino acid precursor. REFERENCES AXELROD, J., AND WEISSBACH, H., (1960); Enzymatic 0-methylation of N-acetyl-serotonin to melatonin. Science, 131, 1312. BOGDANSKI, D. F., WEISSBACH, H., A ~ UDENFRIEND, D S., (1957); The distribution of serotonin, 5hydroxytryptophan decarboxylase, and monoamine oxidase in brain. J . Neurochem., 1 , 272-278. BOGDANSKI, D. F., WEISSBACH, H., AND UDENFRIEND, S., (1958); Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J . Pharmacol. exp. Ther., 122, 182-194. BRODIE, B. B., KUNTZMAN, R., HIRSCH,C. W., AND COSTA,E., (1962); Effects of decarboxylase inhibition on the biosynthesis of brain amines. Life Sciences, 1, 81-84. BRODIE, B. B., TOMICH, E. G., KUNTZMAN, R., AND SHORE, P. A., (1957); On the mechanism of action of reserpine: effect of reserpine on the capacity of tissues to bind serotonin. J . Pharnracol. exp. Ther., 119,461467. CARLSSON, A., LINDQVIST, M., AND MAGNLJSSON, T., ( I 957); 3,4-Dihydroxyphenylalanineand 5hydroxytryptophan as reserpine antagonists. Nature (Lond.), 180, 1200. CLARK, C . T., WEISSBACH, H., AND UDENFRIEND, S., (1954); 5-Hydroxytryptophan decarboxylase: preparation and properties. J . biol. Chem., 210, 139-148. COOPER, J. R., A N D MELCER, I., (1961); The enzymatic oxidation of tryptophan to 5-hydroxytryptophan in the biosynthesis of serotonin. J. Pharrnacol. exp. Ther., 132, 265-268 COSTA,E., HIMWICH, W. A., AND HIMWICH, H. E., (1961); 5-HT content of brain structures of dogs given 5-HTP (5-hydroxytryptophan) compared with the degree of behavioral and neurological changes. Proc. 2nd Coll. Intern. Neuro-Psychopharmacol. Oxford, Pergamon (p. 475-478). COSTA,E., AND RINALDI, F., (1958); Biochemical and electroencephalographic changes in the brain of rabbits injected with 5-hydroxytryptophan (influence of chlorpromazine premedication). Arner. J . Physiol., 194, 214-220. DRAIN, D. J., HORLINGTON, M., LAZARE, R., AND POULTER, G . A., (1962); The effect of a-methyldopa and some other decarboxylase inhibitors on brain 5-hydroxytryptamine. Life Sciences, 1, 93-97. DRUJAN, B.D., SOURKES, T. L., LAYNE, D.S., AND MURPHY, G. F., (1959); The differential determination of catecholamines in urine. Canad. J . Biochem., 37, 1153-1 159, GEY,K. F., AND PLETSCHER, A., (1960); Post-mortem increase of 5-hydroxytryptamine in rat brain after 5-hydroxytryptophan administration. Experientia (Basel), 16, 372-373. GREEN,H., AND ERICKSON, R. W., (1960); Effect of trans-2-phenylcyclopropylamineupon norepinephrine concentration and monoamine oxidase activity of rat brain. J . Pharmacol. exp. Ther., 129,237-242. GREEN, H., AND ERICKSON, R. W., (1962); Further studies with tranylcypromine (monoamine oxidase inhibitor) and its interaction with reserpine in rat brain. Arch. int. Pharrnacodyn., 135, 407425. GREEN,H., AND SAWYER, J. L., (1960); Intracellular distribution of norepinephrine. I. Effect of reserpine and the monoamine oxidase inhibitors, trans-2-phenylcyclopropylamine and I -isonicotinyl-2-isopropylhydrazine.J . Pharinacol. exp. Ther., 129,243-249. GREEN, H., AND SAWYER, J. L., (1962); Intracellular distribution of serotonin in rat brain. I . Effect of reserpine and the monoamine oxidase inhibitors, tranylcypromine and iproniazid. Arch. int. Pharmacodyn., 135,426-441.

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GREEN, H., SAWYER, J. L., ERICKSON, R. W., AND COOK,L., (1962); Effect of repeated oral administration of monoamine oxidase inhibitors on rat brain amines. Proc. SOC.exp. Biol. ( N . Y . ) , 109, 347-349. HESS,s. M., AND DOEPFNER, W., (1961); Behavioral effects and brain amine content in rats. Arch. int. Pharmacodyn., 134, 89-99. HIMWICH, W. A., AND COSTA, E., (1960); Behavioral changes associated with changes in concentrations of brain serotonin. Fed. Proc., 19, 838-845. HIRSCH,C. W., KUNTZMAN, R., AND COSTA,E., (1962); Effects of DOPA-5-HTP decarboxylase inhibition on synthesis of brain amines. Fed. Proc., 21, 364. LERNER, A. B., CASE,J. D., AND HEINZELMAN, L., (1959b); Structure ofmelatonin. J.Amer. chem. Soc., 81, 6084-6085. LERNER, A. B., CASE,J. D., MORI,W., AND WRIGHT,W. (L959a); Melatonin in peripheral nerve. Nature (Lond.), 183, 1821. LERNER, A. B., CASE,J. D., TAKAHASHI, Y., LEE,T. H., AND MORI,W., (1958); Isolation of melatonin, the pineal gland fact01 that lightens melanocytes. J . Amer. chem. Soc., 80, 2587. MCISAAC,W. M., AND PAGE,I. H., (1959); The metabolism of serotonin (5-hydroxytryptamine). J. biol. Chem., 234, 858-864. PLETSCHER, A., SHORE, P. A., AND BRODIE, B. B., (1956); Serotonin as a mediator of reserpine action in brain. J. Pharmacol. exp. Ther., 116, 84-89. SHORE,P. A., AND OLIN,J. S., (1958); Identification and chemical assay of norepinephrine in brain and other tissues. J . Pharmacol. exp. Ther., 122, 295-300. SJOERDSMA, A,, SMITH,T. E., STEVENSON, T. D., AND UDENFRIEND, S., (1955); Metabolism of 5hydroxytryptamine (serotonin) by monoamine oxidase. Proc. SOC.exp. Biol. Med. ( N . Y.), 89,36-38. SMITH,S. E. (1960); The pharmacological actions of 3,4-dihydroxyphenyl-a-methylalanine(amethyl DOPA), an inhibitor of 5-hydroxytryptophan decarboxylase. Brit. J. Pharmacol., 15, 319-327. TEDESCHI, D. H., TEDESCHI, R. E., AND FELLOWS, E. J., (1959); The effects of tryptamine on the central nervous system, including a pharmacological procedure for the evaluation of iproniazid-like drugs. J. Pharmacol. exp. Ther., 126, 223-232. UDENFRIEND, S., WEISSBACH, H., AND BOGDANSKI, D. G., (1957); Increase in tissue serotonin following administration of its precursor, 5-hydroxytryptophan. J . biol. Chem., 224, 803-810.

168

Concept of the Neurochemical Transducer as an Organized Molecular Unit at Sympathetic Nerve Endings E. C O S T A

AND

B. B. B R O D I E

Lahortitory of Cheiiiical Pharmacology, National Heart Institute, National Institirtes of Health, Betliesda, Md. ( U.S.A.)

At one time all drugs that influence sympathztic nerve endings were thought to act directly on the receptor site. Then reserpine was found to exert its pharmacologic effects by impairing the storage of monoamines and the term “storage site” was introduced. When drugs appeared which act on the sites of inactivation, formation and physiological release, we began to think of these sites as parts of single biophysical units responsible for the formation, storage, inactivation and release of the amines. Since these organized molecular units mediate the change of one kind of energy to another, the term “neurochemical transducer” was coined to describe them. Thus, at the nerve ending, electrical impulses act on a neurochemical transducer and are translated into a quantity of free neurohormone. The free amine, in turn, acts on a target organ to produce chemical or mechanical energy, or on an adjacent neuron to produce mor: electrical impulses (Fig. 1). I n biology we are constantly searching for convergence points, where biochemistry and physiology can be bridged. The neurochemical transducer may b: regarded as ELECTRICAL IMPULSES NEUROCHEMICAL TRANSDUCER

..

(STORED NEUROHORMONE 1 /

FREE NEURO~ORMONE

TARGET ORGAN

TARGET NEURON

-CHEMICAL OR MECHANICAL ENERGY

ELECTRICAL IMPULSES

Fig. 1. Schematic representation of the peripheral and central targets for the amines released from the neurochemical transducer (amine storage compartment in ncrve endings).

CONCEPT OF THE NEUROCHEMICAL TRANSDUCER

169

such a convergence point since it contains a store of norepinephrine (NE), epinephrine (E), dopamine or acetylcholine, and is present in each of the billions of synapses and nerve endings in the body. These units control the amount of free neurohormone at receptor sites, thus allowing the organism to respond to environmental change. The structure and mechanism of action of these units may, therefore, be looked upon as one of the critical problems in biology. Since the structure of these organized units is destroyed by homogenization, their function must be studied in the living animal or in the intact organ tissue. In proposing a structure for the neurochemical transducer we have utilized the deductive approach applied so successfully in organic chemistry. Chemists of the early analytical period appreciated the importance of discovering the manner in which atoms are arranged in individual molecules, that is, of determining the structures, but they saw no way of doing it. For many years the problem remained at a standstill until sufficient empirical data were available to propose certain premises, such as that the carbon atom always has a valency of four and is tetrahedral, and that benzene is a ring with resonating double bonds. Today we draw structures like flavin-adenine dinucleotide or adenosine triphosphate, confident that they are “right,” though strict proof is lacking for the original premises on which the structures are based. But the validity of structural theory corresponds so well with theoretical predictions that rigid proof becomes superfluous. Deductive reasoning is also applicable in formulating the structure of the neurochemical transducer. In this approach a tentative structure for the transducer is suggested based on empirical observations. From this model certain inferences are made which are then checked by appropriate experiments. The results often lead to an expansion of the original model. In this approach certain rules are followed: ( I ) the disciplines imposed by physical chemistry must not be ignored; (2) concepts should not be considered unless they can be subjected to experimental proof; (3) the proposed model must be self-developing,that is, suggest experiments that will lead to its own expansion. By following these rules one need not be hampered by the traditional conservatism of pharmacology, since changes in the model made necessary by the acquisition of new data merely mean that the new structure is less “wrong” than the old one. We will now attempt to develop a model of the neurochemical transducer in terms of what we know about NE in nerve endings. Endogenous NE in nerve tissue is present in a relatively inactive form. On energetic grounds it may be presumed that NE is sequestered from inactivating enzymes by a membrane which also prevents free diffusion of the amine onto reactive sites. Since NE is a highly polar compound the barrier may be regarded as lipoid layer (Fig. 2). But a lipoid membrane by itself will not explain the storage of NE since the amine is held in nerve endings at a much higher level than in the extracellular fluid. At this point we shall discuss the uptake and release of serotonin (5HT) in platelets since these studies have a bearing on the general problem of amine storage. Since one molecule of reserpine can release hundreds of 5HT molecules from platelets, it seems References p. 1841185

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logical to suggest that the alkaloid releases 5HT by blocking a carrier mechanism (Carlsson et a/., 1957).

Fig. 2. Norepinephrine (NE) in nerve endings is not freely available to receptors and inactivating enzymes. Therefore, N E must be stored in compartments.

0

10

20

30

40

PLASMA SEROTONIN pg /ml

Fig. 3. 5HT content (+ S.E.) in platelets of normal and reserpine-treated guinea-pigs after incubation (1 h) with various concentrations of amine. The figures in parentheses represent the numker of observations. The broken line represents the proportion of 5HT uptake which is blocked by reserpine.

Kinetic data indicate that reserpine blocks an active transport system that normally maintains a high level of 5HT in platelets (Hughes et al., 1959). The comparison of 5HT uptake by platelets of control and reserpine-treated animals (Fig. 3) shows that there are two components to the uptake of 5HT by platelets: ( I ) a saturable process that is blocked by reserpine and (2) a reserpine-insensitive process, interpreted as a passive diffusion. These data suggest that the efflux of 5HT from platelets occurs by a passive diffusion having a low diffusion constant, so that relatively high internal concentrations are required to drive out 5HT at a rate equal to the active inward flux. In such a system, the inhibition of inward transport by reserpine could account for the depletion of stored amines by this drug. Studies of the accumulation of catecholamines into platelets show that NE and E are also taken up by a reserpine-sensitive process (Hughes et al., 1959).

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171

It is not possible from these data to conclude whether the uptake occurring in the absence of reserpine involves a process of active transport or of intracellular binding since the amine levels in platelets were measured after 1 h of incubation and accordingly are virtually steady state values. In order to show that the uptake of 5HT is carried out by a process of active transport, evidence is needed that reserpine acts at cellular surfaces. This evidence was obtained by measuring the net uptake of 5HT after a short time of incubation. Thus, after 15 and 30 min, 5HT passes into normal platelets much more rapidly than into platelets from guinea-pigs pretreated with reserpine (Fig. 4). Moreover, the rate of uptake does not appreciably change between 15 and 30 min, indicating that the amount of 5HT inside the platelet is too low to produce an appreciable effect on the influx. Since the accumulation of 5HT in 15 min is only a fraction of that at the steady state level, the influx at this time is essentially the same as at zero time. These results indicate that irrespective of the extent of the intracellular

w

0

k

0 25

W W

I-

4

0

30 T I M E MIN

15

45

60

Fig. 4. Uptake of 5HT by platelets of normal and reserpine-treated guinea-pigs after various times of incubation. Concentration of 5HT in medium 6.7 ,ug/ml. Points denote means of 4experiments.

binding of 5HT, reserpine decreases the influx of the amine by acting on cellular surfaces. In additional studies, tracer amounts of labeled 5HT were added to a platelet suspension after a steady state had been attained with non-labeled 5HT. The uptake of labeled 5HT is rapid, although the net uptake of nonlabeled 5HT is zero, indicating that a concentrating mechanism is in continual action (Fuks et al., 1963). Other studies have shown that the active transport is poisoned by ouabain and fluoride (Weissbach et al., 1960). Preliminary results from our laboratory show that N-ethylmaleimide (10-3 M ) not only blocks the active uptake of 5HT and NE by platelets but also causes the amines to be released (Fuks et al., 1963). The storage of NE in nervous tissue must be a more complex process than in platelets since sites of storage, formation and inactivation are close together, not to mention the further complication of the presence of amine-containing granules. Since References p. I841185

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reserpine blocks active transport in platelets, it is not unreasonable to suspect that it blocks a similar process in the nervous system. Favoring this view are kinetic studies by Titus and his associates from this laboratory (Dengler et a]., 1961, 1962) showing that brain and heart slices take up labeled NE against a concentration gradient and that the uptake is blocked by reserpine and poisoned by ouabain (Fig. 5). Again, a passive reserpine-insensitive mechanism is

Min

Fig. 5 . Ratio of isotope concentration in slices of cat cerebral cortex to concentration of [3H]NE in Krebs bicarbonate medium. Vertical bars: standard deviations. Curve 1 : controls; Curve 2 : animals pretreated 24 h before with 3 mg/kg i.p. of reserpine; Curve 3 : in presence of M ouabain.

predominant at high concentrations of NE. Since reserpine blocks the initial rate of entry, these workers conclude that the uptake of N E proceeds by an active transport mechanism with a carrier, perhaps an enzyme carrying the amine across membrane. They argue that if reserpine acted by preventing binding inside the membrane one might expect that the initial rate of entry of labeled amine would be the same for normal and reserpine-treated cells. Included in these experiments was one showing that pretreatment of brain slices with unlabeled N E does not slow the uptake of labeled NE, indicating in another way that the NE uptake system is continually acting. These investigators pointed out that an important difference between the uptake of NE by tissue slices and the uptake of 5HT by platelets is that the isotopic equilibrium between labeled NE in slices and in medium is not reached at a time when [3H]NE levels in slices are no longer increasing. These results indicate that labeled NE is taken up by active transport into one pool, a readily miscible pool and mixes more slowly with a second pool. These results are consistent with the view that endogenous N E is present partly as a mobile pool (Hillarp, 1960) into which the amine is taken up from the circulation and from which it is released by nerve impulses. The mobile pool may be presumed to equilibrate with a larger pool of NE held in granules, presumably by complexing with intragranular components. The NE in granules can be considered to act as a reserve pool of amine.

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We can now add an active transport mechanism to the picture of the neurochemical transducer (Fig. 6). Since the NE is formed inside the storage compartment, the transport system is btst pictured as a pump (heavy arrow) which prevents the ready passage of the amine across the membrane. NE can also leave the compartment by passive diffusion (dotted arrow) ; although the diffusion coefficient of the highly polar molecule NE across the lipoid membrane is relatively low, the concentration gradient will become sufficiently high that the force of outward diffusion will finally balance the inward force of active transport. This combination of an active transport system balanced by passive diffusion is commonly called a “pump and leak” system. How is the amount of stored NE maintained constant without it spilling over onto receptors? A common assumption holds that the synthesis of NE is controlled by some

____,. *....*...

RESERVE POOL (IN

GRANULES1

MOBILE POOL

Fig. 6. Labeled amines are taken up by tissue slices against a gradient and mix with only a portion of total amines. This suggests the existence of two pools: (1) Mobile pool, maintained against a concentration gradient by a specialized transport mechanism (solid arrow); (2) Reserve pool, complexed (ATP?) in granules.

sort of feedback mechanism which stimulates the rate of synthesis as amine stores are expended by nervous stimulation (Von Euler, 1959). But in biology, stored substrates generally undergo continuous synthesis and breakdown, whether the substrate is being utilized or not. Recent studies from our laboratory using [SHINE have established that N E in peripheral nerves is formed continually even in the absence of sympathetic tone (Montanari et al., 1963). As first reported by Axelrod and his group (Whitby et al., 1961) is taken up by various tissues. They conclude that the [SHINE is taken up by sympathetic nerve endings since the labeled amine does not accumulate in the tissues after sympathetic denervation (Hertting et al., 1961). Our results indicate that the [3H]NE is first taken up into a readily miscible pool, and then diffuses more slowly into a second pool (Fig. 7). The initial decline in radioactive [SHINE is relatively rapid but after a few hours the decline becomes exponential (Fig. 7). The exponential decline indicates that the [3H]NE is now uniformly distributed throughout all the stored NE; accordingly, the slope of the decline is a measure of the turnover rate of endogenous NE stores. The decline in radioactivity results from the continual production of new unlabeled NE and the simultaneous removal of NE, labeled and unlabeled. Since the amount of endogenous NE remains constant the rate of NE turnover is determined from the decline in the level of radioactive NE. is initially confined to the mobile pool The assumption is made that the Rqferences p . 184/185

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into which newly formed NE readily enters, and from which endogenous NE diffuses or is released by nerve impulses; it follows, therefore, that the disappearance of labeled NE will be most rapid when it is confined to this pool. Theoretically, the size of this pool should be inversely proportional to the rate of loss of radioactivity. Calculation shows that the rate of synthesis of heart NE is roughly 0.12 pg/h for the mouse, 0.17 pg/h for the guinea-pig, and 0.06 pg/h for the rat. When sympathetic tone is abolished by the administration of the ganglionic blocking agent, mecamylamine, the rate of NE synthesis is not appreciably changed (this laboratory, unpublished).

i-

-I

60,000

I

100

05 5

15

24

Af t e r

FH]NE

48

70

Fig. 7. Time course of [3H]NE content in heart after administration of 100 pC/kg of the labeled amine to rats.

Since NE is formed continually, a regulatory mechanism is needed to ensure that the excess NE diffusing from the neurochemical transducer does not spill over onto receptors. M A 0 has a key role in preventing the uncontrolled release of NE onto receptor sites (Brodie et al., 1959). This role can be explained on kinetic grounds. At the steady state level, the amine diffuses from the compartment at a rate proportional to the concentration gradient. M A 0 (in mitochondria) will inactivate the major part of the amine spilling out before it reaches the receptor site (Fig. 8). A low level of NE will facilitate outward diffusion and maintain the steady state level of the amine inside the compartment well below that which will saturate the transport system. The structure of the chemical transducer must also depict how nerve impulses cause the release of NE onto the receptor sites. On electrical stimulation of sympathetic nerves, containing labeled NE, the radioactivity in the blood coming from nerve endings consists mainly of NE and its normetanephrine product (Hertting et al., 1961a). The nerve impulse may be presumed, therefore, to counteract the pump directly in front of the receptor, thereby releasing some NE, directly or by enlarging pores onto the receptor site (Fig. 9).

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Fig. 8. Normally the concentration of NE stored in brain neurons is constant, but, after blockade or MAO, it increases rapidly. This supports that NE is continuously synthesized but amounts stored are limited by MAO. Physiologically, N E levels increase until leakage by passive diffusion (doticd arrow) onto M A 0 equals rate of formation.

Between successive nerve impulses, the action of the pump will be restored. Since the receptor site is extremely close to the storage membrane, the NE released onto the receptor will be pulled back into the storage compartment by the action of the pump. Studies suggest that even after a train of nerve impulses most of the NE released onto the receptor is returned to storage (Brown, 1960).

Fig. 9. Nerve impulses counteract the mechanism which prevents the extracellular diffusion of N E from the mobile pool, thereby they allow the NE to reach receptors.

Thus, the neurochemical transducer, as described in Fig. 9, provides a reasonable explanation of why the N E content in nerve endings remains constant, even after repeated sympathetic stimulation (Von Euler, 1959); the continual synthesis of NE together with its economical use at the receptor sites ensures that the depots are never depleted under ordinary circumstances. In this way, the small amount of N E stored in nerve endings seems to act as though it were an infinite reservoir. It is also possible that NE suppresses its rate of synthesis by a mass action effect. This factor would explain the rapid formation of heart N E after its level is louered by a short-acting depleter of the amine (Spector et a/.. 1962). Indeed, relatively low levels of dopamine are known to exert a n inhibitory effect o n DOPA decarboxylase (Yuwiler et al., 1960). The chemical transducer concept has been of value in explaining the action of drugs. According to the model shown in Fig. 9, drugs could act on the neurochemical transducer in at least 8 different ways (Table I): Rc9erenct-s p. 1X4/IXS

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TABLE I EFFECTS O F D R U G S O N T H E N O R E P I N E P H R I N E NEUROCHEMICAL TRANSDUCER

1 la 2 3 4 5 6 7

(NE)

Mimics action of NE at reactive sites Mimics action of NE by releasing some of the amine onto reactive sites Blocks action of NE at reactive sites Blocks metabolism of NE at storage compartments Impairs storage of NE by blocking an active transport Blocks synthesis of NE Blocks physiologic release of NF. Activates physiologic release of NE

( I ) Compounds like norsynephrine, structurally related to NE, appear to act by mimicking the action of NE at peripheral nerve endings. (2) Amphetamine and other lipid-soluble analogues of phenylethylamine act on the peripheral sympathetic system through the release of NE. Thus, they do not exert an action in animals depleted of NE stores (Burn et al., 1958). However, the action of these compounds is not completely clear since they act on central adrenergic centers presumably without releasing NE for they exert a central action in animals previously treated with reserpine (Everett et al., 1959). (3) Dibenamine and other adrenergic blocking agents prevent the action of NE by occupying peripheral receptor sites. Chlorpromazine is postulated to block the action of NE in the brain (Fig. 10) (Brodie et al., 1959; Killam, 1959).

IPHERAL

Fig. 10. Drugs can act by blocking the action of NE at receptors.

(4) After blockade of MAO, the content of brain NE is increased; as a result the amine diffuses from the storage compartment into surrounding spaces (Fig. 11). Since the brain extracellular space is small and the blood-brain barrier restrains the free amine from entering the blood stream NE spills over onto receptor sites. Present evidence indicates that the rise in free NE is responsible for the central stimulation after administration of M A 0 blockers (Spector et al., 1960; 1963). When reserpine is given to animals pretreated with a M A 0 inhibitor, the expected decline in brain amines does not occur; the blood-brain barrier prevents the liberated amines from readily diffusing into the blood stream. The conclusion that the inhibitors counteract the reserpine-induced release of NE is contradicted by the pronounced excitation observed in rabbits (Shore et al., 1957).

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177

Fig. 1 1 . M A 0 inhibitors can act by blocking the metabolism of NE a t storage compartments in brain. Concentrations of NE in neurons increase and free NE overflows onto receptors.

TY ROSlN E

1

1

DOPA

1

(ENZYME UNKNOWN)

L- AROMATIC AMINO

ACID DECARBOXYLASE DOPAMINE

0-OX I DASE

Fig. 12. A probable pathway for NE biosynthesis

( 5 ) The main steps postulated for the biosynthesis of NE are as follows (Fig. 12). The synthesis of N E might be prevented by blocking DOPA decarboxylase or by blocking dopamine-/l-oxidase. Attempts to block the synthesis of NE by means of decarboxylase inhibitors are complicated by the fact that DOPA decarboxylase and 5-HTP decarboxylase are the same enzyme (Lovenberg et a/., 1962). Consequently, a decarboxylase inhibitor that blocks the formation of 5HT will also block that of NE. Other difficulties result from the large excess of the decarboxylase enzyme in brain and from the competitive nature of the inhibitors. Two classes of compounds, examples of which are shown below, are particularly potent inhibitors of DOPA decarboxylase (Drain et a/., 1962; Brodie et a]., 1962). HO

NSD 1034 References p . 1841185

NSD 1055

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Administration to mice of NSD 1034 (100 mg/kg) inhibits the activity of brain DOPA decarboxylase by 100 %, as measured in vitro by assay of the enzyme in brain homogenates. However, the rates of endogenous dopamine or serotonin formation in living animals are hardly affected and the brain levels of these amines still rise after the blockade of M A 0 (Kuntzman et al., 1962a). This apparent paradox may be explained by the artificial conditions under which DOPA decarboxylase is measured BRAIN NE AND 5 H T LEVELS AFTER R D 4 1284(7.5mg/kgig 1 INNORMAL AND NSD 1045 (Ilmg/kg I v ITREATEDMICE

20

0

t

0-4NE

i,

I

I

I

0

1

2

3

4

5

0

1

2

3

4

100

40 20

I

0

5

H

Fig. 13. Brain 5HT and N E content of mice were depleted by the benzoquinolizine R o 4-1284 (7.5 mg/kg i.p.) given at 0 time. 1 I mg/kg (i.v.) of the dopamine-/?-oxidase inhibitor NSD 1045 [1(4Bromo-3-hydroxybenzyI)-I, methylhydrazinium dihydrogen phosphate] are given at each arrow. Note that in the treated group (broken line) the return of brain N E to control levels is slower than in the control group (solid line).

in vitro. In the living animal, the enzyme is localized i n the NE storage depots of nerve endings and is presumably greatly diluted when the tissue is homogenized. Thus, the inhibitor, acting competitively, might block the formation of dopamine from DOPA in the homogenate but not the formation of endogenous dopamine in the living organism. In searching for compounds that block the formation of NE it might be more practical to look for inhibitors of dopamine-p-oxidase especially since the hydroxylation of dopamine is a rate-limiting step in the synthesis of N E (Udenfriend et al., 1959). The two classes of compounds mentioned above are not only inhibitors of DOPA decarboxylase but are potent inhibitors of dopamine-P-oxidase, in fact, the most potent yet found (Kuntzman et al., 1962a; Creveling et al., 1962). In mice, they prevent the rapid rise in brain NE level induced by the injection of a potent M A 0 inhibitor (Kuntzman et al., 1962a). Moreover, after releasing brain NE and 5HT with a short-acting benzoquinolizine derivative, these inhibitors prevent the rise in brain NE, but not in 5HT (Fig. 13). At present we do not know the pharmacological consequences of blocking the

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179

formation of NE. The decline in NE might not be rapid, since it will depend on the utilization rate of the stored NE which, in turn, will depend on nervous activity. Unfortunately the inhibitors presently available are short-lasting and do not produce an appreciable exhaustion of endogenous NE stores (Kuntzman et al., 1962a). (6) Certain Rauwolfia alkaloids and synthetic benzoquinolizines inhibit the active pump which maintains NE in the storage compartment (Fig. 14). The loss of NE MA0

MA0

MA0

NORMAL Fig. 14. Reserpine impairs NE storage process and causes a depletion of NE stores. The amine diffuses out of the store and most of it will be degraded by MAO.

stores can be pictured as an uncompensated leaking of the amine onto MAO. Since the two pools are in equilibrium, the intragranular amines coming from the granules will also reach M A 0 by way of the mobile pool. After NE in the heart has been uniformly labeled with [3H]NE, the administration of reserpine releases both endogenous NE and [3H]NE. In our laboratory Nash et al. (unpublished) have shown that the labeled NE leaves the heart mainly as the acid metabolites of NE, indicating that reserpine releases most of the amine onto MAO. (7) Bretylium, a quaternary ammonium compound acts as a sympatholytic agent Br

by interfering with the nerve impulse-induced release of NE (Fig. 15) at peripheral sympathetic nerve endings (Boura et al., 1959). BW 392C60, a strongly basic guanidine

ACi ‘I’

O F - - N = C H

NHCH, ,f

( B W 392C60) “HCH,

derivative, whichalso has a side-chain nitrogen separated fromthering by a singlecarbon atom, acts like bretylium but is far more potent (Costa et al., 1962; Boura et al., 1961). ReJkrences p . 184/IRS

180

E. C O S T A A N D 8. B. B R O D I E

Fig. 15. Guanethidine causes a sustained activation of the process whereby nerve impulses release NE from nerve endings.

