International Review of Neurobiology, Volume 20

INTERNATIONAL REVIEW OF Neurobiology VOLUME 20 Associate Editors W. R. ADEY H . J. EYSENCK D. BOVET C. HEBB JOSE...

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 20

Associate Editors W. R. ADEY

H . J. EYSENCK

D. BOVET

C. HEBB

JOSE

DELGADO

S. KETY

SIR JOHN ECCLES

A. LAJTHA

0. ZANGWILL

Consultant Editors V. AMASSIAN

K. KILLAM

R. BALDESSARINI

C. KORNETSKY

F. BLOOM

B. A . LEBEDEV

P. B. BRADLEY

V. LONGO

0. CREUTZFELDT

P. MANDELL

J. ELKES

H . OSMOND

K. FUXE

S. H . SNYDER

B. HOLMSTEDT

S. SZARA

P. JANSSEN

W. GREYWALTER

INTERNATIONAL REVIEW OF

Neurobiology Edited by JOHN R. SMYTHIES Department of Psychiatry and the Neurosciences Program University of Alabama Medical Center Birmingham, Alabama

RONALD J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birmingham, Alabama

VOLUME 20

1977

ACADEMIC PRESS

New York

San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1977, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

11 1 Fifth Avenue, New York, New York 10003

United Kiirgdom Edition piiblislied by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Koad, London NWI

LIBRARY OF CONGRESS CATALOG CARD NUMBER:59-13822 ISBN 0-12-366820-4 PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTRIBVTORS ..............................................................

ix

Functional Metabolism of Brain Phospholipids

G. BRIAN ANSELLA N D SHEILA SPANNER I . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

11. Structure and Intracellular Distribution of Brain l'hospholipids

111. Origin and iMetabolism of' Brain Phospholipids . . . . IV. Relationship between Phospholipid Composition of Membranes .............. and Brain Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Phospholipids and Synaptic Transmission ...................... .... References . . . . . . . . . . . ......................................

1 2 6

]I 17 26

Isolation a n d Purification of the Nicotine Acetylcholine Receptor a n d Its Functional Reconstitution into a M e m b r a n e Environment

MICHAELs. BRILEYAND JEAN-PIERRE CHANGELIX I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ 11. Isolation and I'iirification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 111. Reconstitution . . . ..................................................

I\'.

Conclusion. .......................... ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31 32 .+I) .iX

3)

Biochemical Aspects of Neurotransmission in the Developing Brain JOSEPH

T. COYLE

I . Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.5

11. Prenatal Development of Central Catecholaminergic Neurons . . . . . . . . . . . . . I I I . Postnatal Development of the Nigrostriatal Circuit .......................

67

IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97 99

X2

The Formation, Degradation, a n d Function of Cyclic Nucleotides i n the Nervous System JOHN

W. DALY

................................... . . . . . . . . . . . . . . . . 105

I. Introduction

................................... III. Cyclic AMP ........................................................... 111. CyclicGMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cyclic Nucleotides and the Function 01' the Central and Peripheral Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. S u m m a r y . . . . . . . . . ......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y

109 13X

1.44 1.x 156

CONTENTS

vi

Fluctuation Analysis in Neurobiology

LOLllSJ. DEFELlCE

.................. ................... ........................ .................. .................. ............... I l l . Results . . . . . . . . . . . . . . ..................... ................... IV. Summary .. . . . . . ........................................ References ............................. I. Introduction . .

11. Methods

169 175 183 206 206

Lipotropin and the Central Nervous System

w. H . G 1 S P E N . J . M. V A N REE,A N D D. DE W l E D I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . 2 09

11. ACTH 4-10 . ............................ 211 . . . . . . . . 230 111. P-MSH . . . . . . .................................. ............................ 232 IV. P-Lipotropin 61–91 ..................................

V. Concluding Remarks .......................... References .............................

. . . . . . . . . . 239

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

242

Tissue Fractionation in Neurobiochernistry: An Analytical Tool or a Source of Artifacts

PIERRE LADURON I. 11. Ill. IV.