(8) Guanethidine, a guanidine derivative, is a sympathomiinetic drug of a peculiar type. We have postulated (Kuntzman et a/., 1962) that it acts oppositely to bretylium, depleting peripheral stores of NE by producing a persistent activation of the physiological release mechanism (Fig. 16). In accord with this view guanethidine produces

Fig. 16. Bretylium prevents the action of nerve impulses on the mechanism whereby NE is released

from nerve endings into receptor sites.

a sympathoinimetic effect lasting 60 to 90 min in the spinal cat (Fig. 17) and for a considerable time in the dog (Abboud et a/., 1961). Again, pretreatment with bretylium inhibits the release of N E by guanethidine but not by reserpine (Table 11). Of particular interest are studies showing that the drug releases NE directly onto the receptors since a considerable fraction of the labeled amine leaves the heart as NE and its o-methylated derivative (Nash et al., unpublished). BW 62-127, a drug in which a benzene

C O N C E P T O F THE N E U R O C H E M I C A L T R A N S D U C E R

181

ring is separated from nitrogen by two carbon atoms, also acts like guanethidine and completely depletes peripheral NE stores (this laboratory, unpublished observations). Thus, guanethidine and BW 62-127 release NE perhaps by activating the process of physiological release while reserpine inhibits the storage process.

30 min after

2 h after

Fig. 17. Effect of guanethidine (10 mg/kg i.v.) (G) and BW 392C60 (10 mg/kg i.v.) (BW) on the blood pressure of adrenalectomized, atropinized, spinal cats. Note that the injection of guanethidine (C) but not that of the bretylium analogue BW 392C60 (BW) causes a sustained increase of sympathetic tonus.

Others have postulated that guanethidine acts primarily by the same mechanism postulated for bretylium (Day et a]., 1962) and that the depletion of N E is a secondary effect (Cass et al., 1961). However, our studies show that a considerable number of compounds which possess a bretylium-like action also prevent the release of NE by guanethidine (Costa et al., 1962). If guanethidine also has a bretylium-like action, it is difficult to understand why it would not prevent its own release of NE! The TABLE 11 C O M P A R I S O N OF EFFECTS O F B R E T Y L I U M A N D RESERPINE O N T H E D E P L E T I O N OF H E A R T N E BY G U A N E T H I D I N E

Bretylium (60 mg/kg i.p.) was given 15 min before reserpine ( I mg/kg i.v.) or guanethidine (I0 mg/kg). The rats were killed 6 h after the last injection. Heart NE content of controls 1.10 pg/g & 0.15 S.D. _ _ _ _ ~

Depletion of heart N E Pretreatment with bretyliiim

Yes No

Cuanethidine

Reserpine

26 78

78 86

statement that guanethidine acts like bretylium is based largely on the finding that the sympathetic nervous system fails to respond to a stimulus shortly after the administration of guanethidine when the NE stores have not yet been depleted (Cass et al., 1961). But other factors must be considered. Immediately after the administration of guanethidine, the nerve ending cannot respond to a nerve stimulus since NE is already being released onto the receptor at a maximal rate. Guanethidine would References p . 1841185

182

E. C O S T A A N D B. E. B R O D I E

first release N E from the mobile pool after which the appearance of NE at receptors will be limited by the rate at which the amine leaves the granules. The pattern of NE release by guanethidine is in accord with this view. During the first hour the amine is released at a relatively rapid rate. Afterwards the release is slow and follows a zero order reaction as though the rate-limiting step is the rate at which the reserve pool is liberated (this laboratory, unpublished). T A B L E 111 EFFECTS O F V A R I O U S D R U G S O N D E P L E T I O N O F HEART

NE

B Y GUANETHIDINE

Potency relative to bretylium in blocking guanethidine

Drug ~~

1 .oo

Bretylium BW 392C60 Harmaline Iproniazid Pargyline Phenelzine Tranylcypromine

7.00

0.72 1.08 1.18 3.30

4.00

Compounds like amphetamine, a-methyl DOPA and metaraminol might release NE by similar processes. In this regard, it seems highly pertinent that small doses of methamphetamine prevent guanethidine from depleting heart NE (Matsumoto et al., 1963). Chang (unpublished), in our laboratory, has shown that amphetamine and ephedrine counteract guanethidine in a similar way. The concept of the neurochemical transducer has been used in studies of the mecha-

5 10 mgpg

50

)O

Fig. 18. Effects of various doses of BW 392C60 on guanethidine depletion of heart NE content and heart M A 0 activity. Broken line: inhibition of MAO; solid line: inhibition of NE release. BW392C60 was injected 2 h before guanethidine (10 mg/kg) and rats were killed 6 h after guanethidine.

CONCEPT OF THE NEURO CHEMICAL TRANSDUCER

183

nism by which M A 0 inhibitors lower blood pressure. A recent report from our laboratory (Gessa et al., 1963) has described how a number of M A 0 inhibitors prevent guanethidine from releasing peripheral NE (Table 111). Since bretylium also prevents the guanethidine-induced release of the amine, the possibility is being entertained that M A 0 inhibitors and bretylium might lower blood pressure by similar mechanisms. We first investigated whether the antagonistic effect of M A 0 inhibitors on guanethidine is related in some way to the blockade of MAO. Our results showed that the I

1

100 -

EQ-

.- 6 0 * .n .L

z-

40-

2QI

, , /

e----. 5

10 mg/kg

,I I

I

50

100

Fig. 19. Effects of various doses of iproniazid on guanethidine-depletion of heart N E content and on heart M A 0 activity. For other explanation see Fig. 18.

two effects are separable. The data in Fig. 18 show that BW 392C60, apotent bretyliumlike compound, has only a slight effect on M A 0 in doses that completely prevent the release of NE by guanethidine. Moreover, pargyline (MO 91 1) and iproniazid (Fig. 19) block the guanethidine effect almost completely in doses that have little effect on MAO. With many of the M A 0 inhibitors, however, the two actions are not easily separable. In preliminary results, the sympatholytic effects of two of the M A 0 inhibitors, phenelzine and pargyline, were compared with those of bretylium and BW 392C60. Electrical stimulation of the celiac ganglia fails to elicit a rise in blood pressure after pretreatment of adrenalectomized cats with bretylium on the one hand, and phenelzine or MO 911, on the other. Again, the administration of the ganglionic stimulant, DMPP, caused a lowering rather than a rise in blood pressure after pretreatment with BW 392C60, phenelzine or pargyline. These results suggest that -the M A 0 inhibitors exert some bretylium-like activity in preventing nerve impulses from releasing NE. The importance of this action can be determined because nerve impulses, produced by preganglionic stimulation of sympathetic nerves, actually reach the nerve ending. In conclusion, the structure of the biophysical unit which stores NE at nerve endings and releases the amine to sympathetic receptors is gradually unfolding. NE is formed continually inside a lipoid compartment regardless of sympathetic tone and is stored in two pools, a readily mobile pool and a reserve granular pool. The NE is maintained in the storage compartment by an energy-requiring active transport or “pump”. Ri>ferencesp . 1841185

184

E. COSTA A N D B. B. B R O D I E

Monoamine oxidase, outside of storage compartment, controls the level of NE so that it does not diffuse onto receptor sites. In this scheme the level of NE in the neurochemical transducer reaches a steady state when the rates of efflux and formation are equal. The amine can leave the storage compartment by discharge onto the receptor site or by diffusion through the membrane of the storage compartment. Nerve impulses presumably antagonize the pump vis-ri-vis the receptor for after stimulation of nerve, the amine appears in blood stream undeaminated. Present studies indicate that the serotoninergic and adrenergic neurochemical transducers function in a similar manner. SUMMARY

A provisional model of a neurochemical transducer has been formulated as an integrated biophysical unit which in nerve endings controls synthesis, storage, release, and metabolism of NE. It is stressed that synthesis, storage, release, and metabolism of NE are not unrelated phenomena and must be considered together in interpreting drug action. The action of a number of drugs that act on sympathetic tone is discussed in relation to the neurochemical transducer. REFERENCES ABBOUD,F. M., ECKSTEIN, J. W. AND PEREDA, S. A., (1961); Acute hemodynamic response to intravenous and intra-arterial guanethidine. Amer. J. Physiol., 201, 462466. BOURA, A. L. A., COPP,F. C., GREEN, A. F., HODSON,H. F., RUFFEL, G. K., SIM,M. F. AND WALTON E., (1961); Adrenergic neurone-blocking agent related to choline 2,6-xyliyl ether bromide (TM lo), bretylium and guanethidine. Nature (Lond.), 191, 1312-13 13. BOURA,A. L. A. AND GREEN,A. F., (1959); The actions of bretylium: adrenergic neurone blocking and other effects. Brit. J . Pharmacol., 14, 536-548. BRODIE,B. B., KUNTZMAN, R., HIRSCH,C. W., AND COSTA,E., (1962); Effects of decarboxylase inhibition on the biosynthesis of brain monoamines. Life Sciences, 81-84. BRODIE, B. B., SPECTOR, S., A N D SHORE, P. A., (1959); Interaction of drugs with norepinephrine in the brain. Pharmacol. Rev., 11, 548-564. BROWN,G. L., (1960); Release of sympathetic transmitter by nerve stimulation. Adrenergic Mechanisms. J. R. Vane, Editor. London, Churchill (p. 116). BURN,J. H., AN D RAND,M. J., (1958); The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. (Lond.), 144, 314-336. CARLSSDN, A., SHORE,P. A., AND BRODIE,B. B., (1957); Release of serotonin from blood platelets by reserpine in vitro. J . Pharmacol. exp. Ther., 120, 334-339. CASS,R., A N D SPRIGGS,T. L. B., (1961); Tissue amine levels and sympathetic blockade after guanethidine and bretylium. Brit. J. Pharmacol., 17, 442450. COSTA,E., KUNTZMAN, R., GESSA,G. L., A N D BRODIE,B. B., (1962); Structural requirements for bretylium and guanethidine-like activity in a series of guanidine derivatives. Life Sciences, 75-80. CREVELING, C. R., VANDER SCHOOT, J. B., AND UDENFRIEND, S., (1962); Phenylethylamine isosteres as inhibitors of dopamine-@-oxidase. Biochem. biophys. Res. Commun., 8, 21 5-219. DAY,M. D., AND RAND,M. J., (1962); Antagonism of guanethidine by dexamphetamine and other related sympathomimetic amines. J. Pharm. Pharmacol., 14, 541-549. DENGLER, H. J., MICHAELSON, I. A., SPIEGEL, H. E., AND TITUS,E., (1962); The uptake of labeled norepinephrine by isolated brain and other tissues of the cat. Int. J. Neuropharmacol., 1, 23-38. DENGLER, H. J., SPIEGEL, H. E., AND TITUS,E. O., (1961); Uptake of tritiumlabeled norepinephrine in brain and other tissues of cat in vitro. Science, 133, 1072-1073. DRAIN,D. J., HORLINGTON, M., LAZARE,R., AND POULTER, G. A., (1962); The effect of a-methyl DOPA and some other decarboxylase inhibitors on brain 5-hydroxytryptamine. Life Sciences, 93-97.

CONCEPT OF THE NEUROCHEMICAL TRANSDUCER

185

EVERETT, G . M., AND TOMAN, J. E. P., (1959); Mode of action of Rauwolfia alkaloids and motor activity. Biological Psychiatry. J . H. Masserman, Editor. New York, Grune and Stratton (p. 75). FUKS,Z . , AND SCHANKER, L., (1963); personal communication. CESSA, G . L., CUENCA, E., AND COSTA,E., (1963); On the mechanism of hypotensive effects of M A 0 inhibitors. Ann. N . Y . Acad. Sci., 107, 935-944. HERTTING, G., AND AXELROD, J., (1961a); Fate of tritiated noradrenaline at the sympathetic nerve endings. Nature (Lond.), 192, 172-173. HERTTING, G . , AXELROD, J., KOPIN,I. J., AND WHITBY,L. G., (1961); Lack of uptake of catecholamines after chronic denervation of sympathetic nerves. Nature (Lond.), 189, 66. HILLARP,N. A., (1960); Catecholamines: mechanisms of storage and release. I Intern. Congr. endocrinol. Abstracts of lectures. Symposium IX,lecture 6, 181-185. HUGHES,F. B., AND BRODIE,B. B., (1959); The mechanism of serotonin and catecholamine uptake by platc!ets. J . Pharmacol. exp. Ther., 127, 103-109. KILLAM,E. K., (1959); Psychopharmacology. National Academy of Sciences. National Research Council Publication (p. 21). KUNTZMAN, R., COSTA,E., CREVELING, C., HIRSCH,C. W., AND BRODIE,B. B., (1962a); Inhibition of norepinephrine synthesis in mouse brain by blockade of dopamine-P(-oxidase. Life Sciences, 85-92. KUNTZMAN, R., COSTA,E., GESSA,G. L., A N D BRODIE,B. B., (1962); Reserpine and guanethidine action on peripheral stores of catecholamines. Life Sciences, 65-74. LOVENBERG, W., WEISSBACH, H., AND UDENFRIEND, S., (1962); Aromatic L-amino acid decarboxylase. J . biol. Chem., 237, 89-93. MATSUMOTO, C., AND HORITA, A., (1963); Studies of the antagonism of guanethidine by methamphetamine. Biochem. Pharmacol., 12, 295-297. MONTANARI, R., BEAVEN, M., COSTA,E., AND BRODIE,B. B.,(1963); Turnover rates of norepinephrine in hearts of intact mice, rats and guinea-pigs using tritiated norepinephrine. Life Sciences, 232-240. SHORE,P. A., AND BRODIE,B. B., (1957); LSD-like effects elicited by reserpine in rabbits pretreated with iproniazid. Proc. Soc. exp. Biol. ( N . Y . ) , 91,433435. SPECTOR, S.,HIRSCH,C. W., AND BRODIE,B. B., (1963); Association of behavioral effects of pargyline, a non-hydrazide M A 0 inhibitor with increase in brain norepinephrine. Int. J. Neuropharmacol., 2, 81-93. SPECTOR,S., KUNTZMAN, R., SHORE,P. A., AND BRODIE,B. B., (1960); Evidence for release of brain amines by reserpine in presence of M A 0 inhibitors: Implication of M A 0 in norepinephrine metabolism in brain. J . Pharmacol. exp. Ther., 130, 256-264. SPECTOR, S., MELMON, K., AND SJOERDSMA, A., (1962); Evidence for rapid turnover of norepinephrine in rat heart and brain. Proc. Sac. exp. Biol. ( N . Y . ) , 111, 79-81. UDENFRIEND, S., AND CREVELING, C. R., (I 959); Localization of dopamine-p-oxidase in brain. J. Neurochem., 4,350-352. VON EULER,U.S., (1959); Autonomic neuroeffector transmission. Handbook ofPhysiology - Section 1 : Neurophysiology Vol. 1. J. Field, Editor. Waverly Press, Baltimore (p. 21 5). WEISSBACH, H., REDFIELD, B. G., AND TITUS,E., (1960); Effect of cardiac glycosides and inorganic ions on binding of serotonin by platelets. Nature (Lond.), 185,99. WHITBY,L. G., AXELROD, J., AND WEIL-MALHERBE, H., (1961); The fate of [3H] norepinephrine in animals. J. Pharmacol. exp. Ther., 132, 193-201. YUWILER, A., GETLER, E,, AND EIDUSON,S.,(1960); Studies on 5-hydroxytryptophan decarboxylase. 11. Additional inhibition studies and suggestions on the nature of the enzymic site. Arch. Biochem., 89, 143-147.

186

Cerebral and other Diseases with Disturbance of Amine Metabolism T H E O D O R E L. S O U R K E S Allan Memorial Institute of Psychiatry, McGill University, Montreal, Quebec (Canada)

INTRODUCTION

We now know of several disorders in which over- or under-production of specific amines plays an outstanding role. Excessive production leads to untoward displays of the biological activities of these amines whenever they are released locally or into the blood; a deficient production registers clinically as abnormal organ function. Two broad groups of such diseases are distinguishable : those with normal numbers of cells, and those with an altered amount of parenchymal tissue. States in which an excess or deficiency of amines occurs as a consequence of increased or decreased nervous activity, for example, fall into the first category. The further causes of such states must as yet be sought at the physiological level of investigation. Minor changes of this kind are, of course, constantly occurring under normal conditions, as homeostasis demands. The diseases with a change in the amount of functioning tissue lend themselves much more readily to histopathological and biochemical research. Some of the gross disorders encompassed by this definition are listed in Table I. They include pheochromocytoma, neuroblastoma, ganglioneuroma and Parkinsonism, which are disorders of catecholamine metabolism, and malignant carcinoid, in which accessory serotoninforming tissue is present. In urticaria pigmentosa and in the mast-cell tumors secn in some animal species, there is overabundant histamine and serotonin. Other diseases may be added to this roster. In the lipidoses, such as amaurotic family idiocy, Niemann-Pick’s disease and Gaucher’s disease, the offending compound is sphingosine, a long-chain dihydroxyaliphatic amine. It occurs in increased amounts in one organ or another, not in the free state but as a component of certain complex lipids - gangliosides, sphingomyelins and cerebrosides, respectively. Although the specific metabolic disorder has not been elucidated, familial dysautonomia (Riley-Day syndrome) may well fit in here. Perhaps there are clinical entities that correspond to excess or deficiency of tyramine, tryptamine, octopamine, y-aminobutyric acid or melatonin. The third column of Table 1 indicates the pharmacological means of reproducing some of the symptoms of the disease, at least the most immediately evident ones.

DISEASES W I T H D I S T U R B A N C E OF AMINE METABOLISM

187

Such comparisons of disease states with an experimental model may have more than heuristic value (Sourkes, 1961; Sourkes et al., 1962). TAB LE I Disease state

Amines concerned

Pharmacological model

Pheochroniocytoma

Adrenaline and noradrenaline Intermittent intravenous injection of adrenaline, noradrenaline or both Neuroblastoma, ganglioneuroma Dopamine Constant infusion of dopa or dopamine Parkinsonism Dopamine Reserpinization Malignant carcinoid Serotonin Sporadic intravenous injection of serotonin Urticaria pigmentosa (localized Histamine and serotonin Intracutaneous injection of histform, generalized rnastocytosis) amine; skin trauma; injection of 48/80; antigen-antibody re action Mast-cell tumors (in animals) Histamine and serotonin As above

PARKINSONISM

Biochemical characteristics Parkinsonism may be taken as the paradigm of cerebral diseases in the context of this Symposium, for it is characterized by the following features. ( I ) It involves some degree of brain pathology, evident in certain parts of the basal ganglia (Martin, 1959; Denny-Brown, 1960). (2) There is subnormal excretion of dopamine, especially in the post-encephalitic and arteriosclerotic types (Barbeau et al., 1961; Barbeau and Sourkes, 1962; Barbeau et al., 1962). (3) Very low concentrations of dopamine are found at post-mortem in the caudate nucleus, putamen and globus pallidus, particularly in the post-encephalitic form (Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1962). (4) There is a reduced ability to convert L-dopa to urinary dopamine and dopac (dihydroxyphenylacetic acid) (Barbeau et al., 1962). ( 5 ) L-Dopa, p-tyrosine and rn-tyrosine have an “anti-Parkinsonism” action in patients with this disease : a brief-lasting reduction in muscular rigidity and akinesia (Barbeau et al., 1962; Birkmayer and Hornykiewicz, 1962; Gerstenbrand and Pateisky, 1962). Some of the data on this subject are presented in Table 11. (6) On the other hand, certain drugs with pronounced effects upon the amines of the brain and other organs evoke, in a reversible manner, specific features of the Parkinsonian syndrome. Reserpine, which affects the storage of amines, and chlorpromazine, which appears to have a second-order effect upon these substances by influencing the membrane permeability of cells or storage granules (Gey and Pletscher, 1961; Guth, 1962), both induce a type of Parkinsonism. a-Methyldopa causes a reversible increase in tremor in Parkinsonian patients (sensitized individuals, as it were), although in normal subjects and in animals it does not have this effect. References p . 197-200

188

TH.

L.

SOURKES

T A B L E 11 EFFECTS OF AMINO ACIDS IN PARKINSONISM

Amino acid

L-Tyrosine, p.0. DL-mta-tyrosine, p.0. L-Dopa, p.0. L-Dopa, i.v. L-Dopa, p.0. i tranylcypromine, p.0. L-Dopa, i.v. t isocarboxazidg, p.0. isocarboxazid, p.0. L-Dopa, i.v.

+ L-Dopa, i.v. + pyridoxine

L-Dopa, p.0. -1 L-a-mcthyldopa, p.0. L-u-methyldopa, p.0. DL-mefa-0-methyldopa, i.v. D-Dopa, p.0. or i.v. ~~-5-Hydroxytryptophan, i.v.

Chief

efetts

References

Lessened rigidity Lessened rigidity Lessened rigidity Anti-akinesia Reduccd rigidity, transient rise in blood pressure, increased tremor Anti-akinesia, nausea, vomiting, collapse Anti-akinesia, reduced rigidity, reduced resting tremor Same as L-dopa alone Increased tremor Increased rigidity and tremor N o change N o change Sedative action in certain patients; checked oculogyric crises in some; no kinetic effect

* * *, * * **

*

** *** **

* *

** * ** **

*

Barbeau et al., 1962. Birkmayer and Hornykiewicz, 1962. Gerstenbrand and Pateisky, 1962. 6 L-Dopa, in combination with certain other inhibitors of monoamine oxidase were also tried, with similar results**.

** ***

(7) In animals with reserpine-induced Parkinsonism, L-dopa and rn-tyrosine, both amine-formers in vivo, have a temporary awakening effect (Carlsson et a/., 1957; Blaschko and Chrusciel, 1960). L-dopa does the same in man (Degkwitz et al., 1960). Some of the biochemical findings in Parkinsonism are in accord with the observation that dopamine is the chief cerebral catecholamine (Montagu, 1957; WeilMalherbe and Bone, 1957; Carlsson et al., 1958), and that it is highly localized in the hypothalamus and basal ganglia (Bertler and Rosengren, 1959; Sano et al., 1960). Other biochemical findings, such as the low urinary excretion of this compound, signify that it is specific for dopamine. Thus, Parkinsonians metabolize exogenous noradrenaline not differently from normal persons (Kirshner, 1961). There are conflicting reports in regard to the urinary excretion of 5-hydroxyindoleacetic acid (Barbeau and Jasmin, 1961 ; Vaisfeld, 1961 ; Resnick et al., 1962). It is unlikely that urinary dopamine reflects directly the levels in the brain. It is more probable that the excretion of this compound is controlled by the liver or kidney, or both, and that whatever the biochemical lesion is that results in a low output of dopamine, it may be a quite general one involving in common the viscera and the basal ganglia. There are, of course, outstanding precedents for such a phenomenon in the combined liver-brain dysfunction of hepatic coma, as well as in two diseases affecting the basal ganglia themselves: hepatolenticular degeneration (Wilson’s disease) and kernicterus.

DISEASES WITH D I S T U R B A N C E OF A M I N E M E T A B O L I S M

189

Parkinsonism and pharmacological models Although the administration of reserpine can reproduce some of the motor and biochemical features of the Parkinsonian syndrome, not all the drugs producing the one set of phenomena necessarily yield the other. Thus, the phenothiazines induce extrapyramidal motor effects, byt they do not cause a depletion of cerebral amines. On the other hand, alpha-methyldopa evokes a long-lasting decrease in the noradrenaline content of the brain (Murphy and Sourkes, 1959; Sourkes et al., 1961), but does not in any way cause the appearance of Parkinsonian signs in man (Sourkes et al., 1962). Degkwitz et a/. (1960) showed that intravenously administered dopa antagonizes some of the effects of reserpine in man, but not those of chlorpromazine. The ineffectiveness of dopa, even in heroic doses in the phenothiazine-treated subject, has since been confirmed (McGeer et al., 1961). It is not unreasonable to conclude that the depletion of certain amines of the basal ganglia, or perhaps the depletion of dopamine alone, reduces the available amounts of a transmitter amine specific to neurons of the basal ganglia. Then, how do the phenothiazines bring about extrapyramidal symptoms? If they are simply interfering with the release of dopamine from a storage granule, the injection of dopa should provide the physiological antagonist. But, as just mentioned, dopa is ineffective. Hence, it can be concluded that the phenothiazines affect transmission in the extrapyramidal system at an altogether different functional level, leaving the neurological substrate of the basal ganglia - dopaminergic fibres, perhaps - to carry on more or less normally. Possible causes of the disorder The investigation of many human diseases is facilitated if the diseased organ is easily accessible; the biochemistry of that organ can then be investigated before and during specific drug therapy or other treatment, enabling one to uncover the correlations between biochemical, physiological and clinical observations. If an analogous disease occurs in an animal species, the research worker is fortunate in having a model that can be intensively studied. However, models of these disorders are hard to find. There are choreas and post-encephalitic neurological syndromes in dogs and other species. Animals with specifically placed lesions in the brain provide another type of model. Parkinsonian symptoms may appear in carbon monoxide poisoning and in certain metal poisonings, such as manganism. Of course, there may be biochemical as well as clinical differences between the natural and experimental diseases, but this is a matter of research. In clinical Parkinsonism, according to Denny-Brown (I 960), the pathological changes are somewhat diffuse and vary as between the different forms of the disease. Perhaps a disorder like hemiballismus, with damage specifically limited to the corpus Luysii, has greater merits for biochemical examination than one with a diffuse histological picture. A biochemical frame of reference, based upon current knowledge of the intermediary metabolism of amines, can be used for the analysis of possible sources of the disease-process. This will permit us to assess our present biochemical knowledge of Referenres p . 197-200

190

TH. L. S O U R K E S

Parkinsonism, obtained for the most part at the periphery, against the various stages in the formation, storage and breakdown of cerebral amines. These steps are shown in Table 111, where they are expressed in a general form. TAB L E 111 Catalysts or other effecting agents

Reactions

Notes

~~

Amino acid

+ Amines

+ COZ

Atnines + Amine,

Amine, various metabolic products --f

Dopa decarboxylase + pyridoxal phosphate

Nervous stimulation; reserpine; a-methyldopa ; guanethidine ; 48/80 Monoamine oxidase; catechol-O-methyl transferase; gaba transaminase; etc.

Amines = a pharmacologically inactive, metabolically stable form of the amine, e.g. held in storage granules offering protection from inactivating enzymes Amine, = the pharmacologically active form of the amine. In this state it is susceptible to theaction of inactivating enzymes

( 1 ) Amino acidprecursors. The immediate precursor of dopamine is dopa, an amino acid derived from tyrosine. It is not known what the physiological pathway for the conversion of tyrosine to dopa is - whether enzymatic or not. It does not appear likely, however, that dopa itself is present in important concentrations in the cell fluids of the brain, and it is conceivable that this situation could occasionally create a difficulty in the maintenance of cerebral stores of amines. Indeed, Weil-Malherbe suggests that the synthesis of brain adrenaline and noradrenaline is limited by the supply of dopa (Weil-Malherbe, 1959). (2) Amino acid decarboxylases. Dopa decarboxylase catalyses the decarboxylation of dopa to dopamine. In ox brain, this reaction proceeds at similar speeds in several parts of the brain and spinal cord; in dog brain 5-hydroxytryptophan, another substrate for the enzyme, is decarboxylated somewhat more rapidly in the caudate nucleus, hypothalamus and mid-brain, than in other portions of the nervous system (for references, see Sourkes, 1962b). Some determinations of dopa decarboxylase have now been made in portions of human brain by Bernheimer and Hornykiewicz (1962). These investigators have been unable to detect a difference in enzymatic activity between the brains of Parkinsonian patients and of others. The matter is worth examining further in view of ( a ) the low activity in all the brains examined, and (b) evidence obtained in our laboratory of a defective conversion by Parkinsonian patients of orally administered L-dopa to urinary dopamine and dopacetic acid (Barbeau et al., 1962). As shown in Table 111, a defect in decarboxylation of the amino acid could arise from insufficient (or inhibited) apodecarboxylase or from insufficient coenzyme, i.e. pyridoxal phosphate. Experiments some years ago in my laboratory showed that one way to reduce the conversion

191

DISEASES W I T H D I S T U R B A N C E OF AMINE METABOLISM

of exogenous dopa to dopamine in vivo is to induce a deficiency of pyridoxine (Sourkes et al., 1960). Whether pyridoxine deficiency also affects the endogenous catecholamines in the mammalian organism is another question; even after our rats had been on the deficient diet for 12 weeks, we could not detect a significant decrease in the cerebral dopamine and noradrenaline. Nevertheless, the results in Parkinsonian patients show that exploration in novel ways of possible functions of pyridoxine in this disease should not be unduly delayed. Another brain decarboxylase acts upon glutamic acid. Glutamic decarboxylase is limited in its distribution essentially to the brain. In their examination of human brains obtained at post-mortem, Bernheimer and Hornykiewicz (1962) noted that the Parkinsonian tissue contained considerably less of this enzyme than the normal brain did. This may signify a role for glutamic decarboxylase in the maintenance of normal voluntary movement. ( 3 ) Storage granules. Adrenal medullary amines are held in a stable form in storage granules that can be separated from other cellular organelles by differential centrifugation in a density gradient. The physiological stimulus for the release of adrenaline is carried along the sphlanchnic nerve, and the final effect is mediated by the transmitter substance, acetylcholine; the use of agents or physical conditions known to alter membrane permeability results in the release of these “bound” amines in vitro. The granules are rich in ATP and lecithin. In other tissues, the catecholamines are stored in granules to a lesser extent than exemplified by the adrenal medulla, e.g. in the brain, spinal cord and neuroblastoma. Some of these tissues are listed in Table IV. TABLE IV BINDING OF CATECHOLAMlNES IN TISSUES

% of .41nine in granular fraction Organ

Brain Spinal cord Heart Adrenal medulla Pheochromocytoma Neuroblastoma

Species

Rabbit Rabbit Cat Dog

~

Dopamine

Noradrenaline

Adrenaline

52 25 53

63 60 38 67-85 75 39

57 47 74 78 42

-

ox

75

Man Man

-

8

References

2

3 4

5

8

7 8

~

3

5

7

Total catecholamines. Weil-Malherbe and Bone, 1957. Weil-Malherbe et al., 1961. McGeer and McGeer, 1962. Wegman and Kako, 1961. Blaschko et a/., 1955; Eade, 1958. Leduc and D’Ioro, 1960. Unpublished, author’s laboratory.

Nevertheless, our knowledge of granules in the adrenal medulla (Blaschko et al., 1956; Eade, 1958; Fortier et al., 1959) and in pheochromocytoma tumors (Leduc and D’Iorio, 1960) is probably applicable to the storage of amines elsewhere in the body. Refcrences p . 197-200

192

T H . L. S O U R K E S

For example, it has been possible to separate two granular fractions from the adrenal medulla, one rich in noradrenaline, the other in adrenaline. Bertler et a]. (1959) have described a special dopamine-containing cell in ruminants. Whittakcr (1 959) and Ryall (1962) have separated serotonin-containing granules of guinea-pig brain. The basophil granules of the mast cells are said to be the storage site for histamine and serotonin (Riley, 1961). Acetylcholine, choline acetylase, and serotonin are contained in the brain fraction labeled “synaptic vesicles” (Whittaker, 1959). Elliott and van Gelder speak of an “occluded” form of y-aminobutyric acid in brain (1 958). There is much evidence for the stoichiometric binding of amiiies by ATP in thc granules, but the lecithin that is present may play an auxiliary role. Indeed, Norlander showed some time ago (1950) that adrenaline and noradrenaline are soluble in ethereal solutions of lecithin. In some current work in my laboratory this has been confirmed. Tyramine, 0-methylnoradrenaline, 0-methyldopamine, serotonin, and tryptamine also partition from aqueous solutions (near neutrality) into a lecithin-ether phase. 5-Hydroxyindoleacetic, p-hydroxyphenylacetic and homovanillic acids are also soluble in ether, but their solubility is not enhanced by the dissolved lecithin. Results of this kind raise the possibility that acidic lipids will yet be found to play some role in amine physiology at the cellular and subcellular level. Woolley (1962) considcrs that the action of serotonin in stimulating smooth muscle to contract depends upon thc solubility of the amine in an acidic lipid of the muscle cell membrane whereby it can transport calcium ions into the cell. There are varied mechanisms of release of amines from their granular locations. Splanchnic stimulation has already been mentioned as the physiological mechanism for release of adrenal medullary adrenaline. Brodie and Shore (1957) have postulated that noradrenaline and serotonin are mediators of two families of neurons in the brain; these amines would be released through specifictypes ofcentral stimulation. Dopamine is probably liberated through the tonic activity of basal ganglia1 neurons, though this awaits neurophysiological study. In this connection, it is of interest that periods of sleep are associated with very low excretion of dopamine in the urine (Bischoff and Torres, 1962). The amines of the mast cells are released through immunochemical reactions, allergic reactions to food items, “drug allergy” reactions and skin trauma (“triple response”). Serotonin is liberated by distension of the gut (Bulbring and Lin, 1958). Reserpine is effective in releasing these amines to one or another extent, but its qualitative discrimination is poor. Tn order to achieve long-lasting depletion of a specific amine, it may be necessary to use other drugs, such as the a-methylamino acids, that permit differential rates of repletion of the lost amines. Of course, if each amine is held in a characteristic granule, we can expect in the future to have drugs that will affect the membrane of one type of granule but not another, so that only one amine will be specifically freed into the cytoplasm. It is conceivable that a disease-state may involve a n inability to form or to maintain a granular membrane. This would mean that amines can be formed but not stored. The Vienna group have shown that dopamine and noradrenaline (Ehringer and Hornykiewicz, 1960), as well as serotonin (Bernheimer et al., 1961), are all present in abnormally low concentrations in the basal ganglia and hypothalamus of Parkinsonian

193

DISEASES WITH D I S T U R B A N C E OF A M l N E METABOLISM

patients, but as yet it has not been determined whether the loss is from the cytoplasmic or from the granular fraction of amines. This is a most important point requiring clarification. In Parkinsonism, we may be dealing with a tissue that is incapable of storing the amines it produces for an appieciable length of time, either in the granular or the dissolved form, as appears to be the case in neuroblastoma (Sourkes et al., 1963). ( 4 ) Monoanline oxidme. Pursuing the subject of biochemical and pharmacological models of Parkinsonism: if the content of monoamine oxidase in the basal ganglia were elevated, one would have a ready explanation for the depressed concentrations of amines. Howevcr, the activity of this enzyme is of the same magnitude in the brains of Parkinsonians as of normals (Bernheimer and Hornykiewicz, 1962). It should be recalled that there are several reports in the literature, some on harmine (banisterine, Beringer, 1928, 1929), having appeared over 30 years ago, asserting that inhibitors of this enzyme have a beneficial effect in Parkinsonism. Some results with monoamine oxidase inhibitors that have been used in Parkinsonism are set out in Table V. It TABLE V M O N O A M I N E O X I D A S E I N H l B I T O R S IN T H E T R E A T M E N T O F P A R K I N S O N I S M

Drugs

Harmine Harmine Harmine Ro-3-1620 (a harmane derivative) Nialamide Nialamide lsocarboxazid 1 Tranylcypromine Tranylcypromine i trifluoperazine

Chief effects reported

Reduced rigidity and akinesia; control of oculogyric crises Increased voluntary movement Reduced rigidity and akinesia; tremor and salivation unaffected Anti-akinesia Anti-depressivc, increase in muscular strength, reduced tremor and paresthesias Anti-akinesia Anti-akincsia Tremor reduced at first, later the rigidity; little effect on akincsia and akathisia Rigidity reduced, but tremor is increased

References

3 4

5 6

n

Two other inhibitory hydrazine derivatives gave similar weak kinetic effects 5 . Beringer, 1929. Pineas, 1929; Rustige, 1929. Fischer, 1929; Schuster, 1929. Birkmayer and Hornykiewicz, 1962. Ramirez, 1960. Doshay, 1961. Barbeau and Duchastel, 1962.

appears that harmine, now known to be a reversible inhibitor of the enzyme, effects a reduction in muscular rigidity and improves movement and spontaneity. Involuntary movements ( e . g . oculogyric crises) are reduced, but tremor is not affected by harmine. A synthetic harmane derivative, Ro-3-1620, is reported to have a kinetic effect in Parkinsonism. Nialamide, used to treat the psychic depression of Parkinsonism, is Rrfrrenscs p . 197-200

194

TH. L. S O U R K E S

said to have additional beneficial effects, in the way of increased muscular strength and reduced tremor and paresthesia. Other hydrazine derivatives also influence the akinesia (Table V). Tranylcypromine is reported to be ofvalue in reducing the muscular rigidity, but not in increasing the voluntary movements of akinetic patients. For one reason or another, these results have not been consolidated into common therapeutic practice, but they are of great interest because of the well-known ability of inhibitors of monoamine oxidase to raise the cerebral content of biogenic amines. There is now direct evidence that this class of compound, administered therapeutically, actually inhibits the enzyme in human brain (Bernheimer et al., 1962; Ganrot et nl., 1962). What do these facts tell us of the state of the enzyme in the brain of the Parkinsonian? There may be a functional excess of monoamine oxidase, so that the concentration of dopamine (and other amines) is subnormal. When a n inhibitor of the enzyme is used, amines are able to increase in concentration and thus restoration of normal function is favored. This technique of bringing the processes of amine production and destruction into better functional agreement ought to be useful when there is an amine deficiency, but may have little bearing on drug-induced Parkinsonism. If reserpinization creates a biochemical model of clinical Parkinsonism, then clinical treatment with these inhibitors ought to be mimicked reasonably well by giving them to reserpinized animals. This is known to cause an increase in brain amines, along with increased motor activity (Udenfriend et a/., 1957; Spector et a/., 1958). However, there is a difficulty with this particular pharmacological model. The specific experimental conditions under which these drugs are given, simultaneously or successively, is critical, as shown in a study of cerebral catecholamines by Weil-Malherbe and his colleagues (1961). Reserpine ought to diminish the proportion of stored amine in the brain; inhibition of monoamine oxidase should then increase the cytoplasmic amines without increasing the granular fraction. These expected results were not seen ; the inhibition of monoamine oxidase actually interfered with the releasing action of reserpine. W I L S O N ’ SD I S E A S E

AND

H U N T I N G T O NC’ SH O R E A

In an earlier report from this laboratory dealing with three cases of hepatolenticular degeneration, it was shown that the dopamine and adrenaline output in the urine was frequently high (Barbeau et al., 1961). Since that time we have measured dopac and homovanillic acid in urines from one of those patients, with the results shown in Table V1. Another case of hepatolenticular degeneration (No. 4, Table VI) has now been studied. The dopamine excretion tended to be elevated above normal in three cases. In Case 2, where the dopamine values were in the normal range, the dopac and homovanillic acid were significantly elevated. The only other case where these compounds were determined (No. 4) showed normal values. Adrenaline output was very high in Case 2. In still another disorder of the basal ganglia, Huntington’s chorea, we have not detected any consistent abnormality in the following endogenous urinary constituents: dopamine, dopac, homovanillic acid, noradrenaline, adrenaline and

195

DISEASES W I T H D I S T U R B A N C E O F A M I N E METABOLISM

T A B L E VI U R I N A R Y AMINES A N D METABOLITES I N W I L S O N ’ S DISEASE*

Dopamine

Subjects

Normal persons

Dopac

Homovanillic acid Noradrenaline (m d (M)

(/4?)

(w)

316 115 (24)

2.99 5 0.33

8.23 1 0 . 7 0

(18)

(18)

9.49, 11.13 (2)

16.10, 20.00 (2)

2.46, I . I 3

5.04, 6.00

Adrenaline (Pg)

Patients :

410 5 42 (7) 335 5 35 (7) 603, 482, 796 (3) 361, 473

1

2 3 4

~

* Values shown were obtained by analysis of 24-h urines. Figures represent the mean f standard error, except where only a few urines were examined; in these cases the individual data are tabulated. The number of individual urines are shown in parentheses; in the normal series (Barbeau et al., 1961; unpublished data, this laboratory) each urine was obtained from a different individual.

TABLE VII URINARY AMINES A N D METABOLITES IN

Subjects

No. of Derns.

Dopamine (pg)

Dopac (mg)

Homovan- Noradre5-Hydroxyindoleillic acid naline Adrenaline acetic acid

18

Patients N.M. G.S. R.M. E.M. Mean

4 4 4 4 16

247 210 236 283 244

0.97 1.21 1.16 1.61 1.24

-

4 Unaffected adult relatives

10

I77

1.34

-

***

(fig)

(w)

18 Normals, ages 18-67

* **

H U N T I N G T O NC’HSO R E A *

236118** 2.22k0.19 6.42*2.28

4.30*** 6.34

(4

3112

1412

2.584918

88 42

21 9 11 18 15

1.30 2.46 3.54 5.72 3.25

11

3.61

31

39 50 34

Values shown are based upon excretion per gram of creatinine. Means 1standard error shown for normal series; means only, for patients and relatives. Three determinations.

5-hydroxyindoleacetic acid (TableVII). This conclusion is based upon repeated analyses for 4 patients, as well as others performed upon samples from their unaffected relatives. The normal excretion of homovanillic acid in 2 patients matches the finding of Williams et al. (1961). It is interesting that Ehringer and Hornykiewicz (1960) found normal values for cerebral dopamine in this disease. Thus, just as in Parkinsonism, References p. 197-200

196

TH. L. S O U R K E S

the ccrebral and peripheral results of dopamine analyses parallel one another i n Huntington’s chorea. PSYCHOSES AND NEUROSES

There is considerable evidence at present for the involvement of the sympathoadrenal system in the manifestations of psychoses and neuroses. The concept of specific metabolic defect has been invoked in some quarters to account for such disordcrs (references cited in Sourkes, 1962~).The adrcnochrome hypothesis is an example of this, but it has been found wanting on several grounds (Smythies, 1960; Sourkes, I962b). These diseases can be discussed profitably at the physiological level (see above). A very extensive study of the urinary excretion of catecholamines has been carried out by Bergsman (1959). His work, together with that of Strom-Olsen and Weil-Malherbe (1958) on manic-depressive psychosis, has revealed abnormal excretory patterns in manic and depressive states. During the manic phase of the manic-depressive psychosis, the excretion of adrenaline and noradrenaline is higher than during the depressive phase (Strom-Olsen and Weil-Malherbe, 1958). In a comparison of manic patients with those suffering from endogenous depression, the former excreted much more of these amines than did the depressives. The excretion of adrenaline was elevated in acute schizophrenic patients, but was normal in the chronic cascs. The rate of excretion of adrenaline was abnormally low in patients with senile dementia (Bergsman, 1959). In psychotic patients studied during alternate periods of drug therapy and placebo administration, Brune et al. (1962) found that the output of catecholamines incrcases when the subjects become more anxious; urinary indolic compounds (5-hydroxyindoleacetic acid, indoleacetic acid and tryptamine) were excreted in larger amounts just preceding and during increased manifestations of the psychosis. Studies in non-psychotic individuals have indicated that personality and behavioural types differ in their pattern of excretion of these amines but, again, there is no reason to postulate that a disturbance in amine metabolism is responsible for these differences. The same conclusion can be drawn from the many studies of the relation of catecholamine metabolism to emotional expression (Sourkes, 1962a, b). It appears from the studies in psychotic subjects that increased urinary output of catecholamines is associated with exaggerated motor activity and mental excitement. The exemplar is the manic patient. Perhaps such hyperactive states are analogous to excitatory actions of monoamine oxidase inhibitors. In fact, it is worth asking the question whether there may not be cerebral diseases with hyperkinesia at the basis of which lie major adaptive changes in the activity of monoamine oxidase in specific portions of the brain. SUMMARY

Of the various cerebral diseases in which some type of disturbance of amine metabolism has been reported, Parkinson’s disease can be taken as a paradigm for it

DISEASES W I T H D I S T U R B A N C E O F AMINE METABOLISM

197

involves ( I ) a central deficiency of dopamine and other amincs, (2) subnormal exaction of dopamine in the urine, and (3) degenerative changes in some of the basal ganglia. A convcrse picture is described for some cases of Wilson’s disease, based upon urinary measurements of catecholamines, dopacctic acid and homovanillic acid. It is not known whether in Parkinsonism the low concentration of dopaminc represents a deficiency of a metabolite, of the dopa-forming system, or of dopa decarboxylase (apoenzyme, pyridoxal phosphate); or whether there is an inability to store the amine as it is formed. In regard to Wilson’s disease, several biochemical defects have been described; an alteration in catecholaminc metabolism in some cases can be added to the list. In seeking models to help illuminate the problem of amine metabolism in the basal ganglia, pertinent information about changed relationships between substrates and enzymes, and bctwcen granule-located and cytoplasmic constituents in “natural” disease-states may be obtained from studies of accessory amine-producing tissues (pheochromocytoma, neuroblastoma, malignant carcinoid). Furthermore, knowlcdge of specific actions of drugs affecting the metabolism of amines makes possible the preparation of animal-experimental models for comparison with clinical states. The evidence for disturbed amine metabolism in Parkinsonism, hepatolenticular degeneration, Huntington’s chorea, schizophrenia, and manic-depressive psychosis has been reviewed and discussed in this light.

ACKNOWLEDGEMENTS

Research in the author’s laboratory on the subjects presented in this paper is currently supported by a Federal-Provincial Mental Health Research Grant, and a grant of the National Vitamin Foundation, Incorporated, New York, as well as by a grant to Dr. R. A. Cleghorn from the United States Public Health Service, National Institutes of Health, Number MY-3050-C3.

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I98

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BERNHEIMER, H., BIRKMAYER, W., AND HORNYKIEWICZ, O., (1961); Verteilung des 5-Hydroxytryptamins (Serotonin) im Gehirn des Menschen und sein Verhalten bei Patienten mit ParkinsonSyndrom. Klin. Wschr., 39, 1056-1059. BERNHEIMER, H., BIRKMAYER, W., AND HORNYKIEWICZ, O., (1962); Verhalten der Monoaminoxydase im Gehirn des Menschen nach Therapie mit Monoaminoxydase Hemmern. Wien. klin. Wschr., 74, 558-559. BERNHEIMER, H., AND HORNYKIEWICZ, O., (1962); Das Verhalten einiger Enzyme im Gehirn normaler und Parkinson-kranker Menschen. Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak, 243, 295. BERTLER, A., AND ROSENOREN, E., (1959); Occurrence and distribution of dopamine in brain and other tissues. Experientia (Basel), 15, 10-1 I . BERTLER, A., FALCK, B., HILLARP,N. A., ROSENGREN, E., A N D TORP,A., (1959); Dopamine and chromaffin cells. Acta physiol. scand., 47, 251-258. BIRKMAYER, W., AND HORNYKIEWICZ, O., (1962); Der L-Dioxyphenylalanin (= L-DOPA)-Effekt beim Parkinson-Syndrom des Menschen : Zur Pathogenese und Behandlung der Parkinson-Akinese. Arch. Psychiar. Nervenkr., 203, 560-574. BISCHOFF, F., AND TORRES,A., (1962); Determination of urine dopamine. Clin. Chem., 8, 370-377. BLASCHKO, H., AND CHRUSCIEL, T. L., (1960); The decarboxylation of amino acids related to tyrosine and their awakening action in reserpine-treated mice. J. Physiol. (Lond.), 151, 272-284. BLASCHKO, H., HAGEN,P., A N D WELCH,A. D., (1955); Observations on the intracellular granules of the adrenal medulla. J . Physiol. (Lond.), 129, 2749. BLASCHKO, H., BORN,G. V. R., D’IORIO,A., AND EADE,N. R., (1956); Observations on the distribution of catechol amines and adenosinetriphosphate in the bovine adrenal medulla. J. Physrol. (Lond.), 133, 548-557. BRODIE,B. B., AND SHORE,P. A., (1957); A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N . Y. Acad. Sci., 66, 631-642. BRUNE,G. G., PSCHEIDT, G. R., AND HIMWICH,H. E., (1962); Correlations between the behaviour of patients with mental disturbances and effects of psychoactive drugs on some urinary products. Proc. 3rd World Congr. Psychiar., Montreal, 1961. Toronto and Montreal, The University of Toronto Press and McGill University Press. Vol. 1 (p. I1 1-1 17). BULBRING, E., AND LIN, R. C. Y., (1958); The effect of intraluminal application of S-hydroxytryptamine and 5-hydroxytryptophan on peristalsis. J. Physiol. (Lond.), 140, 38 1-407. CARLSSON,A., LINDQVIST,M., AND MAGNUSSON, T., (1957); 3,4-Dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists, Nature (Lond.), 180, 1200. CARLSSON, A., LINDQVIST,M., MAONUSSON, T., AND WALDECK,B., (1958); On the presence of 3-hydroxytyramine in brain. Science, 127, 47 I . DEGKWITZ, R., FROWEIN, R., KULENKAMPFF, C., AND Mom, U., (1960); u b e r die Wirkungen des L-DOPA beim Menschen und deren Beeinflussung durch Reserpin, Chlorpromazin, lproniazid und Vitamin B6. Klin. Wschr., 38, 120-123. DENNY-BROWN, D., (1960); Diseases of the basal ganglia: their relation to disorders of movemcnt. Lancet, 2, 1099-1 105 and 1155-1 162. DOSHAY, L. J., (1961); Treatment of Parkinson’s disease. New Engl. J . Med., 261, 1097-1 102. EADE,N. R., (1958); The distribution of the catechol amines in homogenates of the bovine adrenal medulla. J . Physiol. (Lond.), 141, 183-192. EHRINGER, H., AND HORNYKIEWICZ, O., (1960); Verteilung von Noradrenalin und Dopamin (3Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr., 38, 1236-1239. ELLIOTT,K. A. C., AND VAN GELDER, N. M., (1958); Occlusion and metabolism of y-aminobutyric acid by brain tissue. J. Neurochem., 3, 28-40. FISCHER,A., (1929); Untersuchungen iiber die Wirkung des Alkaloides Harmin bei postenzephalitischen ZustHnden. Munch. med. Wschr.,76,451453. FORTIER, A., LEDUC,J., A N D D’IORIO,A,, (1959); Biochemical composition of the chromaffin granules of the medulla. Rev. canad. Biol., 18, 110-1 14. GANROT,P. O., ROSENGREN, E., AND GOTTFRIES, C. G., (1962); Effect of iproniazid on monoamines and monoamine oxidase in human brain. Experientia (Easel), 18, 260. GERSTENBRAND, F., AND PATEISKY, K., (1962); Ueber die Wirkung von L-DOPA auf die motorischen Storungen beim Parkinson-Syndrom. Wien. Z. Nervenheilk., 20, 90-100. GEY,K. F., AND PLETSCHER, A., (1961); Influence of chlorpromazine and chlorprothixene on the cerebral metabolism of 5-hydroxytryptamine, norepinephrine and dopamine. J. Pharmacol. exp. Ther., 133, 18-24.