Introduction .......................................................... Analytical Approach to Tissue Fractionation in the Brain . . . . . . . . . . . . . . . . . Interpretation of Tissue Fractionation Studies ............................ Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25 1 253 269 280 281

Choline Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization JEAN

ROSSIER

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

I. Introduction 11. History

III. Assay IV. Distribution of ChAc in Nonneuronal Tissue'. . . . . . . . . . . . . . . . . . V. Distribution of ChAc in Neuronal Tissue . . . . . ...................... V I . Purification of ChAc . . . . . VII. Biophysical Studies . . . . . . . . . . . . . . . . . ......................... VIII. Mechanism of Action . . . . . . . . . . . . . . . . . . . .......................... ............................. IX. Axonal Transport of ChAc . X. Immunology.. ......................... ...................... XI. Localization at the Cellular Level ..................... ............ XII. Subcellular Localization of ChAc ...............................

284 284 287 29 1 294 296 303 304 312 314 318 324

CONTENTS

vii

XI11 . Choline Transport and ChAc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV . The Role of CI- in the Regulation of Ach Synthesis by ChAc . . . . . . . . . . . . . . X V . Pleiotropic Effect of Nerve Impulses on Ach Synthesis .................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

327 329 330 331

SCBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS O F PREVIOLIS VOLUMES.............................................

339 345

This Page Intentionally Left Blank

C O NTRl B UTO RS Numbers in parentheses indicate the pages on which

the iiiitliors'

contributions begin

G. BRIANANSELL, Department of Pharmacology, Unirvrsity of Birmingliam, Medicul School, Birmingham, England ( 1 )

MICHAELS. BRILEY,Neurobiologie Moliculuire, Institut Pasteur, Puris, France (31) JEAN-PIERRE C H A N G E U X , Neurobiologze M o l h l a i r e , Insthit Pasteur, Paris, Frtim-e ( 3 1 ) T. COYLE, Departments of Pharmacology and Expm'mental Ttrera/writics and Psychiatry and Belimiorul Sciences, The Johns Hopkins Unizwrsity School of Medicine, Baltimore, Maryland (65)

JOSEPH

JOHN W. DALY,Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Disetises, National Institutes of Health, Bethesda, Mnryland ( 105) LOUISJ. DEFELICE, Department of Aniitomy, Emory Uniuersity, Atlanta, Georgza (169)

D. DE WIED,Rud0y'Mugnu.s Institute fM- Phamiacology, Medical Facul(y, Uniriersity of Utrecht, Utrecht, Tlie Netherlaiids (209) W. H . G I S P E N , Rudolf Magnus Institute ,for Pharmacology, Medical Faculty, Uniuersity of Utreckt, Utrecht, TIE Netherlands (209)

PIERRELADURON, Departvnmt of Biochemical Plinrmacology, Jan.s.sen Phcrrmuceutica, Beerse, BelgEu,m ( 2 51) JEANROSSIER,Tlie Salk Institute for Biological Studies, Ln Jolla, California (283) SHE1 LA SPANNER, Department of Pharmacology, I/ nii~ersity of Birmingham, Medical School, Birmingham, E n g l m d ( 1

J. M.

VAN REE, Rudolf Magnus h t i t u t e for Phnrmacology, Medical Faulty, Uni-r)ersityof Utrecht, Utrecht, The Netiierlands (209)

iX


FUNCTIONAL METABOLISM OF BRAIN PHOSPHOLIPIDS By G. Brian Ansell and Sheila Spanner

Department of Pharmacology Univeniiy of Birmingham, Medical School Eirming ham, England

I. Introduction I I . Structure and Intracellular Distribution o f Brain Phosphdipids A. Structure . B. Distribution 111. Origin and Metabolism of Brain Phospholipids A. Transport to the Brain and the Origin of Precursors B. Dr Norm Synthesis-Neurones and Glia . C. Dc Nouo Synthesh-Subcellular Fractions 1V. Relationship between Phospholipid Camposition of Membranes and Brain Function A. Naturally Occurring Modifications of Phospholipid Composition B. Experimental Modifications of Phospholipid Composition C. Effects of Anesthetics V. Phospholipids and Synaptic Transmission A. Relationship Between Lipid-Bound Choline and Acetylcholine Formation in the Brain B, Effects of Neurotransmitters on Phospholipid Metabolism References

11 11 14 16 17

17 20 26

1. Introduction

This brief chapter is intended to cover recent developments in which metabolic processes involving phospholipids may be related to mechanisms important in the functioning of the brain. In general, basic information about the structure and metabolism of brain phospholipids can be found in the relevant chapters in the treatise edited by Ansell et al. (1973) and the review by Ramsey and Nicholas (1972). Such information will not be repeated here in detaiI. More details on the relationship between phospholipid metabolism and function in the nervous system can be found in the proceedings of a recent meeting (Porcellati et al., 1976). 1

2

G . BRIAN ANSELL A N D SHEILA SPANNER

It is unlikely that any new phospholipid structures will be found in the nervous system as a whole, and modern analytical techniques have made possible the identification of the molecular species of all those present. Currently, interest is focusing on, the phospholipid composition and metabolism of relatively homogeneous structural components of the nervous system, for example, myelin and the plasma membranes of nerve endings which are more likely to yield useful information and have, in fact, done so. It is by no means clear, however, why different membranous components of the nervous system have a different phospholipid composition. This comment applies, of course, to other tissues and the rationale for different phospholipid “species” is often totally obscure. The one certainty is that some membrane-bound enzymes need specific phospholipids in order to function normally (Coleman, 1973). What is clear is that when the metabolism of a phospholipid is abnormal then function can be seriously disturbed. In this way an abnormal metabolism may give a clue to normal function, as it has done for other tissue components.

II. Structure and lntracellular Distribution of Brain Phospholipids

A. STRUCTURE In the brain there are four classes of phospholipids; the first three have a glycerol backbone with a fatty acid, usually unsaturated, in the 2-position and a phosphorylated base (choline, ethanolamine, serine) or inositol in the %position. In the l-position is a fatty acid (diacylphospholipid) ( l ) , a long-chain aliphatic O-alk- l-enyl (unsaturated ether) moiety (plasmalogen) (2), or a long-chain aliphatic O-afkyl (saturated ether) moiety (3). Only choline and ethanolamine plasmalogens have been found and only ethanolamine-containing phospholipids with a saturated ether. The fourth class, of which the only representative is ceramide phosphorylcholine (sphingomyelin), contains a long-chain base, sphinganine, linked to phosphorylcholine through its primary hydroxyl group and with its amino group acylated by a long-chain fatty acid (4). There are also small but significant amounts of cardiolipin (phosphatidylglycerol phosphoglyceride) in which phosphatidylglycerol is linked to the basic phosphatidyl unit making it diphosphatidylglycerol (1). The chemistry of phospholipids has been clearly described by Strickland (1973).

FUNCTIONAL METABOLISM OF BRAIN PHOSPHOLIPIDS

3

0

I

X = H , choline, ethanolamine, serine, inositol, inositol monophosphate, inositol diphosphate, or phosphatidylglycerol. RCOO--, CI,-C2, acyl group (mainly saturated; some may be odd numbered or branched) R'COO--, CI4-C*2acyl group (unsaturated predominate)

H,C-OCR'

P I

B

RiCO H2C--O-P-O-X TH II

OH

1,2-diacyl-m-glycero-3-phosphoryl-X H H H2C-O,,and Gregson, N. A. (197 I).J. Cell Sci. 9, 769-789. Hamberger, A,, and Svennerholm, L. (1971).J. Neurochem. 18, 1821-1829. Harvey, M. S., Wirtz, K. W. A., Kamp, H. H., Zegers, B. J. H., and van Deenen, L. L. M. (1973). Biochim. Biophys. Actu 323, 234-239.