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199

GUTH,P. S., (1962); Effect of phenothiazines on neurohumor-containing particles from mammalian brain. Fed. Proc., 21, 1100-1 102. HORNYKIEWICZ, O., (1962); Dopainin (3-Hydroxytyramin) im Zentralnervensystem und seine Beriehung zum Parkinson-Syndrom des Menschen. Dsch. med. Wschr., 87, 1807-1810. KIRSHNER, N., (1961); Discussion of papers. Rev. canad. Biol., 20, 205. LEDUC,J., AND D'IORIO,A., (1960); Etudes biochimiques de deux cas de pheochromocytome. Rev. canad. Biol., 19, 34-52. MARTIN,J. P., (1959); Remarks on the functions of the basal ganglia. Lancet, 1, 999-1005. MCGEER,E. G., AND MCGEER,P. L., (1962); Catecholamine content of spinal cord. Canad. J. Biochem., 40, 1141-1151. J. E., GIBSON,W. C., AND FOULKES, R.G . , (1961); Drug-induced extraMCGEER,P. L., BOULDING, pyramidal reactions: treatment with diphenylhydramine hydrochloride and dihydroxyphenylalanine. J . Amer. metl. Ass., 177, 665-670. MONTAGU, K. A., (1957); Catechol compounds in rat tissues and in brains of different animals. Nature (Lond.), 180, 244245. T. L., (1959); Effect of catecholamino acids on the catecholamine MURPHY,G. F., AND SOURKES, content of rat organs. Rev. canad. Biol., 18, 379-388. O., (1950); Ether-soluble compounds of adrenaline and noradrenaline with lecithin. NORLANDER, Acta physiol. scand., 21, 325-330. PIYEAS,H., (1929); Klinische Beobachtungen iiber die Wirkung von Harmin (Merck). Dtsch. med. Wschr., 1, 910. RAMIREZ, M. P., (1960); Accion de la nialamida en la enfermedada de Parkinson. Abstr. Panamer. Symposium on Inhibicion Enzimarica y su Aplicacion Terapeutica. Mexico, University of Guanajuato (p. 119-120). RESNICK,R. H., GRAY,S. J., KOCH,J . P., AND TIMBERLAKE, W. H., (1962); Serotonin metabolism in paralysis agitans. Proc. SOC.exp. Biol. ( N . Y.), 110, 77-79. RILEY,J. F., (1961); Tissue mast cells: distribution and significance. Canad. J. Biocliem., 39, 633-637. RUSTIGE,E., (1929); Versuche mit Harmin bei Metenzephalitiken. Dtsch. med. Wschr., 55, 613-614. RYALL,R. W., (1962); Sub-cellular distribution of pharmacologically active substances in guinea-pig brain. Nature (Lond.), 196, 680-681. SANO,I., TANIGUCHI, K., GAMO,T., TAKESADA, M., A N D KAKIMOTO, Y . , (1960); Die Katechinamine im Zentralnervensystem. Klin. Wschr., 38, 57-62. SCHUSTER,P., (1929); Ergebnisse von Banisterinversuchen an Nervenkranken. Med. Idin., 25, 562-563. SMYTHIES, J. R., (1960); Recent advances in the biochemistry of psychosis. Lancet, 1, 1287-1289. SOURKES, T. L., (1961); Formation of dopamine it7 vivo; relation to the function of the basal ganglia. Rev. canad. Biol., 20, 186-196. SOURKES, T. L., (1962a); Biochemical changes in the expression of emotion. Canad. psychiat. Ass. J., I, S29-S34. SOURKES, T. L., (1962b); Biochemistry of Mental Disease. New York, Hoeber (Harper and Row). T. L., (1962~);Biochemical abnormalities in mental diseases. Neurochemistry. K . A. C. SOURKES, Elliott, I. H. Page and J . H. Quastel, Editors. Springfield, lll., Charles C. Thomas (p. 990). T. L., DENTON,R. L., MURPHY, G. F., CHAVEZ, B., AND SAINTCYR, S., (1963); The exSOURXES, cretion of dopamine and dihydroxyphenylacetic acid in neuroblastoma. Pediatrics, 31, 660-668. T. L., MURPHY,G. F., AND CHAVEZ-LARA, B., (1962); Experimental and clinical studies of SOURKES, anti-decarboxylases. Proc. 3rd World Congr. Psychiat., Montreal, 1961. Toronto and Montreal, University of Toronto Press and McGill University Press. Vol. 1 (p. 649-653). SOURKES, T. L., MURPHY, G. F., AND WOODFORD, V. R., JR., (1960); Effects of deficiencies of pyridoxine, riboflavin and thiamine upon the catecholamine content of rat tissues. J . Nutr., 72, 145-152. M., (1961); The action of some aSOURKES, T. L., MuRpIiy, G. F., CHAVEZ,B., AND ZIELINSKA, methyl and other amino acids on cerebral catecholamines. J . Neurochem., 8, 109-115. S., PROCKOP, D., SHORE,P. A., A N D BRODIE,B. B., (1958); Effects of iproniazid on brain SPECTOR, levels of norepinephrine and 5-hydroxytryptaniine. Science, 127, 704. STROM-OLSEN, R., AND WEIL-MALHERBE, H., ( I 958); Humoral changes in manic-depressive psychosis with particular reference to the excretion of catechol amines in urine. J . ment. Sci., 102, 696-704. S., WEISSUACH, H., AND BOGDANSKI, D. F., (1957); Biochemical findings relating to the UDENFRIEND, action of serotonin. Ann. N . Y. Acad. Sci., 66, 602-608. VAISFELD, I. L., (1961); Urinary excretion of 5-hydroxyindoleacetic acid in certain diseases of the nervous system. Vop. med. Khim., 7, 309-312.

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WEGMANN,A., AND KAKO,K., (1961); Particle-bound and free catecholamines in dog hearts and the uptake of injected norepinephrine. Nature (Lond.), 192, 978. WEIL-MALHERBE, H., (1959); The effect of reserpine on the intracellular distribution of catecholamines in the brain stem of the rabbit. Biochemistry of the Cenfral Nervous System. F. Briicke, Editor. London, Pergamon (p. 190-195). WEIL-MALHERBE, N.,AND BONE,A. D., (1957); lntracellular distribution of catecholamines in the brain. Nature (Lond.), 180, 1050-1051. WEIL-MALHERBE, H., POSNER,H., AND BOWLES,G. R., (1961); Changes in the concentration and intracellular distribution of brain catecholamines. J . Pharmacol. exp. Ther., 132, 278-286. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine-containing particles from brain. Biochem. J., 72, 634-706. WILLIAMS, C. M., MAURY,S., AND KIBLER,R . F., (1961); Normal excretion of homovanillic acid in the urine of patients with Huntington’s chorea. J . Neurochem., 6, 254-256. WOOLLEY,D. W., (1962); The Biochemical Bases of Psychoses, or The Seroronin Hypothesis About A4ental Diseases. New York and London, Wiley.

20 1

An Effect of Aggregation upon the Metabolism of Dopamine- 1- H BRUCE L. WELCH

AND

A N N MARIE WELCH

Laboratory of Population Ecology, Department of Biology, College of William and Mary, Williamsburg and Departmetit of Pharmacology, Medical College of Virginia, Richmond, Va. ( U . S . A . )

Preliminary reports have been made at symposia (Welch, 1962a, b ) of experiments in which 2-3-month-old white Swiss mice (DUB/ICR) were penned in isolation or in groups of 2, 4 , 8, 16,20, or 32 for a period of 3 weeks, after which time their adrenals were removed, extracted in 5 % trichloroacetic acid, and analyzed fluorimetrically for adrenaline and noradrenaline by a modification of the method of Cohen and Goldenberg ( 1957). A higher percentage of the adrenal medullary catecholamines was present as adrenaline in mice from large groups than in isolates or in mice from relatively smaller groups. This difference was due to a progressively smaller absolute amount of noradrenaline in the adrenal medulla as group density increased. The difference was statistically significant at the 0.001 level of significance in one series of experiments involving 98 mice, and at the 0.01 level of significance in a second series involving 222 mice. There were changes in the adrenal cortex as well as in the medulla. In each experiment the cortex became larger as the number of grouped mice became greater, as indicated by significant increases in adrenal weight. In the second of these experiments the mice were color-coded in order that they might be easily identified, and the number of acts of aggression and the number of receipts of aggression were recorded for each mouse in timed periods daily for the 3-week period. The adrenals of the habitual aggressors were found to be smaller in weight (sig. = O.Ol), but to contain more adrenaline (sig. = 0.005) and more noradrenaline (sig. = 0.05) than the adrenals of the habitual receivers of aggression. The observed differences could not be attributed to differences in the availability of food or water, for these were maintained in superabundance and were equally available to all animals. Indeed, the floor of the pen was wastefully covered with food and the top was covered with water bottles such that an animal had only to lower his head in order to eat, or to raise it in order to drink. Mice in all groups, dominants and subordinants, gained weight during the experimental period. It should also be emphasized that the physiological differences observed between mice in different sized groups were not due to differences in frequency of involvement in fights or to injury from fights per se. Actually there were fewer fights per individual and no more fights per unit time in high-density aggregations than in lower density Rqferences p . 205j206

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ones. And, of course, dominant mice were involved injust as many fights as subordinate ones. These physiological differences were related to more subtle factors than fighting or shortage of food. The animals were apparently responding to differences in the level of stimulation which was characteristic of their environment. It is tempting to endeavor to explain the endocrine differences which exist between relatively crowded and relatively uncrowded mice, and between apparent dominants and subordinants, by assuming that the crowded and subordinate mice should, on the average, experience greater feelings of restriction, frustration of effort, anxiety and unvented emotion than their opposites, the relatively uncrowded and the dominant animals. Such a relationship between the observed endocrine differences and a presumed emotional tendency would agree well with the suggestion of many authors that the quantitative and proportional secretion of the adrenal medulla is correlated with certain emotional states (cf. Goodall, 1951; Ax, 1953; Funkenstein et al., 1957; Elmadjian, 1959; Mason et a/., 1961; Byers et a/., 1962; Cohen, 1962). Generally, emotions of out-directed anger and overt action, and personalities which are prone to such emotions have been associated with high secretions of noradrenaline relative to adrenaline ; conversely, relatively high secretion rates of adrenaline have been associated with emotions of in-directed anger, frustration and anxiety, and with personalities which are prone to these general types of emotions. It is well known that sympathetic secretions are mediated centrally. Notably, stimulation of Hess’ hypothalamic centers which induce ‘flight’ and ‘rage’ response patterns result in secretion of adrenaline and noradrenaline, respectively, from the adrenal medulla (Folkow and Von Euler, 1954). With these considerations, it is interesting to note the rather appreciable number of published papers which demonstrate, in sum, that central nervous excitatory drugs have a markedly greater effect upon grouped mice than isolated mice and that central nervous depressants have a markedly lesser effect in the grouped animals (Gunn and Curd, 1940; Chance, 1946, 1947; Tainter et a/., 1948; Brown, 1958, 1960; Burn and Hobbs, 1958; Lasagna and McCann, 1957; Gray et al., 1960; Piala ef a/., 1959; Hohn and Lasagna, 1960; Fink and Larson, 1962). For instance, Chance (1946) reported that drug dosages which were non-toxic to isolated mice were lethal to mice which were stimulated by the presence of others. He reported that when the area of cage per mouse was kept constant, the presence of nine other mice increased the toxicity of amphetamine tenfold and doubled that of adrenaline, while the increased toxicity of ephedrine and methedrine lay between those of these two substances. The sensitivity of these effects is emphasized by the fact that the median lethal dose of amphetamine for mice in groups of 3 was only one-eighth that for mice in isolation; further, the animals were in groups for only 30 min before injection, and those that died did so within 4 h after injection (Lasagna and McCann, 1957). This difference, then, reflected a very sensitive physiological response to the social environment. It was not due to ‘crowding’per se, but simply to the presence of other animals.

M E T A B O L I S M O F D O P A M I N E - ] -’H

203

With consideration for the possible mechanisms involved in these responses, it should be noted : ( I ) that some central nervous excitatory drugs employed were structural analogues of the catecholamines ; (2) that the administration of tranquilizers such as reserpine, which depletes stores of catecholamines and serotonin, and chlorpromazine which blocks the actions of these brain biogenic amines (Brodie and Costa, 1962) abolishes the difference in response of grouped and isolated mice to excitatory drugs, such that the median lethal dose for grouped mice is no greater than that for isolates (Lasagna and McCann, 1957; Burn and Hobbs, 1958; Piala et al., 1959; Hohn and Lasagna, 1960; Gray et a/., 1960); (3) that the potentiating effect of certain of these excitatory compounds is thought by some (as Von Euler, 1956) to be brought about by elevated levels of brain biogenic amines resulting from the action of monoamine oxidase inhibiting agents. TABLE 1 R E S I D U A L R A D I O A C T I V I T Y (counts/min/mg O F B R A I N T I S S U E ) I N B R A I N O F MICE A F T E R I N T R A M U S C U L A R D O P A M I N E - l - 3 H (12.3 p c , 2 . 2 mg/kg)

24 h (n = 20)

Isolated 7days (n = 20)

Crowded 7days (n = 20)

Replicates (4 pooled brains each)

11.9 17.3 23.3 20.3 18.3

5.8 8.1 8.2

14.5 11.0 14.7 12.8

Mean

19.40

7.31

13.25

sig.

= 0.005

Table 1 gives some preliminary results of work which is now in progress on differences in brain metabolism of radioactive precursors of the catecholamines as influenced by grouping and social interaction. Sixty male white Swiss mice (DUB/ICR), weight 25-31 g, age 2-3 months, which had been isolated for one week, were given intramuscular injections of 12.3 pC (2.2 mg/kg) dopamine-I-3H (New England Nuclear Corporation) in 0.1 ml of physiological saline. Although the ‘blood-brain barrier’ is relatively impermeable to the catecholamine(Udenfriend et a/., 1957; Vogt, 1959), small but significant amounts of even 3H-epinephrine may enter the brain locally, notably in the hypothalamus (Weil-Malherbe et al., 1959), and such indirect evidence for the entry of peripherally administered adrenaline into the brain as the occurrence of changes in cerebral electrical activity and affect state has been reported by a number of authors (Rothballer, 1959; Frankenhaeuser, 196I). Within a few minutes after injection most of the mice exhibited pronounced hypersensitivity and increased psychomotor activity so that it appeared that some of the dopamine may have entered the brain more or less directly. References p . 2051206

204

B. L. W E L C H A N D A. M. W E L C H

Twenty mice were sacrificed by decapitation 24 h after injection, and their brains removed and frozen on dry ice. The brains were pooled in groups of 4, homogenized in 0.4 N perchloric acid, adjusted to pH 6.1 with 0.5 N potassium carbonate, and centrifuged at 10,000 rev./min for 10 min to precipitate out the protein and potassium perchlorate. An aliquot of the extract was counted in a Packard Tri-Carb liquid scintillation counter, the remainder of each sample was chromatogramed on an Amberlite IRC-50 column at pH 6.1, and aliquots of the effluents and eluates were counted in the Tri-Carb. The 40 remaining mice were split into 2 groups, 20 mice to remain in isolation for 7 days, and 20 mice to be housed together in a single group for 7 days. At the end of this time all animals were sacrificed and treated as above. The brains of the mice which were killed 24 h after injection contained about 0.168% of the total 12.3 pC of radioactivity originally administered (9070 c.p.m. of 5.4 * 106 c.p.m., at 20% counting efficiency)*. After 7 days the brains of the crowded mice contained an average of 6776 c.p.m., while the brains of the isolated mice contained 3786 c.p.m., only about half as much. Table I, which gives these figures in counts per min per mg of brain tissue, indicates that the difference in the radioactivity retained by the brains of the crowded mice (1 3.25 c.p.m./mg) and that retained by the brains of the isolated mice (7.37 c.p.m./mg) is significant at the 0.005 level of significance. Further, there was a difference in the percentage distribution of the radioactivity between the eluates and the effluents of the chromatogramed extracts of the brains from the isolated and crowded mice. About 80% of the total radioactivity in the brains from the crowded animals, but only about 65% of that in the brains of the isolates, appeared in the column effluent. However, the absolute amount of radioactivity in the eluates was virtually the same for the 2 experimental conditions. All of the observed difference in retained radioactivity was in the effluent. It was not, therefore, in catecholamines. Interestingly enough, within 24 h of injection over 99 % of the brain radioactivity had been in the column effluent. An interesting effect of aggregation upon metabolism has been observed. It has posed a number of questions which are quite fundamental in nature. Experiments are underway or are planned to more closely define the sensitivity and the temporal aspects of the response to grouping, to study the manner in which it is affected by certain drugs, to localize the radioactivity in specific areas of the brain, to identify the compounds in which the radioactivity is contained, and to elucidate the primary enzymatic mechanisms which are involved. S U M MARY

At 24 h after administration of d ~ p a m i n e - l - ~toH mice, radioactivity was accumulated in the brains in concentrations 60-68 times greater than could be accounted for by

* In mice which were similarly treated and sacrificed 7 h and 27 h after injection, the brains contained an average of 60 and 68 times the amount of radioactivity, respectively, that could be accounted for by radioactivity in the blood alone.

METABOLISM O F D O P A M I N E - 1 - 3 H

205

the radioactivity in the blood alone. At this time the mice were placed in treatment conditions of individual housing and group housing. The radioactivity was depleted from the brains at different rates in the two treatment conditions. After 7 days the amount of radioactivity remaining in the brains of the grouped mice was twice as great as that remaining in the brains of the mice which were individually housed. Whereas virtually all of the radioactivity in the 24-h brain extract failed to be retained on a chromatographic column appropriate for retaining the catecholamines, only 80 % of the radioactivity from the brains of the grouped mice and 65 % of the radioactivity from the brains of the individually-housed mice passed through a similar column without retention after 7 days.

A C K N O W L E D G E MENTS

Dr. Norman Kirshner, Departments of Biochemistry and Experimental Surgery, Duke University Medical Center, Durham, North Carolina, generously provided facilities and counsel during the conducts of these experiments. Supported by U.S. Public Health Service Grants. REFERENCES Ax, A., (1953); The physiological differentiation between fear and anger in humans. Psychosom. Med., 15, 433-442. BRODIE,B. B., AND COSTA,E., (1962); Some current views on brain monoamines. Psychopharmacol. Serv. Cent. Bull., 2, 1-24. BROWN,B., (1958); Influence of inter-animal and environmental stimulation on action of central nervous system drugs. Proc. Western Pharmacol. Soc., San Francisco, January 27-28, 1958 (p. 9-1 I). BROWN,B. B., (1960); CNS drug actions and interaction in mice. Arch. int. Pharmacodyn., 128, 391414. BURN,J. H., AND HOBBS,R., (1958); A test for tranquilizing drugs. Arch. int. Psychodyn., 113,290-295. M., ROSENMAN, R. H., AND FREED,S. C., (1962); Excretion of 3-methoxy-4BYERS,S. D., FRIEDMAN, hydroxymandelic acid in men with high incidence of coronary artery disease. Fed. Proc., 21, Part 11, 99-191. CHANCE,M. R. A., (1946); Aggregation as a factor influencing the toxicity of sympathomimetic amines. J . Pharmacol. exp. Ther., 87, 214-219. CHANCE,M. R. A., (1947); Factors influencing the toxicity of sympathomimetic amines t o solitary mice. J . Pharmacol. exp. Ther., 89, 289-296. COHEN,G., AND GOLDENBERG, M., (1957); The simultaneous fluorimetric determination of adrenaline and noradrenaline in plasma. I. J . Neurochem., 2, 58. COHEN,S. I., (1962); The effects of symbolic stimuli on the cardiovascular system. Symposium on the Rate and Rhythm of the Heart. Annual Meeting of the American Psychosomatic Society, April, 1962. Unpublished. ELMADJIAN, F., (1959) ; Excretion and metabolism of epinephrine. Symposium on Catecholamines. National Institute of Health, Bethesda, Maryland, October 16-18, 1958. Baltimore, Williams and Wilkins Company (p. 409). FINK,G . B., AND LARSON,R. E., (1962); Some determinants of amphetamine toxicity in aggregrated mice. J . Pharmacol. exp. Ther., 137, 361-364. FOLKOW, B., AND VON EULER,US., (1954); Selective activation of noradrenaline and adrenaline producingcells in the cat’s adrenal gland by hypothalamic stimulation. Circulat. Res., 2, 191-195. FRANKENHAEUSER, M., (1961); Psychophysiological Effects of Catecholamine Infusions. Report from the Psychological Laboratory, The University of Stockholm, Number 99, May, 1961.

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FUNKENSTEIN, D. H., KING,S. H., AND CROLETTE, M. E., (1957); Mastery of Stress. Boston, Harvard University Press. GOODALL, McC., (1951); Studies of adrenaline and noradrenaline in mammalian heart and suprarenals. Actaphysiol. scand., 24, Suppl. 8 5 . GRAY,W. D., OSTERBERG, A. C., RAUGH,C. E., AND HILL,R. T., (1960); The behavioral and other pharmacodynamic actions of methoxypromazine (Tentone (R)), a tranquilizing agent. Arch. int. Psychodyn., 125, 101-120. GUNN,J . A., AND CURD,M. R., (1940); The action of some amines related to adrenaline, cyclohexylalkylamines. J . Physiol. (Lond.), 97, 453470. HOHN,R., AND LASAGNA, L., (1960); Effects of aggregation and temperature on amphetamine toxicity in mice. Psychopharmacologia ( B e d . ) , 1, 210-220. LASAGNA, L., AND MCCANN,W., (1957); Effect of tranquilizing drugs on amphetamine toxicity in aggregated mice. Science, 125, 1241-1242. G., JR., BRADY, J. V., CONRAD, D., AND RIOCH,D., (1961); Concurrent plasma MASON,J., MANGAN, epinephrine, norepinephrine and 17-hydroxysteroid levels during conditioned emotional disturbance in monkeys. Psychosom. Med,, 23, 344-353. PIALA,J. J., HIGH,J. T., HASSERT, G. L., JR., BURKE, J. C., A N D CRAVER, B. N., (1959); Pharmacological and acute toxicological comparisons of triflupromazine and chlorpromazine. J . Pharmacol. exp. Ther., 127, 55-65. ROTHBALLER, A. B., (1959); The effects of catecholamines on the central nervous system. S.vmposium on Catecholamines. National Institute of Health, Bethesda, Maryland, October 16-1 8, 1958. Baltimore, Williams and Wilkins Company (p. 494). TAINTER, M. L., TULLER, B. F., AND LUDUENA, F. P., (1948); Levoarterenol. Science, 107, 39-40. UDENFRIEND, S., WEISSBACH, H., AND BOGDANSKI, D. F., (1957); Biochemical findings relating to the action of serotonin. Ann. N . Y. Acad. Sci., 66, 602-608. VOGT,M., (1959); Catecholamines in brain. Symposium on CatecholamineJ. National Institute of Health, Bethesda, Maryland, October 16-1 8, 1958. Baltimore, Williams and Wilkins Company (p. 487). VONEULER,U. S . , (1956); Noradrenuline. Springfield, C. C. Thomas. WEIL-MALHERBE, H., AXELROD, J., AND TOMCHICK, R., (1959); Blood-brain barrier for adrenaline. Science, 129, 1226. WELCH,B. L., (1962a); Discussion of a paper by J. Lloyd. Proceedings of the First International Wildlife Disease Symposium, New York, June 23-27, 1962. Wildlife Dis. Ass. Publ. Chicago. WELCH,B. L., (1962b); Catecholamine content of the adrenal medulla of mice at different densities of grouping. Symposium on Population Physiology. Annual Meeting cf the A n-.erican Association for the Advancement of Science, Philadelphia, Cecember 27-31, 1962. BUN.Ecol. SOC.AKer., 43, 135 (abstract).

207

Effects of Marplan on Catecholamine and Serotonin Metabolism in the Human R . R . SCHOPBACH, A. R . KELLY

AND

J . S . LUKASZEWSKI

Henry Ford Hospital, Detroit 2, Mich. ( U . S . A . )

Despite the widespread use of monoamine oxidase inhibitors and many studies of their actions, the authors are not aware of any report of the comparative effects of their continued administration upon the metabolism of both catecholamines (CA) and serotonin (5-HT) in the human. This study attempts to correlate simultaneous measurements of the parent amines as well as their respective intermediates and metabolites, 3-methoxy-4-hydroxymandelic acid (MOMA), the methoxycatecholamines, metanephrine and normetanephrine (MOCA), and 5-hydroxyindoleacetic acid (5-HIAA). After establishing control levels for these substances, 40 mg Marplan (1 -benzyl-2-[methyl-3-isoxazoylcarbonyl] hydrazine) was ingested each morning for 42 days. The level of 5-HT in the blood and the levels of 5-HTAA, CA, MOMA, and MOCA in the urine were determined during this period and for 92 days subsequently. Indirect evidence of alterations in the central nervous system was obtained by reaction time and critical flicker fusion (CFF) measurements. Within the first few hours after ingesting the Marplan the 5-HIAA excretion was

I: :

300 2 50..

Days on Marplan

i \

Days after discontinuing Marplan

Fig. 1 . Changes in urinary excretion of CA (catecholamines), MOMA (3-methoxy-4-hydroxymandelic acid), and MOCA (metanephrine and normetanephrine) during and subsequent to ingesting 40 mg Marplan daily. References p

210

R. R. S C H O P B A C H et al.

208

decreased, a subjective impairment of sustained attentiveness was noted, CFF was impaired, and disjunctive auditory reaction time improved. This latter may be due to the decreased awareness of extraneous stimuli and the increased ability to attend to pertinent stimuli. The first 24-h urine showed a 40% decrease in 5-HIAA and a 54% increase in MOCA. All of these indicate the very rapid effects that this compound has upon amine metabolism. CA excretion (Fig. 1) began to increase on the 2nd day. This increase continued for 9 days, after which the excretion declined, gradually reaching control levels by the 16th day and stabilizing approximately 35 % below control level from the 20th day until medication was discontinued. The MOCA excretion increased from the first few hours to reach a three-fold level on the 23rd day. The MOMA excretion did not alter until the 6th day, after which it precipitously decreased to about 40% of thecontrollevel. Since we did not quantitate the amounts of 3,4-dihydroxymandelic acid (Fig. 2) or the 3-methoxy-4-hydroxyphenylglycolsulfate, we can not say to what extent each of the pathways to MOMA were affected. Certainly, with greatly increased amounts of MOCA present, there would be a greater possibility of overcoming any block and

"3cO-o" ""0-1 011

0 If

tl-yH

CH-COOH

IIO C -0-M - T 3 , 4 dihydroxymandelic

HO-

3 - methoxy-4-hydroxy-

acid IMAO

H

O

HO

-

\

-

0

\

mandelic acid ( V M A )

4

mo

''-

- C H - C H -NH C - 0 -2M - T 2

Norepinephrine

\ \ \ \

I

3-methoxy-4-hydroxyphenylglycolaldeyhyde

+HZ 1

I

'

I

I

I

OH

V

I

-CH-CH~-NHZ

C-0-M-T

Conjugate

OH H

HO -

Normetanephrine (3-0-methylnorepinephrine)

I

\

H3c0

-8

+ xo-

*

OH

I

-CH-CH2-NH2

THTH

H

/

Conjugate

3 -methoxy-rl-hydroxyphenylglycol sulfate

Fig. 2. Metabolism of norepinephrine.

of forcing the reaction to proceed toward MOMA. Likewise aldehyde dehydrogenases might convert increasing amounts of 3-methoxy-4-hydroxyphenylglycolaldehydeinto MOMA if some of the increased pool of MOCA escaped the block. Weissbach et al. (1961) hasalso describedanother amine oxidase in mice which is not inhibited by M A 0 inhibitors. Such pathways, if also present in man, might become more important when the usual pathways were blocked.

EFFECTS OF M A R P L A N O N

CA

AND

5-HT

209

The sum total of the excreted CA and their measured metabolites increased for the first 6 days, then gradually declined for the remainder of the Marplan period. By the 10th day this total had returned to control levels although the proportion of MOMA : MOCA was quite different. These decreases indicate that the COMT and possibly other metabolic pathways became so increasingly active that they were able to maintain a normal or even subnormal CA level.

I -

lool

I

/ lb

2b

3b

D a y s on Marplan

i

42

l

13

l

20

,

30

,

,

40 50

$0

7'0

ob

912

Days a f t e r discontinuing Marplan

Fig. 3. Changes in blood serotonin and urinary excretion of 5-HIAA during and subsequent to ingesting 40 mg Marplan daily.

The 5-HT blood level (Fig. 3) decreased very slightly after Marplan ingestion. This initial decrease was well within normal variation, but may be meaningful, since decreases were also seen in 5-HIAA and reaction times and since there were subjective symptoms during this period. Subsequently the 5-HT concentration rose rapidly for 3 weeks befora leveling off at about 150 % above control values where it remained as long as the drug was continued. After discontinuing the drug, the blood level did not decrease immediately, probably due to continued effective drug levels. However, after the 3rd day, the level fell rapidly for 3 weeks reaching 0.3 pg/ml or 50 % above control levels where it remained for at least 10 months. A check 2 years later indicated that it had returned to normal during that interval. The 5-HIAA excretion promptly declined but, after the 16th day, increased gradually toward the control level. Surprisingly, after the discontinuance of the drug, the level, instead of rising, fell and continued at a lower rate than the control level for the duration of the study. Again, however, studies done 2 years later indicated that at some time during that interval it had returned to the control level. The prolonged effects suggest that these enzyme systems are quite delicate. These may help explain why Dr. Himwich's (personal communication) reported effects of M A 0 inhibitors upon Parkinsonism persist after the discontinuance of the medication. Subjective insomnia was most prominent on nights 1-10 at which time the CA and total catecholamine excretions were elevated. From the 20th day until 7 days after discontinuing the drug, initiation of micturition, defecation, and ejaculation (but not erection) were severly impaired. These autonomic symptoms correspond with markedly elevated 5-HT combined with decreased CA and decreased total CA metabolite Refcrenccs p . 210

210

R. R. S C H O P B A C H et al.

excretions. If we make the tenuous assumption that these urinary excretion rates mirror the blood levels or even tissue levels, we could suggest that the 5-HT had risen to such a level as to “drown” the parasympathetic system either centrally or peripherally, leaving the sympathetic system to act unopposed.

SUMMARY

The effects of daily ingestion of Marplan upon CA, 5-HT, and their metabolites are reported. These indicate a very rapid action, a secondary decrease in CA excretion, and extremely prolonged effects after discontinuing the drug. Some possible explanations are discussed. ACKNOWLEDGEMENT

Hoffman-La Roche Inc. generously furnished the Marplan (I -benzyl-2-[methyl-3isoxazoylcarbonyl]hydrazine)and grants for this study. REFERENCES

HIMWICH, H. E., Personal communication. WEISSBACH, H., (1961); In vivo metabolism of serotonin and tryptamine; effect of M A 0 inhibition. J . Pharmacol. exp. Ther., 131, 26.

21 1

Effects of Isocarboxazid on Spontaneous and Drug-Induced Ex t rapyramidal A1tera tions CHRISTOPHER B U L L

AND

HANS H . BERLET

Thuclichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, IN. (U.S.A.)

Post-mortem studies of thc brains of patients with parkinsonism have revealed the depletion of the dopamine content of the basal ganglia including the caudate nucleus, putamen, and globus pallidus (Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1962). Noradrenaline (Ehringer and Hornykiewicz, 1960) as well as serotonin (Bernheimer et al., 1961) are also found in abnormally low levels in these patients. A depletion of brain biogenic amines also occurs with reserpine and it is well-known that parkinsonian signs are evoked in patients receiving this drug. The mechanism of action of the phenothiazines is different from that of reserpine for the administration of phenothiazine does not alter the amine content of the brain but instead blocks their actions. The end results, however, are the same in regard to the production of extrapyramidal alterations. The purpose of the present investigation is to determine actions of MA01 on parkinsonian signs whether occurring spontaneously in the course of the disease or evoked either by reserpine or trifluoperazine (Stelazine). It is conceivable that MA01 drugs which raise the levels of biogenic amines may affect favorably the extrapyramidal changes. A reversible MA01 drug, harmine, has been previously reported to have beneficial effects on parkinsonian changes (Beringer, 1929). We proposed to pursue this idea further with an irreversible isocarboxazid (Marplan). Although this investigation is not completed the time seems appropriate to present some of our findings, since we have just heard a discussion of some relationships between brain amine levels and disorders of the extrapyramidal system (Sourkes, this Symposium). METHOD

In our study of schizophrenic patients, we produced extrapyramidal symptoms, first with trifluoperazine in doses ranging from 5 to 100 mg daily and later with reserpine in doses ranging from 4 to 10 mg daily. After clinically rating the intensity of the extrapyramidal symptoms (tremor, muscular rigidity, cogwheeling, and changes in gait and handwriting) on a basis of 1 to 4 plus, and waiting until we had a stable baseline of these ratings, we added the isocarboxazid in doses ranging from 30 to References p . 214

212

C. B U L L A N D H . H . B E R L E T

40 mg daily, and noted any changes in intensity that occurred during the ensuing weeks. RESULTS

A group of 4 patients with organic, that is non-drug-induced extrapyramidal symptoms of arteriosclerotic or senile origin, was trcated with isocarboxazid in 2 separate courses to determine the effects on their symptoms. Table I represents the changes TABLE I EFFECT OF I S O C A R B O X A Z I D O N E X T R A P Y R A M I D A L S I G N S

Organic (4 patients)

-

Effect on spontancous alterations

Total no. of’ plumes*

Baseline

Placebo Isocarboxazid Placebo lsocarboxazid Placebo

Difference after Percent change in isocarboxazid symptoms

12 1.5 I1

-4.5 -

-38

5.5

-5.5

-50

7.5

-

-

* Rating intensity of presenting symptoms of tremor only in 3 patients and choreoathetoid movements only in 1 patient. i n intensity of the presenting complaints of tremor (3 patients) or choreoathetoid movements (one patient) in the organic group when isocarboxazid was given. Improvements of 38 and 50 % were found in the total of the symptoms of 4 patients. Of a total of 21 schizophrenics started on the drug-induced extrapyramidal altcrations 1.5 completed the study of trifluoperazine-induced symptoms and 7 of these T A B L E 11 E F F E C T OF I S O C A R B O X A Z I D O N E X T R A P Y R A M I D A L S I G N S

Drug-induced

-

Effect on trifluoperazine-induced and reserpine-induced changes (7 patients)

Ba reline

Placebo Trifluoperazine Trifluoperazine and isocarboxazid Placebo Reserpine Reserpine and isocarboxazid

Total no. of plusses *

Difference after Percent change in isocarboxazid symptoms

I 80 84 18.5 69 57

-I2

-I7

* Rating intensity of rigidity, cogwheeling, tremor, and changes in gait or handwriting, when symptoms occurred.

213

EFFECTS O F ISOCARBOXAZID

went on to finish the study of reserpine-induced extrapyramidal symptoms, the others having been dropped from the group in most instances because of too great a drop in blood pressure. Table I1 represents the response of drug-induced symptoms in the group of 7 patients who completed two phases in which both trifluoperazine-induced symptoms and reserpine-induced symptoms were treated with isocarboxazid. The increase of 5 % of the trifluoperazine group cannot be considered significant while the improvement of 17% of the reserpine group may represent a trend. Table 111 T A B L E 111 EFFECT OF ISOCARBOXAZID ON EXTRAPYRAMIDAL SIGNS

Drug-induced

-

Effects on trifluoperazine-induced changes (8 patients) Total no. of p fusses*

Baseline

Placebo Trifluoperazine Trifluoperazine and isocarboxazid

12 78.5 93

Diflerence after Percent change in isocarboxazid symptoms

-

-

+13.5

$18

* Rating intensity of rigidity, cogwheeling, tremoi, and changes in gait handwriting, when symptoms occurred.

01

represents a response of 8 patients who received trifluoperazine but not reserpine. A worsening of 18 % was observed and may also be regarded as indicating a trend. DISCUSSION AND CONCLUSIONS

In contrast to the marked improvement of the extrapyramidal alterations in the organic patients (Table I) is the absence of a change in the trifluoperazine-induced extrapyramidal signs (Table 11) in 7 patients and a trend for the worsening of the other 8 patients with trifluoperazine-induced symptomatology (Table 111). The sum of all the changes induced by reserpine is lowered indicating only a suggestive improvement with isocarboxazid (Table 11). It should be mentioned however, that in 2 of these 7 patients receiving reserpine a dramatic reduction in the intensity of the extrapyramidal symptoms was induced by isocarboxazid. One of the patients had developed extrapyramidal symptoms in one parameter, namely, cogwheeling, with a 3+ intensity and the other had developed rigidity with a 3" intensity. Both were restored towards normal in these 2 parameters when treated with isocarboxazid. In the latter patient there was a concomitant change in behavior. He became very overactive, overtalkative and euphoric. The former patient also became more euphoric, more outgoing and admitted to hallucinations he had carefully denied. No such marked changes were noted in the other patients of this group who had not shown marked changes in extrapyramidal symptoms. We hope that the repetition of the experiments with the 2 patients whose reserpineinduced symptoms improved with isocarboxazid administration will help to determine References p . 214

214

C. B U L L A N D H . H. B E R L E T

whether the response is reproduceable and if there is a relationship between the mental and neurological response. We would thus conclude at this point that the trifluoperazine-induced symptoms do not improve with isocarboxazid and may even worsen. The reserpine-induced symptoms showed a trend in improvement which was definite in 2 of the 7 patients. This pilot study indicates that drug-induced extrapyramidal signs are more refractory to isocarboxazid than those of organic etiology. SUMMARY

The monoamine oxidase inhibitor isocarboxazid was given to 4 patients with nondrug-induced (arteriosclerotic) extrapyramidal symptoms, 15 with trifluoperazineinduced extrapyramidal symptoms and 7 with reserpine-induced extrapyramidal symptoms. There was marked improvement of the extrapyramidal alterations in the ‘organic’ patients. Trifluoperazine-induced symptoms did not improve with isocarboxazid and even worsened. Reserpine-induced symptoms showed a trend i n improvement which was definite in 2 of the 7 patients. REFERENCES K., ( I 929); Zur Banisterin- und Harminfrage, Nervenarzt, 2, 548-549. BERINGER. BERNHEIMER, H., BIRKMAYER, W., AND HORNYKIEWICZ, O., (1961); Verteilung des 5-Hydroxytryptamin (Serotonin) im Gehirn des Menschen und sein Verhalten bei Patienten mit ParkinsonSyndrom. Klin, Wschr., 39, 1056-1059. EHRINGER,H., A N D HORNYKIEWICZ, O., (1960); Verteilung von Noradrenalin und Dopamin (3Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr., 38, 1236-1239. HORNYKIEWICZ, O., (1962); Dopamin (3-Hydroxytyramin) im Zentralnervensystem und seine Beziehung zum Parkinson-Syndrom des Menschen. Dmh. med. Wschr., 87, 1807-1 810.

215

y-Aminobutyric Acid Binding and Content in Density Gradient Subfractions of Mouse Brain H . WEINSTEIN, S . VARON, E. ROBERTS

AND

TSUYOSHI KAKEFUDA

City of Hope Medical Center, Duarte, Calif. (U.S.A.)

Sano and Roberts (1963) have demonstrated that particulate material of mouse brain homogenates will accumulate added I -14C-y-aminobutyric acid (yABA) at 0” in 0.2 M NaCl. This phenomenon is specific for brain tissue. In recent work in our laboratory the distribution between supernatant and particles was determined of the total yABA and the I-14C-yABA after a binding experiment under standard conditions. The particles were found to contain yABA even prior to resuspension in the test medium. At the end of the incubation period, a steady state prevails for the distribution between supernatant and particles of the 1-14C-yABA. If, at this time, all the yABA originally present throughout the test system had been involved in the binding process, the specific activity of the yABA isolated from the particles should have been the same as that from the medium or the entire system. The concentration of 1-14C-yABA in the particles was approximately 10 times that in the supernatant while the concentration ratio of the nonradioactive yABA was found to be about 30 to 1. These experiments showed clearly that all the yABA originally contained in the particles was not involved in the ‘binding’ process under the experimental conditions. Further support for the occurrence of distinct yABA pools at the end of the experiment has been gained from studies of the effect of inhibitors on the yABA binding system. This will be reported elsewhere. In the above experiments the radioactivity and total yABA content of the particles and supernatant was determined. The quantity of bound yABA which would have the same specific activity as that of the supernatant will be referred to as the ‘traceable ’pool. This consists of endogenous and exogenous yABA. The amount of yABA in the pellet in excess of this value consists entirely of endogenous yABA, and will be referred to as the ‘non-traceable’ pool. The validity and implications of such a distinction are discussed elsewhere (Varon, Weinstein and Roberts, in preparation). In the present experiments an attempt was made to determine whether the different compartments could be separated physically by means of density gradient centrifugation, and, if possible, to identify by electron microscopic examination the morphological entities in which they might exist. References p . 218

216

H. W E I N S T E I Net

al.

METHODS

Whole mouse brains were homogenized in 0.25 M sucrose. The particles which sediment between 1500 x g (10 min) and 15,000 x g (15 min) were allowed to accumulate 1-14C-yABA by incubation for 30 min at 0” in 0.2 M NaCl and 0.05 M Tris-HC1 buffer, pH 7.3. After sedimentation and removal of the supernatant solution the particles were rehomogenized in 2 M sucrose and the suspension centrifuged 5 min at 5000 rev./min in the Spinco SW39 rotor. Small masses of clumped material rose to the surface and were discarded. An aliquot (0.75 ml) of the remainder was placed at the bottom of a density gradient tube, 0.5 ml layers of progressively less concentrated sucrose were added (see Fig. I), and th tubes were centrifuged in the Spinco SW39 rotor at 38,000 rev./min for 90 min. A hole was pierced in the bottom of the tube and 0.5-mi fractions were obtained from each tube. Equivalent inaterials from 3 tubes were pooled and aliquots of the suspensions were used for the determination of protein (Lowry et al., 1951), total yABA (Weinstein, Roberts and Kakefuda, 1963) and I-14C-yABA. The content of exogenous yABA in each fraction was calculated from the specific activity of the 1-14C-yABA used (2.71 pC/pM). The endogenous yABA was the value obtained by subtracting the exogenous yABA from the total content of yABA determined enzymatically. RESULTS A N D DISCUSSION

Fig. 1 shows the distribution of exogenous (1-14C)-yABA and endogenous yABA throughout the density gradient tube and the distribution of total protein. The protein distribution is an approximate index of the particle distribution in fractions #2 through #lo. Fraction #1 was clear and devoid of particles. Large quantities of exogenous and endogenous yABA were found in fraction # 1. The amount of free yABA carried into the sucrose by resuspension of the original pellet was calculated and found insufficient to account for the exogenous and endogenous yABA of fraction # 1. This indicates that a sizable loss from the particles occurred during the rehomogenization in the 2 M sucrose. Fraction #3 contained very little of the exogenous yABA and considerable quantities of endogenous yABA. Thus, the yABA in this fraction would appear to be related mostly to the ‘nontraceable’ pool. Electron microscopy showed this fraction to be composed almost exclusively of mitochondria (see Fig. 1). Fractions # 4 through #6 contained large amounts of exogenous and endogenous yABA. These fractions consisted of numerous nerve ending fragments, some free mitochondria, and small membranous elements of undetermined origin (Fig. 1). Fractions #7 and #8 were primarily composed of membrane-like fragments of undetermined origin and some fragments of nerve end’ngs and myelin sheaths. Fraction #I0 was made up of numerous myelinated fragments, and fraction #9 appeared to be a mixture of the membrane-like material of fraction # 8 and the myelinated fragments of fraction # 10. Fractions #7 through # 10 contained relatively small quantities of exogenous yABA compared to endogenous yABA levels of these fractions. These fractions may contribute to the ‘nontraceable’ pool.

yABA M.1

2

BINDING A N D CONTENT IN MOUSE BRAIN

I

1.4

1

1.3

I

1.2

I

1.1

I

1.0

I

0.9

I

0.8

I 0.7

217

10.351

Fig. 1 . Distribution of exogenous 1-14C-yABA and endogenous yABA on a sucrose density gradient. The designations across the top of the graph indicate the molar concentrations of sucrose used to establish the gradient. The intervals are proportional to the volumes of the sucrose layers. Fractions # 1 through # 10 represent the successive 0.5-ml samples which were collected at the end of the centrifugation. The interpretations of the electron micrograph of fractions #3, # 5 , #8, and #10 are discussed in the text. These interpretations are based on those of Gray and Whittaker (1962) and = endogenous yABA; o . . . . . . o 1-14C-yABA; De Robertis et al. (1962). 0-0 . . . . . . = mg protein.

The small quantities of exogenous yABA in fractions #3 and #7-#I0 indicate either that they never contained a ‘traceable’ pool or that the ‘traceable’ pool of these fractions was readily removed from these fractions during the procedures employed, contributing to the high residual yABA of fraction# 1. Fractions #4 through #6 were found to be relatively rich in isotope. The following two experiments excluded the possibility that large amounts of isotope might have been entrained by the large quantities of material which equilibrated in this region of the gradient. Aliquots of all fractions from the gradient were diluted with an equal References p . 218

218

H. W E I N S T E I N

et nl.

volume of water to permit the sedimentation of the particles. It was found that as much as 40% of the exogenous yABA could still be sedimented and that the maximal quantity of sedimentable isotope was still in fraction #S. The other experiment was carried out without the addition of I-14C-yABA. The nonradioactive fractions were collected from the gradient and subsequently were exposed to 1-14C-yABA in the presence of added saline. Under these conditions binding capacity was still demonstrable in fractions #4 through #6 with the maximal binding capacity being exhibited by fraction #S. In studies which are currently being carried out, a similar analysis is being made of a post-mitochondria1 fraction which sediments between 15,000 x g (15 min) and 80,000 x g (30 min). This material contains both ‘traceable’ and ‘nontraceable’ pools. SUMMARY

Studies of Elliott and Van Gelder (1960) which indicated the presence of yABA in the sedimentable material of brain homogenates were extended by the demonstration that exogenous yABA can be accumulated by brain particulate fractions (Sano and Roberts, 1963) in the presence of NaCl. More recent studies in our laboratory have suggested that some of the fractions of the saline-treated particulates may have two or perhaps more pools of yABA, which may reside in different morphological structures. ACKNOWLEDGEMENTS

This work was supported in part by grants NB-2655 and NB-1615 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, and by a grant from the National Association for Mental Health. REFERENCES E., DE TRALDI, A. P., ARNAIZ,G. R.DELORES, AND SALGANICOFF, L., (1962); Cholinergic DEROBERTIS, and non-cholinergic nerve endings in rat brain. J . Neurochern., 9, 23-35. ELLIOTT, K. A. C., A N D VANGELDER, N. M., (1960); The state of Factor I in rat brain; the effects of metabolic conditions and drugs. J . Physiol. (Lond.), 153, 423432. GRAY,E. G., AND WHITTAKER, V. P., (1962); The isolation of nerve endings from brain; an electron microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.), 96,79-88. LOWRY,0 . H., ROSEBROUGH, N. J., FARR,A. L., AN D RANDALL, R. J., (1951); Protein measurement with the Folin phenol reagent. J . biol. Cliern., 193, 265-275. SANO,K., AND ROBERTS, E., (1963); Binding of y-aminobutyric acid by mouse brain preparations. Biochem. Pharmacol., 12, 489-502. VARON,S., WEINSTEIN, H., AND ROBERTS, E., (1964); Exogenous and endogenous 11-aminobutyric acid of mouse brain particulates in an in vifro binding system. Biochem. Pharmacol., in the press. WEINSTEIN, H., ROBERTS, E., AND KAKEFUDA, T., (1963); Studies of subcellular distribution of y-aminobutyric acid and glutamic decarboxylase in mouse brain. Biochern. Pharmacol., 12 503-509.

219

General Discussion *

HOLTZ:Before the general discussion starts, I would like to make some brief remarks

in regard to the excellent review given by Dr. Sourkes on the correlation between Parkinsonism and a disturbance of amine metabolism. I want to draw your attention to a paper just published by Ernst in Holland (Acta pharmacol. neerl., 11 (1962) 48-53). This author was able to show that tyramine and dopamine which are omethylated in the 4-position of the ring instead of in the 3-position will produce Parkinson-like symptoms, i.e. tremor and muscular rigidity in cats. Thus, a disturbance of the enzymatic mechanism of o-methylation discovered by Dr. Axelrod could be involved in the development of the human disease. May we have additional discussion? EVERETT: I would like to comment on Dr. Green’s paper and his most excellent studies concerning the rise and fall of serotonin in the brain over a period of time and also his findings in regard to the high levels of serotonin that were obtained, by giving repeated doses of a monoamine oxidase inhibitor, without causing behavioral effects. We must keep in mind the important point that the total amines and the amount available for physiological function may be quite different in various circumstances. Several previous speakers have pointed out that high levels of the amines as they are extracted and determined may not indeed necessarily be correlated with behavior. In some ways it would be surprising if they were. The amines determined may be bound and therefore not necessarily active nor available to affect behavior. So it appears the problem we are up against as Dr. Costa and others have pointed out, concerns not only the level of these various amines at any given time, but also the availability of these substances at receptor sites to produce behavioral states. Thus, we still have the general problem of deciding not only how much of these various biogenic amines are present but what part of them is available to the organism for use. Returning to Dr. Green’s study, you will note that there is a major difference between the animals that showed behavioral change and the animals that did not, even though the total levels of amines in the brain were the same. The ones that were showing the behavioral effects had been given a large dose of 5-HTP. They undoubtedly had a very high level of these amines in their blood stream as well as in the brain and, of course, it is an entirely different picture, from the dynamic point of view. The 5-HTP is going into the brain and being formed into 5-HT. These amines are not at that time necessarily bound and it is not surprising that more of the 5-HT formed during this period is available to produce physiological effects. In contrast

*

This discussion refers t o the papers of Dr. H. Green, Dr. E. Costa and Dr. Th. L. Sourkes.

220

DISCUSSION

to the other group, the amines accumulated slowly and are very likely more completely bound and unavailable to produce behavioral effects. HOLTZ: I would like to point out that an increase or a decrease of the amine content of the brain by itself is not necessarily the deciding factor for the physiological or pharmacological action of a drug. Some years ago we found that iproniazid injected about 20 h before hexobarbital in mice significantly shortened the duration of anesthesia (Holtz, Balzer, Westermann and Wezler (Naunyn-Schmiedeberg’sArch. exp. Path. Pharmak., 231 (1957) 348). We explained the effect as being due to an increase of the amine content in the brain. That this explanation was too simple has now been shown by my colleagues Westermann and Stock (Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 4 (1962) 329). Pretreatment of the animals with harmaline, a short acting reversible M A 0 inhibitor, protected M A 0 against the long-lasting irreversible blockade by iproniazid or nialamid, and 24 h later there was no increase of the amine content in the brain. Nevertheless the period of hexobarbital anesthesia was shortened to the same degree, indicating that some M A 0 inhibitors may exert pharmacological actions which cannot be explained only by the blockade of this enzyme. Another example reported by my co-workers Balzer and Palm and myself (Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 239 (1960) 520) is afforded by reserpine which decreases not only the amine in mice but also the GABA content of the brain. A M A 0 inhibitor like iproniazid is able to prevent both the decrease in amine and in GABA content, although GABA is not metabolized by MAO. Lastly, as first shown by Pfeiffer and by Bain and Killam, many hydrazides like semicarbazid, thiosemicarbazid and others will produce seizures and at the same time lower the GABA content of the brain, because they inactivate pyridoxal-5-phosphate, the coenzyme of glutamic acid decarboxylase. Pyridoxine protects the animals against the convulsive action of the hydrazides. Though we were able to confirm these results, we did find however that a dose of pyridoxine which protected against seizures, failed to normalize GABA, for the GABA content of the brain suffered further decreases (Balzer, Holtz and Palm, 1960). Obviously pyridoxine catalyzed the metabolic disappearance of GABA to a greater extent than its synthesis from glutamic acid and by that means enhanced the turnover in the glutamic acid-(GABA)-a-ketoglutaric acid cycles. Not the GABA content per se but the rate of the turnover seems to be of decisive importance for the excitability of cortical motor cortex. Therefore we should consider not only the content of amines in the brain as being influenced by drugs but also realize that the turnover of amines in the brain may be of greater importance. In that case, the most important function of M A 0 in brain perhaps would be to maintain the turnover of the biogenic amines. GIARMAN: I would like to comment on Dr. Costa’s paper. I would like first to congratulate him on an elegant presentation of a very provocative and panoramic scheme. However, I have noticed from his title that he made it all-encompassing to include the monoamines, and it occurred to me, if I understood correctly, that one of the criteria for this scheme would be that the amine would have an active transport into brain. He showed this for noradrenaline. As far as I know there is no demonstration that serotonin has such an active transport into brain. We have attempted to d o this

DISCUSSION

22 1

and Schanberg from my laboratory has recently published a paper (J. Pharmacol. exp. Ther., 139 (1963) 191) showing that there is no active transport for serotonin in brain slices. I wonder whether Dr. Costa feels that serotonin fits into this scheme. COSTA:Several investigators have studied the uptake of 5-HT by brain slices. In these experiments labelled 5-HT was used; but the specific activity of the available material was not high enough to detect the presence of an active transport for this amine in brain slices. Since I have not read Dr. Schanberg’s paper I cannot say whether his data permit the conclusion that brain cells do not possess a n active transport for 5-HT*. From the data on 5-HT uptake into platelets and on N E uptake into brain slices mentioned in our presentation we have presumed that brain slices also contain an active transport for 5-HT. HOLTZ:I would like to make a contribution on two points of the paper delivered by Dr. Green (ths Symposium). First, as pointed out by Dr. Green, the injection of 5-HTP increased the content of 5-HTP as well as of serotonin in the brain. This effect was enhanced by pretreatment of the animals with iproniazid or tranylcypromine. He thinks it possible that iproniazid enhances the influx of the precursor amino acid through the blood-brain barrier; thus the increase of serotonin in the brain would partly be caused by a higher availability of the precursor amino acid. Under entirely different considerations we obtained similar results. Studying the effect of reserpine and M A 0 inhibitors on the carbohydrate and protein metabolism in mice (Balzer and Palm, Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 243 (1962) 65) we found that 1%-y-aminoisobutyric acid (I4C-AIB), which is not metabolized but transported

* I have read Dr. Schanberg’s paper (J. Pharmacol. exp. Ther., 139 (1963) 191) but I cannot agree with Dr. Giarman that the evidence presented proves that brain slices lack an active transport for 5-HT. Unfortunately, the 5-HT available to Dr. Schanberg was not very ‘hot’ and so he used 5-HT at concentrations of 2.5 ,ug/ml and higher. In our laboratory Titus and collaborators (Znt. J. Neuropharn;acol., 1 (1962) 23) have shown that concentrations of NE, greater than 0.005 pg/ml are close to that which saturate the N E active transport system in brain slices. 1suspect that the concentration of 5-HT used by Dr. Schanberg was 500 times greater than that which might have saturated the transport system so that the uptake now appeared to be a process of passive diffusion. As a matter of fact the results of Dr. Schanberg suggest that some other component to uptake is involved since extrapolation of his plot of 5-HT uptake back to zero time shows a positive value of considerable magnitude. GIARMAN: What Dr. Costa seems to be saying here is that the uptake of characteristics of 5-HT in brain is something quite special in contrast to uptake characteristics of other substances in brain and of 5-HT in other tissues (e.g., mast cells and platelets). Schanberg and I could not agree more. As a matter of fact, Schanberg points out, in the paper alluded to, that 5-HT enters brain slices by a process quite different from that which accounts for a 5-fold concentration of 5-HTP by the same slices. In the case of 5-HTP we are clearly dealing with facilitated transport; but what we are dealing with in the case of 5-HT is questionable (incidentally, Schanberg and I have tested uptake of 5-HT at 0.5 ,ug/ml with no difference in results). In fact, it is hardly possible to accept the conclusion by Titus and collaborators (Int. J . Neuropharmacol., 1 (1962) 23) that N E is actively transported by brain slices, when they show that such transport is saturated by concentrations greater than 0.005 pglml. At such concentrations it is almost impossible to differentiate true uptake by transport from non-specific adsorption. The point to bear in mind here is that when true facilitated transport exists for 5-HT (as in platelets and mast cells), it is demonstrable at levels of 5-HT much greater than 1 ,ug/ml(Day and Green, J. Physiol. (Lond.), 164 (1962) 227). This, of course, is also true for facilitated transport of certain amino acids by brain.

222

DISCUSSiON

like natural occurring amino acids, enters the brain more rapidly in animals pretreated with iproniazid while the reverse holds true for animals pretreated with reserpine (Fig. 1). This is in agreement with the results presented by Dr. Green with 5-HTP. However, in his experiments this effect lasted only for 2 h while in our experiments increased levels of 14C-AIB were observed for more than 2 days. This difference in duration is probably due to the fact that 5-HTP is metabolized quite rapidly, while AIB is not. A second point: In Dr. Green’s experiments the injection of 5-HTP produced pharmacological actions (forepaw clonus, body tremor, etc.) only when the animals _..