28

G. BRIAN ANSELL AND SHEILA SPANNER

Hauser, H., and Dawson, R. M. C. (1968).Bi0chem.J. 109,909-916. Hawthorne, J. N., and Bleasdale, J. E. (1975).Mol. Cell. Biochem. 8 , 83-87. Helmkamp, G . M. Jr., Harvey, M. S., Wirtz, K. W. A., and van Deenen, L. L. M. (1974).J. Biol. Chem. 249, 6382-6389. Hendrickson, H. S., and Reinertsen, J. L. (1969). Biochernktry 8, 48554858. Hokin, L. E.. and Hokin, M. R. (1955).Biochim. Biophys. Acta 18, 102-110. Hokin, L. E., and Hokin, M. R. (1958).J . Biol. C h m . 233, 818-821. Hokin, M. R. (1969).]. Neurochem. 16, 127-134. Hokin, M. R. (1970).J. Neurochmn. 17, 357-364. Horrocks, L. A. (1969).J. Neurochtm. 16, 13-18. Horrocks, L. A. (1971). Int. SOC. Neurochem., 3rd Int. Meet., Budapest p. 312. (Abstr.) Horrocks, L. A., and Radominska-Pyrek, A. (1972). FEBS Lett. 22, 190-194. lllingworth, D. R., and Portman, 0. W. (1972). Biochem. J. 130,557-567. Johnson, S. M., and Bangham, A. D. (1969). Biocbim. Biophyr. Actn 193,92-104. Jungalwala, F. B. (1974). Brain Res. 78,99-108. Jungalwala, F. B., and Dawson, R. M. C. (1971). Biochem. J. 123, 683-693. Kanfer, J. N. (1972).J. Lipid Res. 13, 468-476. Kishimoto, Y., Agranoff, B. W., Radin, N. S., and Burton, R. M. (1969).J. Netirochem. 16, 397404. KLawans. H. L., and Rubovits, R. (1970),In “L-Dopa and Parkinsonism” (A. Barbeau and R. H. McDowall, eds.), pp. 107-1 16. Davis, Philadelphia, Pennsylvania. Klenk, E., and Kahlke, W. (1963). Hoppe-Seyler’s Z. Physiol. Chem. 333, 133-139. Kohlschutter, A., and Herschkowitz, N. N. (1973). Brain Res. 50,379-385. Kunze, H., Nahas, N., Traynor, J. R., and Wurl, M. (1976). Biochim. Ewphys. Acta 441, 93-102. Lapetina, E. G., and Hawthorne, J. N. (1971). Biochem. J. 122, 171-179. Lapetina, E. G., and Michell, R. H. (1973a). FEBS Lett. 31, 1-10. Lapetina, E. G., and Michell, R. H. (1973b). Bi0chem.J. 131,433-442. Lapetina, E. G., and Michell, R. H. (1974).J. Neurocha. 23, 283-287. Leibowitz, Z., and Gatt, S. (1968). Biochim. Biophys. Acta 164,439-441. Lennon, A. M., and Steinberg, H. R. (1973).J . Neurochem. 20,337-345. Lunt, G. G . , and Pickard, M. R. (1975).J. Neurochmn. 24, 1203-1208. MacBrinn, M. C., and OBrien, J. S. (1968).J. Lipid Res. 9, 552-561. McDonald, W. I., and Sears, T. A. (1970a). Brain 93,575-582. McDonald, W. I., and Sears, T. A. (1970b). Brain 93,583-598. McGeer, P. L., McGeer, E. G., and Fibiger, H. C. (1973). Lani-et ii, 623-624. McMartin, D. N., Horrocks, L. A., and Koestner,A. (1972).Acta Neuropathd. 22,288-294. Mann, S. P. (1975). Experientia 31, 1256-1258. Mantovani, P., Pepeu, G., and Amaducci, L. (1976). In “Function and Metabolism of Phospholipids in the Central and Peripheral Nervous Systems” (G. Porcellati, L. Amaducci, and C. Galli, eds.), pp. 285-292. Plenum, New York. Marchbanks, R. M. (1975). Int. J. Bwchem. 6, 303-312. Michell, R. H. (1975). Bwchim. Biophys. Acta 415, 81-147. Michell, R. H., and Lapetina, E. G . (1972). Nature (London) New Biol. 240,258-259. Michell, R. H., Jones, L. M., and Jafferji, S. S. (1976).1n “Stimulus-Secretion Coupling in the Gastrointestinal Tract” (R. M. Case and H. Goebell, eds.), pp. 89-103. MTP Press, Lancaster, England. Miller, E. K., and Dawson, R. M. C. (1972a).Eiochem.J. 126,805-821. Miller, E. K., and Dawson, R. M. C. (1972b). Biochem.J. 126,825-835. Norton, W. T., and Poduslo, S. E. (1971).J. Lipid Res. 12, 84-90. Nussbaum, J. L., Neskovic, N., and Mandel, P. (1971).J. Neurochem. 18, 1529-1543. OBrien, J. S. (1967).J. Theor. Biol. 15, 307-324.