Concentrations in Mice 2.105-

. ..

..... .. ..... .

,,

2.104

.

...

../,/’ .Q’

Q i

blood

/ /

? .

/ Q ’

104

15

30

60

90

200

500

lOOO(17hrs.)

1OOOMin

Fig. I . Action of reserpine and iproniazid on the influx of 14C-AIB into brain (mice). Balzer and Palm. Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 243 (1962) 65. A . . . . . . . . A = iproniazid; 0-0 = controls; 0 - - - 0 = reserpine.

were pretreated with a M A 0 inhibitor. I think one has to keep in mind that the precursor amino acid was injected intraperitoneally. Thus it can be assumed that most of the serotonin originating from 5-HTP by decarboxylation in the liver was inactivated immediately by M A 0 in the liver mitochondria. After blockade of M A 0 in the liver a great deal of the ‘liver born’ serotonin will enter the systemic circulation and produce pharmacological actions, e.g. a long-lasting bronchoconstriction (Fig. 2). Similarly the activity of another amine, dopamine is greatly reduced when injected intraperitoneally : dopamine produces a sustained pressor response when injected i.m. (Fig. 3A) but has virtually no effect on the blood pressure when injected i.p. (Fig. 3B). However, after blockade of M A 0 the i.p. injection is as effective as the i.m. injection (Fig. 3C). Therefore the i.p. injection of 5-HTP after blockade of M A 0 may cause long-lasting peripheral effects, e.g. bronchoconstriction and anoxemia, and the question arises whether the pharmacological effects described by Dr. Green are only centrally caused or partly due to peripheral actions of serotonin.

DISCUSSION

223

ANOKHIN:We have heard at this session many interesting remarks about different chemical mechanisms involved in nervous activity. There is no doubt nowadays that the investigation of nervous mechanism on the molecular level is a logical and an extremely important test of neurophysiology. Yet, the question arises immediately, how do these very fine neurochemical processes, interacting among themselves and involved in so many different positive and negative connections, form the resulting

Fig. 2. Bronchoconstriction (method of Konzett and Rossler, in the guinea-pig after i.v. injection of either serotonin (S) or 5-hydroxytryptophan (5-HT). a-Methyldopa (a-MD) abolishes the action of 5-HT. Westerman et al., Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 234 (1958) 194.

activity of the entire brain, and therefore, the adaptive features of the whole organism. This question is not a fanciful one, because we have now a quite real basis, a real background for performing such kinds of experiments and realizing that final point of neurophysiological synthesis (Fig. 4). I should like you to pay attention to one example of such kinds of investigations performed recently in our laboratory. It was shown on cats that starvation of 2 days duration yields a marked EEG activation in frontal lobes of cerebral hemispheres, while slow electrical activity takes place in other cortical regions. This pattern of electrical activity can be seen when the cat is under urethane anesthesia. This EEG activation is connected with the influence from the digestive apparatus ascending from hypothalamus, as demonstrated in numerous control experiments (Sudakov, 1960). It was discovered then that the cat which is examined after 5 but not 2 days of starvation (also under urethane anesthesia), showed generalized activation which is now expressed throughout all regions of the cortical hemispheres. It was quite natural to think that this generalization possesses the same

224

DISCUSSION

Fig. 3. Pressor effect of dopamine in rats after intramuscular and intraperitoneal injection. Rats: 260-290 g; urethane anesthesia. Blood pressure recorded in carotid artery. Timing (lowest trace) = 1 min. A = 10 mg/kg dopamine i.m.; B = 10 mg/kg dopamine i.p.; C = 10 mg/kg dopamine i.p.; blockade of monoamine oxidase by iproniazid (100 mg/kg i.m.) 6 h before dopamine injection. Holtz and Westermann, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 237 (1959) 21 1 .

A

C

B

AMINAZINE 5 mg/kg

URETHANE 1.5 g / k g FR F LSMR -SML-

PR

PL - -R O OL

I I _

--

'

--

-

-

I - DAY STARVATION

-

..-

4 -DAY STARVATION

SEC

Fig. 4. Three groups of tracings of brain electrical activity: (A) after 1 day starvation, (B) after 4days starvation, and (C) after giving aminazine (5 mg/kg) (chlorpromazine). It is evident that the primary activation occurred first in the right and left frontal lobes (FR, FL) and then became generalized and extended to the other cortical areas studied, sensory motor right and left (SMR, SML), parietal right and left (PR, PL), and occipital right and left (OR, OL) after 4 days of starvation. After the injection of aminazine this generalized activation disappeared in all cortical areas except the frontal lobes where it remained constant.

DISCUSSION

225

biological quality, i.e. of the digestive system. At first it was thought that the excitation spread from the primary cortical area of activation in the frontal lobes to other cortical regions together with an increase of hypothalamic excitation. Yet it has turned out that tht: aminazine (chlorpromazine) injections block only the secondary generalized activation and leave unchanged the activation in frontal lobes. We have good reasons to think, due to the adrenolytic character of chlorpromazine, that the generalized activation appears as a result of the excitation of adrenergic substrate of the brain stem reticular formation, and since the frontal lobe activation is very well preserved we have concluded that the hypothalamic substrate of this activation is of another chemical nature which resists the blocking action of chlorpromazine. In evaluating the integrated function of these two different subcortical substrates with different chemical quality we must believe that this coordination possesses great physiological meaning. It should be thought that the primary specific digestive EEG activation takes place only in frontal cortical regions, while the increase of digestive excitation includes the excitation of subcortical mechanisms of another nature, apparently adrenergic in character. This adrenergic reaction promotes the development of stress situation which involves the participation of the sympathicoadrenal system of the entire organism. With this example you can see how complicated the interrelationships are between nervous substrates of different chemical natures as they participate in the process of the behavioral integration of the whole organism.

226

Summary H A R O L D E. H I M W I C H Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. ( U . S . A . )

To introduce my summary, it is appropriate to point out that the large volume of new and important data on binding sites of brain biogenic amines, as exemplificd by the papers presented in this Symposium, are the flowerings of seeds planted in the not too distant past, The first disclosure of the presence of serotonin in the brain came from Page’s laboratory (Twarog and Page, 1953) where serotonin had been previously purified and given its present name (Rapport et al., 1948). Systcmatic studies of the levels of serotonin (Amin et al., 1954) and noradrenaline (Vogt, 1954) in various parts of mammalian brain followed shortly thereafter. It should be emphasized, however, that earlier discoveries, not made on brain tissues, have shaped our thoughts and guided our investigations even up to the present t:me. Long before any work had been done on the brain, Holtz’s discovery of DOPA decarboxylase (Holtz, 1939) had been made. Another important conception was that obtained from Blaschko’s work on the specificity of interactions betwcen enzymes and substrates which began in 1939 (Blaschko, 1939) and has sharpened our ideas of enzyme specificity in general and of the aromatic amino acid decarboxylase in particular (Blaschko, 1950). Blaschko (this Symposium, p. 1) points out that pharmacologists study active sites in order to make synthetic compounds which react with these specific sites. As he says, pharmacologists pick the lock instead of opening it with the proper key. He points to the observations of Schiimann and Philippu (1962) revealing that some sympathomimetics including tyramine, displace catecholamines in isolated medullary granules of cattle in vitro thus indicating that these sympathomimetics may not necessarily act directly on receptor sites and suggesting that the latter structures possess greater specificity than had been previously believed. A fertile observation also originally not made on brain tissue is Zeller’s discovery (1952) that iproniazid inhibits MAO. It is true that inhibitors of other enzymes have since been found and among them is a-methyl-DOPA and its congeners as reported by Sourkes (1954) as well as the more specific decarboxylase inhibitors about which you have just heard from Carlsson (this Symposium, p. 9). It must also be remembered that almost at the beginning of the period of systematic investigations on brain biogenic amines, the action of reserpine to lower their levels was announced in two series of papers which were published practically simultaneously from Brodie’s (Pletscher et al., 1955; Shore et al., 1956; Brodie et al., 1957) and Vogt’s (Paasonen and Vogt, 1956; Holzbauer and Vogt,

SUMMARY

227

1956-57; Muscholl and Vogt, 1958) laboratories. Some of the ideas which are under intense investigation today similarly developed practically with the very inception of our attack on brain biogenic amines. In 1954 Marthe Vogt wrote, ‘It might be tempting to assign to cerebral sympathin a transmitter role like that which we assigned to the sympathin found in the sympathetic ganglion and the postganglionic fibers. There are, however, a number of facts which call for caution.’ Moreover, our first ideas on storage sites were likewise enunciated independently from Sweden (Bertler et al., 1956; Carlsson et al., 1957) and England (Muscholl and Vogt, 1957, 1958) when it was found that under the influence of reserpine, peripheral adrenergic neurons lose a large fraction of their noradrenaline and simultaneously fail to evoke responses from their effector organs on electrical stimulation of these neurons. Again in 1957 Brodie and Shore forwarded the possibility of serotonergic fibers in the brain, and Brodie et al. (1959) suggested that both noradrenaline and serotonin might function as neurotransmitters in the brain. It is not my purpose to give a complete history of our field but rather to see how these seminal observations have been brought to fruition and especially as expressed in the papers we have heard in this Symposium. You will have noticed our speakers have presented areas of disagreement and they have also presented areas of agreement. Both are advantageous for the advance of knowledge but advantageous in different ways. Areas of agreement suggest a plateau from which further advances can be made. On the other hand by introducing an element of controversy the speakers bring a contested point to sharp focus and as a result research is stimulated in order to see to what extent each view is correct. I am glad to say that our discussions revealed a greater dissemination of light than heat. A step toward the elucidation of binding sites of brain biogenic amines was made by employing the method of centrifugation of brain homogenates in order to separate the biogenic amines into the portions occurring in the supernatant and the particulate fractions. As pointed out by Giarman (this Symposium, p. 72), a single centrifugation permits a more accurate estimation of amine fractions than procedures requiring multiple centrifugations. And here we find an area of agreement par excellence for Giarman has resolved a contradiction in the literature. Weil-Malherbe and Bone (1957) have reported that the chief decreases of catecholamines wrought by reserpine in rabbit brain stem occurred in the cytoplasmic fraction. But Green and Erickson (1962) have noted that the noradrenaline passed from the particulate to the supernatant. Schanberg and Giarman (1962) have found that serotonin also decreases chiefly in the particulate. The new observations of Giarman, Freedman and Schanberg (this Symposium, p. 72) disclose both types of results also occurring with serotonin depending upon the duration of reserpine action. During the early stages, depletion of the supernatant occurs at a more rapid rate than that of the particulate fractions of 5-HT. Later, however, a greater depletion of bound than unbound 5-HT was seen when the characteristic pharmacologic effects of reserpine were fully developed (Fig. 2 : Giarman, this Symposium, p. 76). Though the mechanism of action for the early changes is not determined, that of the latter is understood (Carlsson, this Symposium, p. 9) and reveals why the particulate is finally depleted more than the supernatant. References p . 238-240

228

SUMMARY

During the following repletion phase, the rate of return to normal levels is more rapid iii the bound than in the unbound fraction. Thus the effects of reserpine appear first on the unbound fraction and disappear last in the same fraction. Using gradient centrifugation in order to subfractionate the particulate, Hillarp (1960) delved deeper into the question of binding sites than was possible by differential centrifugation and found two pools of catecholamines in the cells of the adrenal medulla, a smaller one with lighter granules and a larger one with catecholamines comparatively tightly bound with ATP in heavier granules. Here we find a concurrence of ideas in regard to two pools though there are some differences in regard to the particulars. For example, Axelrod agrees with Carlsson in regard to the granular sites of the labile pool but both Costa and Holtz confine the labile pool to the cytoplasm in general rather than to granules within the cytoplasm (Fig. 6: Costa and Brodie, p. 173). Studies of labeled noradrenaline in brain tissue show that it mixes freely only with a fraction of the noradrenaline in brain tissue slices and, therefore, indicate that the endogenous noradrenaline is present in at least two pools. In one of these noradrenaline is readily available and is, therefore, called the mobile pool which can take up noradrenaline from the circulation as well as release noradrenaline to it. The other pool is larger and in it the amines are more tightly bound by intragranular components. This Costa and Brodie regard as a reserve pool, which, however, is in equilibrium with the mobile pool. In peripheral nerves the noradrenaline enters the mobile pool as a result of active transport and the position of these two workers in regard to the action of reserpine and similar drugs, for example the benzoquinolizines, is that suchcompounds inhibit the active transport which is necessary to maintain noradrenaline in the mobile pool. Costa and Brodie further report that noradrenaline enters brain slices by active transport (Dengler et al., 1962). It is true that Schanberg (1963) has failed to find active transport of 5-HT in brain slices, but Costa and Brodie feel that the problem is still open and that such active transport could prevent the amine found in the intracellular compartment from escaping (see pages 168-185). They further report their ideas of a neurochemical transducer (Fig. 1 : Costa and Brodie, this Symposium, p. 168) which is part of a biophysical system. Noradrenaline is regarded as a neurochemical transducer. The system involves the discharge of the amine due to the stimulating effect of the nerve potential and in turn the released amine either evokes an action by an effector organ or transmits the impulse to the next neuron. They illustrate one of the many virtues of their useful concept in affording a paradigm for analysis for the mechanism of action of several drugs. To continue with our main theme on binding sites, the ideas of Costa and Brodie are in broad concurrence with those of Axelrod as shown in the latter’s working model of sympathetic nerve endings (Fig. 1 : Axelrod, this Symposium, p. 86) disclosing noradrenaline-containing vesicles within the nerves. He too holds that there are two pools of stored catecholamines, one which is more tightly bound and is sensitive to reserpine but not to tyramine nor to the nerve impulse, while the more lightly bound amine can be released by all three. The majority of the storage vesicles are deep within the nerves. Reserpine releases noradrenaline from the deep vesicles and the catecholamine is thus exposed to the action of the M A 0 enzyme, deaminated, and

SUMMARY

229

leaves the nerve in the form of inactive metabolites. On the other hand, some vesicles close to the synaptic terminals release noradrenaline outside the nerve cell whether as a result of the action of sympathomimetic amines or nerve impulses. Noradrenaline so liberated reacts with the receptor and is then metabolized by COMT. It may also however, be discharged into the circdation or returned to the storage vesicle. According to Carlsson and Lindqvist (1962) the concept obtained from analysis of peripheral sympathetic nerve seems to apply in broad detail to the brain as well. These workers showed that the dopamine formed in the brain, after the i.v. administration of DOPA, first reaches the sites of M A 0 within the cell, and is then deaminized and oxidized to form DOPAC. Only after that catecholamine leaves the cell is it metabolized by COMT to form homovanillic acid (Fig. 4: Carlsson, p. 14). These workers suggest that M A 0 is situated close to the area of catecholamine synthesis while COMT is localized outside the amine-producing cells. Thus brain dopamine is not attacked by COMT until it is released from the cell in which it is produced. In his new experiments presented in this Symposium, Carlsson uses powerful COMT inhibitors, including NSD-1015, which is less toxic than pyrolgallol and, therefore, can be given in vivo with greater safety. He points out that normetadrenaline and 3-methoxytyramine disappear from the brain with these new COMT inhibitors (Fig. 8 : Carlsson, this Symposium, p. 19), and he concludes that in the brain as in the rest of the body COMT is largely responsible for the degradation of extracellular catecholamines while the intracellular metabolism occurs via M A 0 within the cell near the site of synthesis and storage. Just as in peripheral nerve reserpine causes increases of acidic products formed in the M A 0 pathway and diminishes the 3-0-methylated metabolites. Because reserpine prevents the binding of catecholamines they remain in the cytoplasmic sap where they penetrate into the mitochondria and become substrates for MAO. Thus there are lesser amounts of catecholamines available for liberation into the extracellular space and to receptor sites. Pletscher, Gey and Kunz (this Symposium, p. 45) did not study reserpine on brain but another drug with similar actions, namely the benzoquinolizine derivative, RO 4-1284. They administered D L - [ ~ - ~ ~ C ] - D OtoP rats A and rabbits and found that their drug diminished radioactivity in the stored amine fraction but increased that of phenolcarboxylic acid as less of the exogenous amine is stored and instead undergoes metabolism by MAO, an observation in general agreement with the results of Carlsson mentioned above. Unlike reserpine, chlorpromazine and chlorprothixene failed to affect the total content of endogenous amines but reduced the amine fraction and increased that of the phenolcarboxylic acids derived from the endogenous precursor. Pletscher et al. suggest that the latter two drugs impair the penetration of the amines into the storage sites. The authors believe that the effect of these two neuroleptics might be caused in part by a decrease in body temperature which these drugs produced in rats. When they maintained body temperature at normal levels the neuroleptics no longer affected the levels of the amines nor of the phenolcarboxylic acids. Pletscher and Gey (1960) had previously reported that in rats and mice but not in guinea-pigs and rabbits, chlorpromazine impaired the rise of brain biogenic amines induced by administration of MAOI. It must be remembered that though rats and References p . 238-240

230

SUMMARY

mice are homeothermic animals they still have relatively poor temperature control. Moreover, the French surgeons were the first to use chlorpromazine as part of the lytic cocktail when they lower the body temperature for some operative procedures on human patients. While Dr. Clara Morpurgo was still at the Galesburg laboratory (1962), she used rats and showed that the effects of tranylcypromine to increase the level of serotonin in the brain were impeded both by chlorpromazine and phenobarbital and under the influence of both of these drugs the rectal temperatures of rats fell (Table 1). When the temperature was maintained, however, chlorpromazine lost the ability to diminish the rise of serotonin evoked by tranylcypromine. Thus this action of chlorpromazine applies not only to catecholamines as shown by Pletscher and co-workers, but also to indoleamines. TABLE I INFLUENCE O F PHENOBARBITAL A N D CHLORPROMAZINE O N T H E INCREASE O F B R A I N S E R O T O N I N I N D U C E D BY T R A N Y L C Y P R O M I N E I N R A T S

Rats sacrificed 4 hours after administration of tranylcypromine. Phenobarbital and chlorpromazine injected I hour before tranylcypromine. p. versus tranylcypromine alone.

Treatment

Tranylcypromine I mg/kg i.p. Tranylcypromine I mg/kg i.p. pentobarbital 32 mg/kg i.p. Tranylcypromine 1 mg/kg i.p. chlorpromazine 20 mg/kg i.p. Tranylcypromine 1 mg/kg i.p. -1chlorpromazine 20 rng/kg i.p. (at 36 "C)

+ +

*

Rectal temperature of control rats

=

Serotonin

Rectal temp. *

No. of Rats

P: ofcontrols

12

171 5 4

6

135+4

<0.0 1

33.7"

12

143+4

tO.O1

33.7"

6

168*4

>0.50

37.1"

P

36.9"

36.4 "C.

Carlsson (this Symposium, p. 9), continuing his studies of reserpine noted the effect of that drug on adrenal medullary granules at different intervals following injection of a single dose of reserpine ( 5 mg/kg intravenously) to rabbits and observed that the incorporation of labeled amine is blocked for 12 to 24 h following the injection. After about 48 h the incorporation is restored to normal despite the fact that adrenal medulla is stilI depleted of catecholamines (Fig. 3 : Carlsson, this Symposium, p. 13). In other words, restoration of the storage function precedes that of aminc levels. If there is one idea held in general agreement in this Symposium it is that there may be discrepancies in the correlations between the level of brain biogenic amines and behavior. In Carlsson's experiments storage function rather than amine levels is correlated with sedation and other pharmacological effects of reserpine. His experiments show that the amine must be taken up by the labile granules before it can be released by nerve activity. Normally the endogenous precursor, an amino acid, is decarboxylated in the neuron. The amine is then incorporated in the labile granular fraction. There is also general agreement among the participants of this Symposium that an important action of drugs of the reserpine type is to interfere with storage of amines and particularly in the labile pool. Now Carlsson goes further and presents evidence

SUMMARY

23 1

for the consequences of this action of reserpine on the labile fraction. Because of previous storage, nerve transmission is still possible for some time after reserpine administration. But adrenergic neurotransmission ceases when the labile fraction is exhausted. Di Carlo (this Symposium, p. 64) presents evidence indicating that serotonin or a serotonin-like compound plays a physiological role in conduction of peripheral nerves and quotes Nachmansohn in support of his ideas on the chemical basis for conduction though the latter’s formulation concerns acetylcholine and does not include a serotonin-like compound. The present viewpoint on nerve conduction is well expressed by Fourtes (1958): ‘The impulse proceeds along a nerve fiber because the action currents accompanying activity at a point are anadequatestimulus for producing activity of a neighboring point’ (Tasaki, 1953). The concensus, which confines the chemical hypothesis to synapses and the phenomena of transmission, is illustrated by the papers of Axelrod, Carlsson, Costa and Brodie, Falck, De Robertis and Whittaker in this Symposium. A suggestion of Carlsson (this Symposium) that a-methyl-DOPA inhibits the first step in the synthesis of 5-HT is rendered even more attractive by the disclosure of Gal and Marshall (this Symposium, p. 56) that the hydroxylation of tryptophan in the position 5 may take place in the brain. Gal injected labeled tryptophan intracerebrally and observed the production of labeled 5-HTP and serotonin in the brain of pigeons. In addition, labeled tryptamine was formed. There is not only more than one pool for endogenous brain biogenic amines but also for exogenous. Weinstein, Varon, Roberts and Kakefuda (this Symposium, p. 21 5) conclude that there are at least two pools for endogenous GABA and two pools for exogenous GABA. Another example of exogenous and endogenous pools is afforded by Pletscher and co-workers (this Symposium, p. 45) who noted that chlorpromazine and chlorprothixene did not affect the storing capacity of the endogenous amines but did lower the exogenous amines derived from injected D L - ( ~ - ~ ~ C ) - D O P A . Proof of a separate exogenous pool was afforded by Green and Sawyer (this Symposium, p. 150), who found that the administration of iproniazid or tranylcypromine induced complete inactivation of M A 0 for approximately 16 h and produced continued increases of endogenous serotonin during that period. In animals premedicated with MAO-I and then injected with 5-HTP, serotonin rose to a maximum in 2 h and disappeared within 6-12 h, at a time when M A 0 was still completely inhibited. This serotonin is, therefore, metabolized by some other pathway than that of MAO. The author concluded that it belongs to a separate pool, the exogenous one, which exhibits catabolic processes even when M A 0 is inhibited. According to their work extracranial tryptophan, or 5-HTP, may not be the immediate precursors of endogenous 5-HT. On the contrary, the brain proteins themselves may provide the amino acid directly. In any event, the versatile aromatic amino acid decarboxylase not only acts on endogenous 5-HTP and DOPA but also on the exogenous precursors. Green and Sawyer (this Symposium, p. 150)observed not only biochemical differences between the endogenous and exogenous serotonin pools but also in their behavioral effects. Forepaw clonus, body tremors and increased motor activity occurred after combined MAO-I and 5-HTP administration but not with MAO-I alone. With these ReJrrences p . 238-240

232

SUMMARY

bases of biochemical and behavioral differences, they make their main point that exogenous 5-HT goes to a different pool than that formed endogenously. Similar experiments have been previously reported on dogs which are better adapted for certain kinds of behavioral studies than are rats. Bogdmski et al. (1958) found that with 5-30 mg/kg 5-HTP, dogs exhibited a decrease of spontaneous activity while in contrast the administration of larger doses of 40-60 mg/kg did not cause increased sedation but on the contrary evoked excitement, disorientation and changes of motor and sensory function. This biphasic action was also observed by W. A. Himwich and Costa (1960), in dogs pretreated with the MAO-1, tranylcypromine 2 mg/kg and remaining without observable behavioral changes for 6 h thereafter. At the end of that period an injection of 2 mg/kg 5-HTP induced sedation, but when the dose of that amino acid was increased to 5-7 mg/kg 5-HTP a relatively brief calming action was followed by signs of excitement, including continuous hyperactivity and obstinate progression. In these experiments serotonin increases were observed in all parts of the brain studied with the largest rise on a percentage basis in the caudate nucleus, a structure which is otherwise predominantly dopamine in character. Bertler and Rosengren (1959) had previously reported similar results for rabbit brain. W. A. Himwich and Costa (1 960) concluded that the ‘situation is extremely unphysiological and, therefore, that the syndrome described is the result of a reversible biochemical lesion.’ In the experiments of Pletscher and co-workers (this Symposium, p. 45) in which radio-DOPA was injected it might be expected that catecholamines would accumulate not only in its usual sites but also in locations previously occupied predominantly by serotonin and thus again we would have an abnormal storage. But the exogenous pool would be formed of catecholamines rather than of indole substances as in the experiments of Green and Sawyer (this Symposium, p. 150). Carlsson (this Symposium, p. 9) points out that the syndromes produced by 5-HTP and DOPA are markedly different indicating that different receptors are activated by their respective decarboxylation products and his observation and thought are in agreement with Blaschko’s ideas of specificity (this Symposium, p. 1). To what extent, however, are the observed symptoms the result of the phenomenon of imbalance between the various amines in the brain? We have h e x d the paper of Guroff and Udenfriend (1964) who have shown that the administration of tyrosine interferes with the transport of tryptophan into the brain. In some experiments performed by Costa et a/. (1960), the effects of various M A 0 drugs on the EEG of rabbit brain was studied. When this slowly acting MAO-I, iproniazid, was used in doses less than 400 mg/kg (Himwich et al., 1959) there is little lag in the rise of noradrenaline compared with that of serotonin and the EEG of the rabbit showed its usual variations from sleep to alert patterns and back to sleep again. But the more rapidly acting MAO-I drug tranylcypromine (Parnate) and phenylisopropylhydrazine (Catron) induced a persistent desynchronization and much more rapid rise of serotonin than noradrenaline. This EEG alerting may be regarded as due to the imbalance between an increase of serotonin larger than that of noradrenaline. Abnormalities in behavior may not only be associated with excessive levels of brain amines but also by their decreases. Sourkes (this Symposium, p. 186) has pointed out

SUMMARY

233

the similarity between the effects of reserpine and the disease process of Parkinsonism in reducing the level of brain amines. His review revealed that there are low concentrations of dopamine particularly in the postencephalitic form in the caudate nucleus, putamen and globus pallidus (Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1962). In the hypothalamus in particular both noradrenaline and serotonin are reduced. On the basis of his data Sourkes suggests that the deficiency in dopamine is probably a general phenomenon and not only limited to the basal ganglia, because the urinary excretion of that amine is low especially in the postencephalitic and arteriosclerotic forms of Parkinsonism (Barbeau, Murphy et al., 1961 ; Barbeau and Sourkes, 1961; Barbeau, Sourkes et al., 1962). Furthermore, there is a reduced ability to convert DOPA to urinary dopamine and DOPAC (Barbsau, Sourkes et al., 1962). Suggestive evidence is also supplied by the briefly acting antiparkinson effect observed in patients with this disease as the administration of L-DOPA may result in a reduction of muscular rigidity. One etiological factor for Parkinsonism could be an inadequate activity of DOPA decarboxylase. At present, however, the evidence does not support this possibility. The opposite condition, namely that of excessive M A 0 activities can also reduce brain amines but that alternative has not been found to be the case in Parkinsonism (Bernheimer and Hornykiewicz, 1962). Thus the administration of a MAOI, with the possibility of remedying the defect, does not reverse the pathology due to a failure of an enzyme but rather acts as a drug to raise subnormal levels of brain amines. This conclusion is substantiated by data that MA01 drugs increase brain biogenic amines in man (Ganrot et al., 1962; Bernheimer et al., 1962). Harmine (Beringer, 1928, 1929) has been used in the past with beneficial effects and you have heard the results of Bull and Berlet (this Symposium, p. 21 1) who inform us that we have seen marked improvement in tremor of Parkinsonian patients on the administration of isocarboxazid. They have found that the tremors produced by trifluoperazine (Stelazine) were not improved by isocarboxazid. On the other hand their work disclosed a suggestive improvement with patients who were placed first on reserpine alone and then on reserpine and isocarboxazid. 1 would like to recount the results on two of these patients. In one, an extreme degree of rigidity was developed due to reserpine medication. There was a n involuntary resistance to extension of the patient’s arm by the examining physician. This rigidity disappeared completely under isocarboxazid. In another patient a marked cogwheel phenomenon developed. As the examining physician placed his hand on the biceps muscle he felt that the muscle, instead of extending and flexing smoothly, underwent a series of small, separate movements as if moving from cog to cog. Marked cogwheel in this patient was dramatically reduced after the administration of isocarboxazid. Schopbach (this Symposium, p. 207) reports a surprising observation that the administration of 40 mg of isocarboxazid daily to a normal male subject for a period of 42 days evoked remarkably long-lasting changes continuing long after medication had ceased. These alterations included an elevation of serotonin in the blood for 139 days and a decrease in urinary 5-HIAA for 92 days. In addition, urinary normetanephrine was above normal for 66 days while VMA was lowered for 48 days. Because these results were established in a single individual, it would be well to study this ReJerenres p . 238-240

234

SUMMARY

same problem on several persons. Schopbach's work not only revealed alterations in amine metabolism but also in behavior as a result of isocarboxazid. But Welch and Welch (this Symposium, p. 201) noted the reverse condition, namely that behavioral variations are reflected in monoamine metabolism. They showed that 7 days after the i.m. injections of labeled DOPA into mice the levels of radioactivity of the brains of the grouped animals were practically twice as high as in those of the isolated animals, a result attributed to the stressing effects caused by the presence of other animals. The influence of psychological factors is also seen in our observations of schizophrenic patients which discloses that rises in urinary adrenaline and noradrenaline levels were observed with increased anxiety and were not associated with psychotic activation unless the latter were accompanied by anxiety. On the other hand levels of urinary indoles correlated with the intensity of the schizophrenic symptoms, as increases of indoles accompanied aggravation of the psychotic symptomatologies (Himwich and Brune, 1961 ; Brune and Pscheidt, 1961 ; Brune and Himwich, 1962). Another way to approach the problem of the binding sites of brain biogenic amines is by actual visual examination by means of a light microscope and in a great forward step such a method has been developed by Falck (1962), who transforms amines into intensely fluorescent isoquinolizine derivatives by reacting dry frozen brain tissue with formaldehyde gas. He has thus been able to bring the study of brain biogenic amines to a finer focus than was possible by means of bio-assay or chemical analysis, and examine amounts of brain biogenic amines too small to be determined chemically or by bio-assay. Another great advantage of this method is that it discloses the intracellular locations of brain biogenic amines while the spatial relationships of the structures within the cell are maintained. He has thus demonstrated noradrenergic fibers in the hypothalamus which in general exhibits fine varicose fibers occurring in greatest concentrations in the preoptic region, supraoptic, paraventricular nuclei and the pariventricular nuclei of the hypothalamus, and concludes that these fibers represent the terminal parts of axons forming synaptic contacts. Evidence is, therefore, afforded for the idea that noradrenaline serves as a synaptic transmitter in the brain. With the aid of Carlsson et al. (1962) he also found that dopamine is present in the caudate nucleus, with dopamincrgic fibers probably localized in the neuropil. Falck has similarly demonstrated the presence of serotonergic fibers indicating that 5-HT is a transmitter in the brain. And now we have additional suggestive evidence by Heller and co-workers (this Symposium, p. 53) that the destruction of the medial forebrain bundle within the lateral hypothalamus produced a significant fall of brain serotonin levels as compared with normals while destruction of areas not related to the medial forebrain bundle did not result in significant decreases. Carlsson's work (this Symposium, p. 9) has shown similar diminutions of serotonin and noradrenaline caudal to the transection of the spinal cord of rabbit. In another portion of the central nervous system, the medial geniculate body of the cat (Utley, 1963) has found an important difference between the serotonin contents in neurons and glial cells. Employing the method of operative interference he has produced secondary degeneration of the neurons and could thus distinguish between the neurons and the glial cells which

235

SUMMARY

were left unchanged. By this differential method he showed that most of the serotonin was found in the glial cells. Quay (this Symposium, p. 61), using the spectrophotofluorometer, studied serotonin levels in the pineal gland of rat and Falck with his new method was able to clarify the sites of this monoamine when he reported that in rat and mouse pineal an intense yellowish fluorescence develops not only in the nerves as in other species but also in the parenchymal cells. Quay finds that the pineal is only moderately sensitive to reserpine and again Falck agrees, noting that reserpine reduces serotonin to about 1/2 of the control levels. He shows further that pineal nerves are readily depleted but the parenchymal cells are much more resistant, thus accounting for the partial retention of serotonin despite the use of reserpine. Finally, Quay finds a circadian cycle with serotonin levels lowest at night and highest approximately at mid-day, a physiological observation indicating that the time of day is an essential consideration in such studies. It is possible to probe even further into ultrastructure with the electron microscope than with light microscopy and Whittaker and De Robertis have combined the former with biochemical methods thus coming to the point where ultrastructure and function meet on common ground, a combination of methods which may lead to the actual discovery of the precise binding sites of the brain biogenic amines. As Whittaker has brought out in his discussion there is basic agreement between his observations and those ofDe Robertis. A nice correlation was observed in both laboratories when bound acetylcholine and the enzyme facilitating its formation, cholineacetylase, were observed in the same compartment while cholinesterase occurred in another subfraction. It is true, however, that there were some differences in the conclusions of the two workers when they used different procedures. In both laboratories homogenates of brain tissue were used to produce primary fractions by a series of differential centrifugations with increasing speeds. Table I1 presents the characteristic of a primary fraction, T A B L E I1 P R I M A R Y F R A C T I O N S F R O M B R A I N HOMOGENATE

Fraction

Cenfrijiugation

De Robertis

MIT

Differential

Whittaker

PZ

Differential

Ultrastructure

Myelin Mitochondria Nerve endings Myelin Mitochondria NEPs Microsomes

chiefly mitochondria1 in content, called MIT by De Robertis and PZ by Whittaker. These were produced under similar conditions, reveal similar ultrastructures and from them the subfractions are derived. For the results presented in Table 111 both De Robertis and Whittaker employed gradient centrifugation to subfractionate the References p . 238-240

236

SUMMARY

T A B L E 111 S U B F R A C T I O N S OF M I T O C H O N D R I A L F R A C T I O N

De Robertis

Whittaker

Subfraction

Centrifugation

Ultrastructure

C

Gradient

Nerve endings

D

Gradient

Nerve endings

B

Gradient

NEPs

Other contents

AChE AChA

ACh 5-HT Noncholinergic

AChE AChA Dopamine Histamine Substance P

ACh 5-HT

TABLE IV H Y P O S M O T I C L Y D E R I V E D S U B F R A C T I O N S FROM M I T O C H O N D R I A L F R A C T I O N

De Robertis

Whittaker

Subfraction

Centrifugation

MI

Differential

Mz

Differential

0

Gradient

D

Gradient

F

Gradient

H

Gradient

Ultrastructure

Myelin Mitochondria NE ghosts Synaptic vesicles Membranes No organized structure Synaptic vesicles NEP ghosts PS M NEP ghosts NEP damaged

Other contents

AChE MA 0

5-HT

AChA

ACh

AChA ACh AChE ACh 5-HT

mitochondria1 fractions. De Robertis’ layer C exhibits nerve endings which contain all three components of the acetylcholine system as well as 5-HT, while the 4th layer, D, presents nerve endings which are only non-cholinergic. Thus he finds a subfraction in which noncholinergic nerve endings can be separated from the cholinergic ones. Whittaker’s 2nd subfraction B contained both acetylcholine as well as 5-HT. In view of the fact that De Robertis separated his mitochondria1 fraction into 5 layers while Whittaker’s gradient was divided into 3, it is not improbable that Whittaker’s layer B contained the elements of De Robertis’ layers C and D. De Robertis (Table IV) derived his 3 hyposmotic subfractions by differential centrifugation of his primary MIT fraction. MI contains 5-HT while the acetylcholine system is present in M2. The Mz and M3 subfractions together correspond to the 7 layers obtained by density gradient centrifugation from Whittaker’s water treated primary PZWSfraction. Acetylcholine occurs in the 2nd layer, D, while the 6th layer, H, contains both acetylcholine and 5-HT. Thus again Whittaker does not separate 5-HT from acetylcholine. Whittaker has

SUMMARY

237

informed us (this Symposium, p. 90) that he is interested in exploring this difference between his work and De Robertis’. He is, therefore, going to analyze further the D layer containing only synaptic vesicles in order to determine whether or not he can separate the non-cholinergic vesicles from the cholinergic ones. In De Robertis’ figures we have seen that 5-HT is present in the primary microsomal fraction and in this regard it is well to recall the remarks of Potter and Axelrod (1962) that both 5-HT and noradrenaline are found in the microsomal fraction. In any event, the solution of the character of the amine occupying the non-cholinergic vesicles of the brain is a pressing problem. Finally, Koenig (this Symposium, p. 137) makes the unorthodox suggestion that biogenic amines are continued in lysosomes which he finds to be glycolipoprotein granules. According to Duve (1959) lysosomes contain acid phosphitase, cathepsin and a number of other potentially destructive hydrolytic enzymes. In partial support of his hypothesis, Koenig points out that Whittaker found acid phosphatase along with acetylcholine and serotonin in the B layer, and a glance at figure 3 reveals some of the other substances contained in that layer. In brief review, we have heard evidence suggesting not only central cholinergic synapses but also others of noradrenergic, dopaminergic and serotonergic character from Carlsson and Falck (this Symposium), the latter using his new methods of fluorescent microscopy. The experiments of De Robertis and Whittaker have revealed cholinergic and non-cholinergic vesicles in the synapses of the brain with indications that the binding sites of acetylcholine are contained within the cholinergic vesicles. But conviction for the truth of a conclusion is gained slowly as data accumulate, and let us see what additional evidence we have for these conceptions. The work on the central neurotransmitter function of acetylcholine is buttressed by a large amount of biochemical data on the cholineacetylase-acetylcholine-cholinesterase system. In support of the ideas of the Swedish investigators on noradrenergic and dopaminergic neurotransmission is the biochemical evidence on the aromatic amino acid decarboxylase in the formation of these neurohumors and the interrelationships between M A 0 and COMT in the termination of neurotransmission (Axelrold, Carlsson, Costa and Brodie, Pletscher, Gey and Kunz in this Symposium). In regard to the problem of noncholinergic neurotransmission, we would like to have more information on the neurohormonal contents of the non-cholinergic vesicles as well as additional data on the catabolic pathways of serotonin. It is known that when labelled adrenaline is administered 97-98% can be traced in the urine in the form of known metabolic products (Cohen et a/., 1962). Such a balance has not been attained with serotonin for the metabolic fate of at least 50% of this serotonin is still undisclosed after the administration of this serotonin creatine phosphate (Gessner et a]., 1960). The gap in our knowledge in regard to serotonin metabolism has been strikingly demonstrated by the experiments of Green and Sawyer (this Symposium, p. 150) when they found that exogenous serotonin can be catabolized rapidly at a time when M A 0 is completely inhibited. Thus still another goal has been set for us. In contrast, Giarman (this Symposium, p. 72) resolves a contradiction in the literature on the mechanism of action of reserpine i n depleting brain amines, and Sourkes (this Symposium, p. 186) completes the picture with a paper on disturbances of amine metabolism and disease. RefcrmceA p . 238-240

238

SUMMARY

The papers presented in this Symposium have demonstrated the increasing acceleration in the growth of our information on these basic problems. Each investigator who has taken part in this Symposium has added in his own way important data to enlarge and refine our information. But curiosity is eternal - we must look to the future not only for experiments to test our present viewpoints but to new vistas and from the participants in this Symposium much of that information will come. REFERENCES AMIN,A. H., CRAWFORD, T. B. B., AND GADDUM, J. H., (1954); The distribution of substance P and 5-hydroxytryptamine in the central nervous system of dog. J. Physiol., 126, 596-61 8. BARBEAU, A., MURPHY,C . F., AND SOURKES, T. L., (1961); Excretion of dopamine in diseases of basal ganglia. Science, 133, 1706-1707. BARBEAU, A., AND SOURKES, T. L., (1961); Some biochemical aspects of extrapyramidal diseases. Rev. canad. Biol., 20, 197-203. BARBEAU, A., SOURKES, T. L., AND MURPHY, G . F., (1962); Les catecholamines dans la maladie de Parkinson. Monoamine et Systerne nerveux central. J. de Ajuriaguerra, Editor. Paris. Masson (p. 247-262). K., (1928); Uber ein neues, auf das extrapyramidal-motorische System wirkendes Alkaloid BERINGER, (Banesterin). Nervenarzt, 1, 265-275. BERINGER, K., (1 929); Zur Banesterin- und Harminfrage. Nervenarzf, 2, 548-549. BERNHEIMER, H., BIRKMAYER, W., AND HORNYKIEWICZ, O., (1962); Verhalten der Monoamineoxydase im Gehirn des Menschen nach Therapie mit Monoaminoxydase Hemmern. W e n . klin. Wschr., 74, 558-559. BERNHEIMER, H., AND HORNYKIEWICZ, O., (1962); Das Verhalten einiger Enzyme im Gehirn normaler und Parkinson-kranker Menschen. Arch. exp. Pathol. Pharmakol., 243, 295. BERTLER, A., CARLSSON, A., AND ROSENGREN, E., (1956); Release by reserpine of catecholamines from rabbits’ hearts. Naturwissenschaften, 22, 1-2. BERTLER, A., AND ROSENGREN, E., (1959); On the distribution in brain of monoamines and of enzymes responsible for their formation. Experientia (Basel), 15, 382-388. BLASCHKO, H., (1939); The specific action of L-DOPA decarboxylase. J. Physiol., 96, 5OP-51P. BLASCHKO, H., (1950); Substrate specificity of amino acid decarboxylases. Biochim. biophys. Acra, 4, 130-1 37. BOGDANSKI, D. F., WEISSBACH, H., AND UDENFRIEND, S., ( I 958); Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharmacol. exp. Ther., 122, 182-194. R. G., AND SHORE,P. A., (1957); Possible interrelationships BRODIE,B. B., OLIN,J., KUNTZMAN, between release of brain norepinephrine and serotonin by reserpine. Science, 125, 1293-1294. BRODIE,B. B., AND SHORE,P. A., (1957); A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N. Y. Acad. Sci., 66, 631-642. BRODIE,B. B., SPECTOR, S., AND SHORE,P. A., (1959); Interaction of drugs with norepinephrine in the brain. Pharmacol. Rev., 11, 548-564. BRUNE,G . G., AND HIMWICH,H. E., (1962); Indole metabolites in schizophrenic patients. Arch.gen. Psychiat., 6, 324-328. BRUNE, G.G., AND PSCHEIDT, G.R., (1 961);Correlations between behavior and urinary excretion of indoleamines and catecholamines in schizophrenic patientsas affected by drugs. Fed. Proc., 20,889-893. CARLSSON, A., FALCK,B., AND HILLARP, N. A,, (1962); Cellular localization of brain monoamines. Acta physiol. scand., Suppl. 196, 56, 1-28. CARLSSON, A., AND LINDQVIST, M., (1962); In vivo decarboxylation of a-methyl-DOPA and u-methylm-tyrosine. Acta physiol. scand., 54, 87-94. CARLSSON, A., ROSENGREN, E., BERTLER, A., AND NILSSON,J., (1957); Effect of reserpine on the metabolism of catecholamines. Psychotropic Drugs. S. Garattini and V. Ghetti, Editors. Amsterdam Elsevier (p. 363-372). COHEN, R . A., BRIDGERS, W. F., AXELROD, J., WEIL-MALHERBE, H., LABROSSE, E. H., BUNNEY, W. E., CARDON, P. V. AND KETY,S. S., (1962); The metabolism of the catecholamines (clinical implications). Combined Clinical Staff Conference of the National Institutes of Health, Ann. intern. Med., 56, 960-987.

SUMMARY

239

COSTA,E., PSCHEIDT, G . R., VANMETER,W. G . , AND HIMWICH,H. E., (1960); Brain concentrations of biogenic amines and EEG patterns of rabbits. 1.Pharmacol. exp. Ther., 130, 81-88. DENGLER, H. J., MICHAELSON, I. A., SPIEGEL,I. E., AND TITUS,E., (1962); The uptake of labeled norepinephrine by isolated brain and other tissues of the cat. f n t . J . Neurophysiol., 1, 23-38. DUVE, C. DE, (1959); Histological distribution of brain gangliosides. Lysosomes as glycolipoprotein granules. Nature, 195, 782-784. EHRINGER, H., AND HORNYKIEWICZ, o., (1960); Verteihng von Noradrenalin und Dopamin (3Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. WJchr., 38, 1236-1239. FALCK,B., (1962); Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand., Suppl. 197, 56, 1-25. FOURTES,M. G . F., (1958); Generation, conduction and transmission of nerve impulses. Arch. ital. Biol., 96, 285-293. GANROT,P. D., ROSENGREN, E., AND GOTTFRIES, C. G . , (1962); Effect of iproniazid on monoamines and monoamine oxidase in human brain. Experientiu (Busel), 18, 260. GESSNER, P. K . , KHAIRALLAH, P. A., MCISAAC,W. M., A N D PAGE,I. H., (1960); The relationship between the metabolic fate and pharmacological actions of serotonin, bufotenin and psilocybin. J. Pharmacol. exp. Ther., 130, 126-133. GREEN,H., AND ERICKSON, R. W., (1962); Further studies with tranylcypromine (monoamine oxidase inhibitor) and its interaction with reserpine in rat brain. Arch. int. Pharmacodyn., 135, 407-425. GUROFF,G., A N D UDENFRIEND, S., (1964); The uptake of aromatic amino acids by brain of mature and newborn rats. The Developing Brain, Progress in Brain Research, Vol. 9. H. E. Himwich and W. A. Himwich, Editors. New York and Amsterdam, Elsevier. In the press. HILLARP,N. A., (1960); Different pools of catecholamines stored in the adrenal medulla. Acta physiol. scand., 50, 8-22. HIMWICH,H. E., AND BRUNE,G . G . , (1961); Correlations between amine metabolism and activity of psychosis in schizophrenic patients. Abstracfs Les Risumth Vortragsuusziige Los Temas. The Third Congress of Psychiatry, part 1, Montreal, June 4-10, pp. 124. HIMWICH,H.E., VANMETER,W. G . ,A N D OWENS,H. F., (1959); An EEG analysis of psychotomimetic drugs. Neuro-fsychopharmacology.P. B. Bradley, P. Deniker and C. Radouco-Thomas, Editors. New York. Elsevier. (pp. 329-333). HIMWICH, W. A., AND COSTA, E., (1960); Behavioral changes associated with changes in concentrations of brain serotonin. Fed. froc., 18, 838-845. HOLTZ,P., (1939); DOPA decarboxylase. Naturwissenschafren, 27, 724-725. HOLZBAUER, M., AND VOGT,M., (1956-57); Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat. J . Neurochem., 1, 8-1 1. HORNYKIEWICZ, O., (1962); Dopamin (3-hydroxytyramin) im Zentralnervensystem und seine Beziehung zum Parkinson-Syndrom des Menschen. Dtsch. med. Wschr., 87, 1807-1 8 10. MORPURGO, C., (1962); Influence of phenothiazine derivatives on the accumulation of brain amines induced by monoamine oxidase inhibitors. Biochem. Pharmacol., 11, 967-972. MUSCHOLL, E., AND VOGT, M., (1957); The action of reserpine on sympathetic ganglia. J. Physiol., 136, 7P. MUSCHOLL, E., AND VOGT,M., (1958); The action of reserpine on the peripheral sympathetic system. J . Physiol., 141, 132-155. PAASONEN, M. K . , AND VOGT,M., (1956); The effect of drugs on the amounts of substance P and 5-hydroxytryptamine in mammalian brain. J . Physiol., 131, 617-626. PLETSCHER, A,, AND GEY,K. F., (1960); Wirkung von Chlorpromazin auf pharmakologische Veranderungen des 5-Hydroxytryptamin und Noradrenalin-Gehaltes im Gehirn. Med. exp. (Basel), 2, 259-265. PLETSCHER, A., SHORE,P., AND BRODIE,B., (1955); Serotonin release as a possible mechanism of reserpine action. Science, 122, 374-375. POTTER,L. T., AND AXELROD,J., (1962); Intracellular localization of catecholamines in tissues of the rat. Nature (Lond.), 194, 581-582. RAPPORT,M. M., GREEN,A. A., AND PAGE,I. H., (1948); Crystalline serotonin. Science, 108, 329-330. SCHANBERG, S. M.. (1963); A study of the transport of 5-hydroxytryptophan and 5-hydroxytryptamine (serotonin) into brain. J. fharmacol. exp. Ther., 139, 191-200. SCHANBERG, S. M., AND GIARMAN, N. J., (1962); Drug-induced alterations in the sub-cellular distribution of 5-hydroxytryptamine in rat’s brain. Biochem. Pharmacol., 11, 187-194.

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SCHUMANN, H. J., AND PHILIPPU,A., (1962); The mechanism of catecholamine release by tyramine. Int. J . Neuropharmacol., 1, 179-182. SHORE, P. A., CARLSSON, A., AND BRODIE,B. B., (1956); Mechanism of serotonin-release by reserpine. Fed. Proc., 15, 483. SOURKES,T. L., (1954); Inhibition of dihydroxyphenylalanine decarboxylase by derivatkes of phenylalanine. Arch. Biochem. Biophys., 51, 444-456. TASAKI, I., (1953); Nervous Transmission. Springfield, Thomas. TWAROG, B. M., AND PAGE,I. H., (1953); Serotonin content of some mammalian tissues and urine. Amer. J. Physiol., 175, 157-161. UTLEY,J., (1963); y-Aminobutyric acid and 5-hydroxytryptamine concentrations in neurons and glial cells in the medial geniculate body of the cat. Biochem. Pharmacol., 12, 1228-1230. VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. 1. Physiol., 123, 451-481. WEIL-MALHERBE, H., AND BONE,A. D., (1957); Intracellular distribution of catecholamines in the brain. Nature (Lond.), 180, 1050-1051. ZELLER,E. A., BARSKY,J., BERMAN,E. R., AND FOUTS,J. R., (1952); Action of isonicotinic acid hydrazide and related compounds on enzymes involved in the autonomic nervous system. J . Pharmacol. exp. Ther., 106, 427428.

24 1

Author Index* Abboud, F. M., 180 Abood, L. G., 72, 130 Adam, H. M., 100 Albers, R. W., 128, 133 Albrecht, P., 62 Aldridge, W. H., 97, 128 Amin, A. H., 54, 226 A n d h , O., 11 Aprison, M. H., 128 Ashcroft, 0. W., 20 Augustinsson, K. B., 138 Ax, A., 202 Axelrod, J., 18, 45, 50, 81-89, 133, 162, 163, 174, 203, 237 Bain, S. A., 128 Barbeau, A., 17, 187, 188, 190, 193-195, 233 Barger, G., 6 Barrnett, R., 77, 139 Barsky, J., 84 Beaven, N., 173 Bellamy, D., 124, 128 Bennett, H. S., 113, 120 Bergsman, H., 196 Beringer, K., 193, 21 I , 233 Berlet, H. H., 211-214, 233 Bernheimer, H., 190, 194, 21 I , 233 Bertaccini, G., 56 Bertler, A., 17, 31, 32, 38, 133, 188, 192, 227, 232 Besendorf, H., 24 Birkrnayer, W., 17, 187,188, 192-194, 211, 233 Bischoff, F., 192 Bittner, J. J., 62 Blaschko, H., 1-8, 72, 93, 100, 188, 191, 226 Blessing, J. A., 10 Bloom, G., 32 Bodian, D., 93 Bogdanski, D. F., 53, 56, 74, 150-152, 160, 162-164, 194, 203, 232 Boigne, N., 3 Bone, A. D., 72, 132, 188, 191, 227 Born, G. V. R., 191 Boura, A. L. A., 179 Bowles, G. R., 76, 191, 194 Boyd, J. D., 1 Bradley, P. B., 91 Brady, J. V., 202 Braganca, B. M., 92

* Italics

Bridgers, W. F., 237 Brightman, M. W., 128 Brodie, B. B., 5, 11, 12, 15, 17, 18, 20, 39, 53, 54, 56, 73, 84, 128, 133, 151, 152, 164, 165, 168-185, 192, 194, 203, 226, 227 Brossi, A., 48 Brown, B., 202 Brown, G. L., 54, 175 Brune, G. G., 196, 234 Biilbring, E., 65, 192 Bull, C., 211-214, 233 Bunney, W. E., 237 Burgen, A. S. V., 126 Burke, J. C., 202, 203 Burn, J. H., 6, 83, 86, 176, 202, 203 Byer, S. S., 21 Byers, S. D., 202 Cannon, C., 87 Cannon, W. B., 34 Cardon, P. V., 237 Carlini, G., 77 Carlsson, A., 5 , 6, 9-27, 28, 35, 38, 39, 118, 132, 133, 164, 170, 188, 226,227, 229, 234 Case, J. D., 162 Cass, R., 181 Chanee, M. R. A., 202 Chavez-Lara, B., 187, 189, 193 Chipman, L. M., 126 Chrusciel, T. L., 6, 132, 188 Cilento, A., 65 Clark, C. T., 151, 162 Clark, W. G., 20 Clemente, C. D., 133 Cleugh, J., 100 Cohen, L. H., 5, 201 Cohen, R. A., 237 Connamacher, R. F., 9 Conrad, D., 202 Cook, L., 150, 161, 163 Cooper, J. R., 56, 163 Copp, F. C., 179 Corrodi, H., 18, 22, 28 Costa, E., 5, 11, 12, 15, 17, 18, 20, 53, 150, 162, 165,168-185,203,232 Coupland, R. E., 32 Cowan, F. F., 87 Craver, B. N., 202, 203

indicate the pages on which the paper of the author in these proceedings is printed.

242

AUTHOR INDEX

Crawford, T. B. B., 54, 226 Creveling, C. R., 18, 82, 178, 179 Crolette, M. E., 202 Crout, J. R., 18, 82 Cuenca, E., 183 Curtis, D. R., 91 Dahl, E., 33 Dale, H. H., 6 Davis, R., 91 Davison, A. N., 128, 133 Day, M. D., 181 DeDuve, C., 77, 93, 139, 237 Degkwitz, R., 188, 189 De Iralda, A. P., 95, I l l , 118, 120, 121, 123, 128-130, 133, 217 Del Castillo, J., 113 De Lores Arnaiz, G. R., 95, I I I , 217 Dengler, H. J., 50, 172, 228 Denny-Brown, D., 187, 189 Denton, R. L., 193 De Robertis, E., 95, 1 1 I , 118-136, 217 De Schaepdryver, A. F., 18 Di Carlo, R., 64 Di Carlo, V., 64-66 D’Iorio, A., 191 Dixon, W. E., 6 Doepfner, W., 150 Doshay, L. J., 19 3 Dowe, G., 99 Dragstedt, C . A., 84 Drain, D. J., 20, 177 Drujan, B. D., 151 Duchastel, Y . , 193 Eade, N. R., 191 Eckstein, J . W., 180 Ehringer, H., 17, 187, 192, 195,211,233 EhrlCn, I., 28 Eiduson, S., 175 Elliott, K. A., 192, 218 Elmadjian, F., 202 Erickson, K . W., 150, 151, 160, 161, 163, 165, 227 Erspamer, V., 4 Essner, E., 139 Everett, G. M., 176 Falck, B., 28-44, 65, 118, 192, 234 Fange, R., 138 Farini Duggan, H. J., 118, 123, 133 Farr, A. L., 216 Feldberg, W., 54, 93, 123, 124 Fellows, E. J., 150 Fernandez-Moran, H.. 1 13 Fila-Hrornadko, S., 18, 22 Finger, K . F., 24 Fink, G. B., 202

Fischer, E. H., 3 Fischer, G., 77, 193 Florey, E., 65 Folkow, B., 202 Fortier, A., 191 Foulkes, R. G., 189 Fourtes, M. G. F., 231 Fouts, J. R., 226 Frankenhaeuser, M., 203 Freed, S. C . , 202 Freedland, R. A., 56 Freedman, D. X., 72-80, 227 Friedman, M., 202 Frowein, R., 188, 189 Fuks, Z . , 171 Funkenstein, D. H., 202 Furchgott, R. F., 31 Gaddum, J. H., 54,90, 100,226 Gaines, 139 Gal. E. M., 56-60 Gamo, T., 188 Ganrot, P. O . , 194, 233 Gaskell, W. H., 1 Gerstenbrand, F., 188, 189 Gessa, G. L., 11, 12, 1.5, 53, 179-181, 184 Gessner, P. K., 237 Getler, E., 175 Gey, K. F., 24, 45-52, 151, 187, 229 Giarrnan, N. J., 53, 72-80, 132, 227 Gibson, W. C., 189 Giuditta, A., 95 Glasser, A., 4 Goldenberg, M., 204 Gomez, J., 95, 120, 121, 128, 129 Gonnard, P., 3 Goodall, McM., 54, 202 Gordon, E. K., 78, 82, 84 Gottfries, C. G., 194, 233 Gray, E. G., 90, 91, 95, 102-104, 121, 128, 139, 217 Gray, S. J., 188 Gray, W. D., 202, 203 Green, A. A., 226 Green, A. F., 179 Green, H., 150-167, 227 Grieserner, E. C., 84 Guillery, R. W., 54 Gunn, J. A., 202 Curd, M. R., 202 Guroff, G., 232 Guth, P. S., 187 Hagen, J. M., 6, 191 Hagen, P., 1, 5, 6, 132 Haggendal, J., 10, 12, 17, 20, 33 Halberg, F., 62 Halevy, A., 61, 63

AUTHOR INDEX

Hamill, P., 6 Hanzon, V., 128 Hartree, E. F., 5 Harvey, J. A., 23, 53-55 Hassert, G. L., Jr, 202, 203 Hattori, T., 57 Heath, J. D., 32 Hebb, C. O., 72, 93, 123, 126-128 Heinzelman, L., 162 Heller, A., 23, 53-55 Hellman, B., 31 Heneaque, P., 5 Hertting, G., 45, 50, 82-86, 173, 174 Hess, S. M., 9, 150 High, J. T., 202, 203 Hill. R. T.. 202. 203 Hillarp, N:-A.,’9, 13, 20, 28, 31, 32, 35, 39, 72, 118. 132. 172. 192.228.234 Himwich, H. E:, 150, 162, 196, 209, 226-240 Himwich, W. A., 150, 162, 232 Hiroshi, Y., 82 Hirsch, C. W., 20, 53, 165, 176, 178, 179 Hobbs, R., 202, 203 Hodson, H. F., 179 Holm, R., 202, 203 Holton, P., 100 Holtz, P., 3, 4, 226 Holzbauer, M., 85, 227 Horita, A., 182 Horlington, M., 20, 177 Horne, R. W., 103, 104 Hornykiewicz, O., 17, 187, 190, 195, 211, 233 Hughes, R. C., 3, 170 Hullin. R. P., 128 Ikawa, M., 3 Inouye, A., 130 Inscoe, P. K., 18 Jasmin, G., 188 Jenkins, W. T., 3 Jibril, A., 138, 139 Johnels, A., 138 Johnson, M. K., 93, 97, 105, 109, 128 Kahimoto, Y., 188 Kakefuda, T., 215-218 Kako, K., 191 Kappers, J. A., 32 Kataoka, K., 130 Katz, B., 113 Keilin, D., 5 Kelly, A. R., 207-210 Kelly, J. W., 32 Kent, A. B., 3 Kety, S. S., 237 Khairallah, P. A., 237 Kibler, R. F., 195

243

Killam, E. K., 176 King, S. H., 202 Kirkland, R. J., 107, 109, 114 Kirshner, N., 13, 18, 82, 188 Koch, J. P., 188 Koenig, H., 137-141 Koizumi, K., 91 Kopin, I. J., 45, 78, 82-84, 86, 173 Koppanyi, T., 87 Krebs, E. G., 3 Krnjevid, K., 91 Kuhlenkampff, C., 188, 189 Kuntzman, R., 11, 12, 15, 20, 53, 84, 133, 152, 164, 165, 176, 178-181 Kunz, E., 45-52 La Brosse, E. H., 237 Lagerstedt, S., 72 Langemann, H., 4 Laroche, M. J., 82 Larson, R. E., 202 Lasagna, L., 202, 203 Laverty, R., 99, 102 Layne, D. S., 151 Lazare, R., 20, 177 Leach, E., 100 Leduc, J., 191 Lee, F. L., 132 Lee, W. C., 138, 162 Leiby, C. M., 9 Lerner, A. B., 162 Lessin, A. W., 50 Lin, R. C. Y., 192 Lindqvist, M., 6, 10-12, 15, 17, 18, 20, 22 , 33, 164, 188, 229 Lishajko, F., 32 Lissak, K., 54 Loewi, O., 123 Long, R. F., 50 Lovenberg, W. M., 5, 177 Lovry, 0. H., 216 Ludden, C. T., 10 Luduena, F. P., 202 Lukaszewski, J. S., 207-210 Lundborg, P., 13 Maary, S., 195 MacIntosh, F. C., 54 MacLean, P. D., 53 Maengwyn-Davies, G. D., 87 Magnusson, T., 6, 11, 15, 20, 22, 23, 164, 188 Maickel, R. P., 38 Malrnfors, T., 43 Mangan, G., Jr, 202 Mann, P. J. G., 92 Mannarino, E., 82 Marrazzi, A. S., 91 Marshall, F. D., Jr, 56-60

244

AUTHOR INDEX

Martin, J. P., 187 Maruo, B., 57 Masami, M. 82 Mason, J., 202 Matsumoto, C., 182 McCann, W., 202, 203 McCarty, L. P., 138 McGeer, E. G., 189, 191 McGeer, P. L., 191 McIsaac, W. M., 152, 237 Mead, J. A. R., 84 133 Melcer, I., 56, 163 Melrnon K . 175 Meltzer, H. Y., 77 Metzler, D. E., 3 Michaelson, I. A., 74, 75, 99-102, 107, 109, 113, 114, 130, 172, 228 Mitchell, J. F., 90, 100 Mohs, U., 188, 189 Montagu, K. A., 188 Montanari, R., 173 Moore, R. Y., 23, 53-55 Moorhead, M., 33 Mori, W., 162 Morpurgo, C., 230 Murphy, G. F., 17, 151, 187-190, 193, 195, 233 Muscholl, E., 227 Nachmansohn, D., 64 Nashold, B. S., Jr, 82 Nathan, P., 128 Nguyen-Chi, J. P., 3 Nguyen-Philippon, L., 3 Nilson, B., 72 Nilsson, J., 38, 227 Norlander, O., 192 Novikoff, A. B., 139 Nyman, M., 100 O h , J. S., 151 Ono, S., 132 Osterberg, A. C., 202, 203 Ostlund, E., 138 Owens, H. F., 232 Owman, Ch., 32, 33 Ozaki, M., 9 Paassonen, M. K., 53, 73, 226 Page, I. H., 152, 226, 237 Palm, D., 3 Pasini, A., 4 Pateisky, K., 187, 188 Patrick, R. W., 45, 50, 83, 85 Pellegrino de Iraldo, A., 118, 123, 133 Pereda, S. A., 180 Perkins, M., 31 Petrushka, E., 95 Philippu, A., 6, 226

Phillis, J. W., 91 Piala, J. W., 202, 203 Pineas, H., 193 Pletscher, A., 24, 45-52, 56, 74, 151, 187, 226, 229 Poll, H., 1 Porter, C. C., 9, 10, 21 Posner, H. S., 76, 191, 194 Potter, L. T., 33, 45, 82-86, 237 Poulter, G. A., 20, 177 Prockop, D., 194 Pscheidt, G. R., 196, 232, 234 Purkhold, A., 54 Quastel, J. H., 92 Quay, W. B., 61-63 Ramirez, M. P., 193 Rand, M. J., 6, 83, 86, 176, 181 Randall, R. J., 216 Rapport, M. M., 226 Raugh, C. E., 202, 203 Redfield, B. G., 171 Rehn, N. O., 31 Renson, J., 56 Resnick, R. H., 188 Reyi, I., 82 Richardson, K. G., 33, 83 Richter, D., 128 Riley, J. F., 192 Rinaldi, F., 150 Rioch, D., 202 Roberts, E., 215-218 ROOS,B.-E., 20-22 Rosebrough, N. J. 216 Rosell, S., 85 Rosengren, E., 17, 22, 23, 31, 32, 38, 133, 188, 192, 194,227,232,233 Rosenman, R. H., 202 Ross, C. A., 10 Rothballer, A. B., 203 Ruffel, G. K., 179 Rustige, E., 193 Ryall, R. W., 192 Ryd, G., 85 Saint-Cyr, S., 193 Salganicoff, L., 95, 1 11, 120, 121, 123, 128-130, 133,217 Sano, I., 188, 215 Sawyer, J. L., 150-167 Schanberg, S. M., 72-80, 227 Schanker, L., 272 Schopbach, R. R., 207-210 Schiimann, H. J., 6, 31, 32,226 Schuster, P., 193 Scott, D., 139 Seiden, O., 15

AUTHOR INDEX

Senoh, S., 18 Sharman, D. F., 20, 21, 99, 102 Shaw, E., 54 Shideman, F. E., 138 Shinagawa, J., 130 Shore, P. A., 13, 53, 54, 73, 84, 133, 151, 164, 170, 174, 176, 192, 226, 227 Sidman, R. L., 31 Silver, S. L., 73, 126 Sim, M. F., 179 Sjoerdsma, A., 133, 152, 175 Smallman, B. N., 93, 128 Smith, M. J., 100 Smith, S. E., 5, 21, 151 Smith, T. E., 133, 152 Smythies, J. R., 196 Snell, E. E., 3-5 Snyder, E. R., 3 Sommer, A., 1 Sourkes, T., 4, 5, 17, 151, 186-200, 226, 233 Spector, S., 84, 133, 174-176, 194, 227 Spiegel, H. E., 50, 172, 228 Spriggs, T. L. B., 181 Stacey, R. S., 50 Stedman, E., 92 Steiner, F. A., 24 Stevenson, T. D., 133, 152 Stone, C. A., 10 Stoppani, G., 4 Strom-Olsen, R., 196 Szerb, J. C., 113 Tainter, M. L., 202 Takahashi, H., 57, 162 Takesada, M., 188 Taniguchi, K., 188 Tasaki, L., 23 I Tedeschi, D. H., 150 Tedeschi, R. E., 150 Tennenbaum, M., 92 Thieme, G., 28 Timberlake, W. H., 188 Titus, D. C., 21, 50, 171, 172, 228 Toman, J. E. P., 176 Tomchick, R. 81-83, 85, 86, 152, 164, 165, 203 Torack, R. M., 139 Torp, A., 28, 32, I92 Torres, A., 192 Toschi, G., 97, 128 Totaro, J. A., 9, 10, 21 Trano, Y, 5 Tuller, B. F., 202 Twarog, B. M., 226 Udenfriend, S., 5, 9, 1 I, 18, 53, 56, 74, 82, 133, 150-152, 160, 162-164, 177, 178,194, 203,232

245

Utley, J., 234 Vaisfeld, I. L., 188 Van Der Schoot, J. B., 178 Van Gelder, M. M., 192, 218 Van Meter, W. G., 232 Varon, S., 215-218 Visscher, M. B., 62 Vogt, M., 73, 82, 85, 118, 121, 132, 203, 226, 227 Von Euler, U. S., 32-34, 54, 132, 173, 175, 202, 203 Von Mecklenburg, C., 33 Wadzinsky, I. M., 56 Waisman, H. A., 56 Wajda, I., 65 Walaszek, E. J., 72, 130 Waldeck, B., 13, 17, 18, 188 Walton, E., 179 Watkins, J. C., 91 Watson, L. S., 21 Wegmann, A., 191 Weil-Malherbe, H., 72, 76, 81-83, 128, 173, 188, 190, 191, 194, 196, 203, 227, 237 Weiner, N., 31, 132, 133 Weinstein, H., 215-218 Weissbach, H., 5, 53, 56, 150-152, 160, 162-164, 171, 177, 194, 203, 209, 232 Weissman, A., 24 Welch, A. D., 1, 72, I91 Welch, A. M., 201-206 Welch, B. L., 201-206 Wells, J. A., 84 Welsh, H. J., 33 Wengen, H. C., 10 Werdinius, B., 20-22 Westerman, E. O., 38 Whitby, L. G., 45, 50, 81, 83, 85, 86, 173 Whittaker, V. P., 72,74,75, 83, 90-117, 121, 128, 130, 132, 139, 192, 217 Williams, C. M., 195 Wilson, E. M., 5 Witkop, B., 18 Wolfe, D. E., 33, 83, 128 Wolstencroft, J. H., 91 Woolley, D. W., 54, 192 Wright, W., 162 Yuwiler, A., 175 Zaltzman-Nirenberg, P., 1 1 Zeller, E. A,, 84, 226 Zieher, L. M., 120, 123, 128-133 Zielinska, M., 189 Zodrow, W. W., 138 Zylberger, R., 133

246

Subject Index Acetylcholine, bound, nature of, 92-97, 113 content in synaptic vesicles, 11 3, I30 distribution, 123-128, 147 isolation, from nerve ending, 128, 129 localization in synaptic complex, 129, 130 mechanism of release, 93 reaction, enzyme cholineacetylase, 92 synthesis, 1 13 Acetylcholinesterase, bound to nerve ending membrane, 130 distribution in microsome fraction, 128,147, 148 Adenosine triphosphate, distribution, 100, 145 Adrenal medulla, catecholamine concentration, 201-206 granules, relation to Parkinson syndrome, 191,196 storage, 13 hormones, 1 metabolic products, catecholamine, 149 mitochondria, concentration of ATP, 69, 70, 72 Adrenaline (see also Catecholamines) pathways of formation, 1 release, influence of drugs, 81, 83 uptake from blood, 81 Amino acid decarboxylase, relation to Parkinson syndrome, 190 Amino acids, decarboxylation, 5, 7, 71 a-methyl amino acids, inhibition, 5 a-methylation, 4 Amphetamine, effect on, peripheral sympathetic nerve, norepinephrine, 176 release of norepinephrine, 182 toxicity dose, sensitivity, 202 Antidepressant drug, effect on catecholamine concentration, 84-87 Basal ganglia, catecholamine concentration in Parkinson syndrome, 188, 189 depletion of dopamine, 17, 21 1 occurrence of Huntington’s chorea, 194, 195 Behavior, concentration, decline in serotonin, 219, 231 depression, depletion of monoamine stores, 24

effect, brain monoamine, 9, 234 effect, isocarboxazid treated, 21 3 relation to brain GABA content, 220 Benzoquinolizine, depletion action on monoamines, 9, 229 inhibition of adrenalin storage, 179 Blood-brain barrier, accumulation of serotonin, iproniazid, 221 diffusion of 5-HT, 5-HTP, 160, 162, 163 epinephrine, hypothalamus, 203 penetration of amines, 45 Brain, accumulation of exogenous monoamines, 45-52 concentration, 5-HTP, 152 monoamines, 9-27,227 reserpine, effect on monoamines, 12, 14 con tent, GABA, mouse, 215-218 norepinephrine, after blockage of inhibitor, I76 distiribution, 5-HT in subcellular fraction, 74 inhibitor of monoamines, 133 norepinephrine, 132, 133 serotonin, medial forebrain bundle, rat, 53-55, 61-63 enzymes, monoamine synthesis, 20-22 granules, storage of catecholamines, 191 lesions, localization of monoamines, 22, 23 particles, containing serotonin, 74 response to 5-HT injection, 150-167 Calcium, liberator of amines, 70 Catecholamines (see also Adrenaline, Epinephrine, Noradrenaline and Norepinephrine) binding sites, lysosomes, 137, 141 brain, localization of hypothalamus, 14 cellular localization, 28, 44 concentration in mitochondria, 72 effect of drugs on depletion, 30 effect of metabolism on storage, 45 excretion pattern in patients, 196 influence of reserpine on storage, 13 inhibitory effects, 24 interaction with tissue, historical, 1-8 intracellular and extracellular, reserpine, 20 intraneuronal localization, 81 localization in chromaffin cells, 71

SUBJECT INDEX

metabolism, correlation to Parkinson syndrome, 219 emotional expression, 196 in diseases, 186-200 Marplan effect on, 207-210 pharmacological effect, 220 pools, differences in biochemical properties, 164 precursors, 15-20, 24 relation to enzyme sites, 2 release from storage in Parkinson syndrome, 192 releasers, 48 subcellular localization, 82 urinary content in patients, 196 Catechol-0-methyltransferase, activity in rat brain, 81-83, 87 influence on circulating norepinephrine, 16-22 inhibitors, 18, 20, 22, 82, 229 side effects, 18 Caudate nucleus, concentration of dopamine, 42 Cell fractionation, subcellular particles, 1 18 Cerebellar cortex, homogenate, nerve ending particles, 145-147 Cerebral cortex, concentration of gangliosides, 138 distribution of cholinesterase, 124-128 fluorescent structures, 3 5 4 3 isolation nerve endings, 120-122 lesions, effect on serotonin, 53-56 monoamines as transmitter, 24 penetration of amino acids, 6 storage sites of biogenic amines, 90-1 17 Chlorpromazine, action on reticular formation, 225 effect of hypothermia, 50, 51 effect on endogenous amines, 48, 49, 229-230 influence of tryptamine uptake, 50 Choline acetylase, distribution in homogenate, 105-1 15 localization in mitochondria, 113, 128 Cholinergic ending, identification with electron microscopy, 90 Cholinesterase, localization in microsomes, 113 Choroid plexus, storage of monoamines, 70 Chromaffin cells, concentration of catecholamines, 7 I hormonal content, 1 origin from neural crest, 1 DOPA, inhibitory effect, 20 radioactivity, concentration in brain, 46 relation to Parkinson syndrome, 190

247

DOPA decarboxylase, inhibition, a-methylated amino acids, 5 monoamine decline, 20 prevention of norepinephrine synthesis, 177 relation to Parkinson syndrome, 190, 233 site of active, 2 Dopamine, distribution, basal ganglia, 132, 133 metabolic activity, 201-206 urinary output, Parkinson syndrome, 188 Wilson’s disease, 194, 196, 197 EEG, relation to starvation, cat, 223-225 Emotion, correlation to adrenal medulla secretion, 202 correlation to hypothalamic centers, 202 expression, catecholamine metabolism, 196 Enzyme, inhibitory effect, 17-22 sites of action, 2 Epinephrine (see also Catecholamines) penetration into blood-barrier, 203 transmitter function, sympathetic system, 33 urinary output, Wilson’s disease, 194

Fluorescence, difference between primary and secondary amines, 30 method of localization in CNS, 31, 35-43 presence of catecholamines, 28, 30 GABA, binding sites, 215 distribution, mouse brain, 215-21 8 Ganglia, decarboxylase activity in sympathetic, 70 Guanethidine, effect on norepinephrine storage, 180-183 Histamine, concentration in mast cells, 100 distribution in brain, 99 Homogenation, biogenic amines in CNS, 121 cortex, human, 128 isolation of 5-HT, 151-167 isolation of storage sites, amines, 90-1 17 mitochondria1 fraction, 128 5-HT (5-Hydroxytryptamine), (see also Serotonin) bound and unbound forms, 73-75 distribution of mitochondria, microsome fraction, 130-132, 147 endogenous and exogenous pools, I65 function as transmitter in brain, 9 intracellular distribution, 157

248

SUBJECT INDEX

metabolic activity, 162 occurrence in synaptic vesicles, 128 presence in soluble cytoplasm and particular material, 158 releaser of reserpine, 33 storage in pineal body, 33 uptake, brain slices, 221 5-HT decarboxylase, subcellular distribution, I32 5-HTP (5-Hydroxytryptophan), concentration in CNS, 157, 158 effect of administration, 157, 160 pharmacological action, 222 precursors to 5-HT, 150-167 rate of penetration in CNS, 160 synthesis in brain, 56 5-HTP decarboxylase, activity in homogenates, I52 Huntington’s chorea, metabolic parameters, 194, 195 5-Hydroxytryptamine (see 5-HT) 5-Hydroxytryptophan (see 5-HTP) Hypothalamus, catecholamines, Parkinson syndrome, 188, 192, 193 concentration of monoamines, 3 5 4 3 , 82 indication by fluorescence, 42 penetration into blood-barrier, 203 LSD, effects on distribution of 5-HT, 75 Lysosomes, binding sites, biogenic amines, 137-141 content, acid phosphatase, 149 granules, aminc-carrying, 147 Marplan, effect on catecholamine metabolism, 207-210 Mast cell, content of histamine, 100 storage of histamine, Parkinson syndrome, 192 Membrane, localization of acetylcholinesterase, 130-1 33 Metabolic pathway, formation of adrenaline, I hydroxylation of tryptophan, 56-60 inhibition of 5-HT, 162 methylation of norepinephrine, 82 Metaraminol, intermediary action, 10 presence in heart tissue, 68 Microsomes, presence of acetylcholinesterase, 129, 147 Mitochondria, content of synaptic vesicles, 142-145 distribution of 5-HT, 130-1 32 fraction containing nerve endings, 128 presence of bound norepinephrine, 133

Monoamine oxidase, activity in homogenate, I52 distribution in mitochondria1 fraction, 133 effect of inhibition on S H T , 160, 161 function as norepinephrine pump, 183, 184 inhibitor, effect on blood pressure, 183 effect on peripheral nerve, 64-66 isocarboxazid, behavior, 21 2-21 4, 233 response to iproniazid, 48, 51, 75, 153, 154, 183, 226 relation to Parkinson syndrome, 193, 194 Monoamines, cellulai localization, 28-43 in area postrema, 70 in caudate nucleus, 42 in hypothalamus, 35 in mast cells, 30, 32 in median eminence, 39 in neurohypophysis, 39 in neurons, 30 in pancreatic ,!?-cells, 3 I in paraventricular muscle, 35 in peripheral axons, 31 in periventricular muscle, 35 in pineal body, 32 in platelets, 30 in preoptic region, 35 in retina, 43 in spinal cord, 42 in supra-optic muscles, 35 Motoneuron, effect of 5-HTP, 15 Nerve-ending particles, content of synaptic vesicles, 123 distribution of acetylcholine system, 128, 129 distribution, brain, 104-1 15, 147 formation and properties, 101-103 from caudate nucleus, 102 from cortex granule fibers, 102 isolation, from CNS, 120-122 presence in homogenate fraction, 95, 97, 145-147 Neurohypophysis, fluorescence effect, mouse, 39, 40 Neuron, amine release, Parkinson syndrome, 192 catecholamine secretion, 1 hormone activation, end organ, 6 influence of sympathetic denervation, 86 presence of fluorescence, 34 presence of sympatholytic agent, 179 relation to chromaffin tissue, 1 sympathetic nerve ending, norepinephrine uptake, 173, 229 structural component, 174, 175

SUBJECT INDEX

Neurotransmitter, as chemical transducer, sympathetic nerve ending, 168-186 drug interference, 90 proof for acetylcholine, 90 Noradrenaline (see also Catecholamines) distribution, 132 metabolic pathways, 84 presence in granular vesicles, I33 system of release, 83 Norepinephrine (see also Catecholamines) accumulation in nerve ending, sympathetic stimuli, 175 action of benzoquinolizine, 9 adrenergic blocking agent, 176 biosynthesis, I77 distribution in CNS, 132-133 function as chemical trdnsducer, 168-1 86 localization after brain lesion, 23 pool, mobile and reserve, 172 presence in nerve ending, 169, I7 I synthesis, in heart rate, 174 turnover rate in hyperthyroidism, 148 uptake and release of tryptamine, 83 Parkinson syndrome, catecholamine metabolic disturbance, 187, 189-194, 196, 197 caused by reserpine, 17, 233 dopamine depletion, basal ganglia, 21 I relation to amine metabolism, 219 Phosphorylase, detection of activity sites, 3 Pineal body, content of, 149 5-HT and drug depletion, 33, 61-63 presence of fluorescence, 32, 82 release of reserpine by 5-HT, 32 storage of monoamines, 32, 82 Pseudo cholinesterase, presence in brain, 128 Psychoses, effect in sympathoadrenal system, 196 reserpine administration, schizophrenic, 21 1, 234 Radioactivity, distribution, 46, 47 of adrenaline, 8 I of amines, 45 of amino acids, 45 of noradrenaline, 8 1 of phenolcarboxylic acids, 45 Reserpine, action on platelets, I 3 action on serotonin, 72-80 blockage of 5-HT active transport, 170, 172 depletion of norepinephrine, 9, 10, 84

249

effect on monoamine storage, 168, 176 effect on unbound 5-HT, 75, 76 fluorescence in hypothalamus, 42 inhibition of monoamine uptake, 67 inhibition of norepinephrine pump, 179 liberatory effect of catecholamine, 23 relation to level of brain monoamines, 12 relation to metaraminol, 68 relation to Parkinson syndrome, 187, 188,194 Reticular formation, action of chlorpromazine, 225 Sedation, function of reserpine, 12 Serotonin (see also 5-Hydroxytryptamine) active transport, 220 behavioral correlates, 219 distribution in brain, 53-55, 61-63, 226 effect on peripheral nerve, 64-66 fall of CNS lesions, 53 influence on 5-HTP, 221 in oestrous fluctuation, 61 in pineal body, 61 metabolic mechanism, 237 platelet, uptake and release, 169 relation to circadian rhythm, 62, 63, 235 reserpine, effect on brain distribution, 72-80 subcellular distribution, 72 Spinal cord, presence of fluorescence, 42 Synapse, presence of chemical transducer, 168-1 86 presynaptic, mitochondria content of, 144, 145 structure of vesicles, 123 presence of 5-HT, 128 Transmission mechanism, blockage by reserpine, 17 effect of norepinephrine, 10 Transmitter, adrenaline in heart tissue, 33 monoamine precursors, I6 relation to norepinephrine, 234 storage, 129 structure of excitatory, 16 substitutes for norepinephrine, 1 I synthetic activity, 129 Tryptamine, relation to chlorpromazine uptake, 50, 51 release of norepinephrine, 83 Tryptophan, metabolic pathways, pigeon brain, to 5-HTP, 59 transport mechanisms, 232 Vesicles, concentration of mitochondria, 69, 70 isolation procedure, 104-1 15, 123, 142

250

SUBJECT INDEX

Vesicles (continuation) metabolic pathway, 145-147 monoamine storage, 23, 148, 237 reserpine blockage, 24, 228

presence of acetylcholine in synaptic, 130 Wilson’s disease, pathologic products in urine, 194, 197

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