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29

OBrien, J. F., and Geison, R. L. ( 1 974). J. Lipid Res. 15,44-49. Pasquini, J. M., Krawiec, L., and Soto, E. F. (1973).J. Neurochem. 21, 6 4 7 4 5 3 . Poduslo, S. E., and Norton, W. T. (1972).J. Neurochem. 19, 727-736. Porcellati, G., Arienti, G., Pirotta, M., and Giorgini, D. (1971).]. Neurochem. 18,13951417. Porcellati, G., Amaducci, L., and Galli, C., eds. (1976). “Function and Metabolism of Phospholipids in the Central and Peripheral Nervous Systems.” Plenum, New York. Possmayer, F. (1974). Brain Res. 74, 167-174. Radominska-Pyrek, A., and Horrocks, L. A. (1972).J. Lipzd Res. 13, 580-587. Raghaven, S..Rhoads, D., and Kanfer, J. (1973). J . B i d . Chem. 247, 7153-7156. Ramsey, R. B., and Nicholas, H. J. (1972). Adv. Lipid Res. 10, 144-232. Rasminsky, M., and Sears, T. A. (1972).J. Physiol. (London) 227, 323-350. Rasmussen, H., Goodman. D. B. P., and Tenenhouse, A. (1972).CRC Crir. Rn).Hiodirtn. 1, 95-148. Roberti, R., Binaglia, L., Francescangeli, E., Goracci, G., and Porcellati, G. (I975).Lipids 10, 121-127. Rosenberg, P. (1970). Toxicon 8,235-243. Rosenberg, P., and Hoskin, F. C. G. (1963).J. Gen. Physiol. 46, 1065-1073. Rosenberg, P., and Ng, K. Y. (1963). Biochim. Biophys. Acta 75, 116-128. Schacht, J., and Agranoff, B. W. (1972).J. Bwl. Chem. 247,771-777. Schacht, J., and Agranoff, B. W. (1973). Biochem. Biophys. Res. Commun. 50, 934-941. Schacht, J., Neale, E. A., and Agranoff, B. W. (1974).J. Neurochem. 23, 211-218. Scherphof, G., and Westenberg, H. (1975). Biochim. Biophp. Acta 398,442451. Schmid, H. H. 0..and Takahashi, T. (1970).J. LipidRes. 11,412-419. Seeman, P. (1972). Pharmacol. Rev. 24, 583-655. Segall, H. J., and Wood, J. M. (1974). Nature (London) 248, 456-458. Sidman, R. L., Dickie, M. M., and Appel, S. H . (1964). Science 144, 309-31 1 . Singh, H., Spritz, N., and Geyer, B. (1971).J. Lzpzd Res. 1 2 , 4 7 3 4 8 1 . Sneddon, J. M., and Keen, P. (1970). Biochem. Phannacol. 19, 1297-1306. Snyder, F., ed. (1972). “Ether Lipids: Chemistry and Biology.” Academic Press, New York. Spanner, S., Hall, R. C., and Ansell, G. B. (1976). Biochem. J . 154, 133-140. Stavinoha, W. B., and Weintraub, S. T. (1974). Science 183, 964-965. Strickland, K. P. (1973). In “Form and Function of Phospholipids” (G. B. Ansell, R. M. C. Dawson, and J. N. Hawthorne, eds.), pp. 9-42. Elsevier, Amsterdam. Strickland, K. P., Thompson, R. H. S., and Webster, G. R. (1956).J. Neurol., Neurosurg. Psychiatty 19, 12-16. Sun, G. Y., and Horrocks, L. A. (1969).J. Neurochem. 16, 181-189. Sun, G. Y., and Sun, A. Y. (1972). Biochim. Biophys. Actn 280, 306-315. Takahashi, T., and Schmid, H. H. 0. (1970). C h m . Phys. Lip& 4, 243-246. Takeuchi, T. (196X). In “Minamata Disease” ( M . Kirksnna, ed.), p. 141. Kumamoto Univ. Press, Kumamoto, Japan. Tasaki, I. (1952).Jnp.J. Physiol. 3, 73-74. Tyson, C. A., Zande, H. V., and Green, D. E. (1976).J. Biol. Chem. 251, 1326-1332. Webster, G. R., Marples, E. A., and Thompson, R. H. S. (1957).Biochem. J. 65, 374-377. Whittaker, V. P. (1966). Ann. N . Y. h a d . Sci. 137, 982-998. Wirtz, K. W. A., Jolles, J., Westerman, J., and Neys, F. (1976). Nature (London) 260, 354355. Woelk, H., and Porcellati, G. (1973). Hoppe-Seyler’s 2. Physiol. Chem. 354, 90-100. Wu, Y.-C., Cho, T. M., Lok, H. H., and Way, E. L. (1976). Biochem. Pharmucol. 25, 15511553. Yagihara, Y., and Hawthorne, J. N. (1972).J. Neurochem. 19, 355-367. Yagihara, Y., Bleasdale, J. E., and Hawthorne, J. N. (1973).J. Neurochem. 21, 173-190.


ISOLATION AND PURIFICATION OF THE NICOTINIC ACETYLCHOLINE RECEPTOR AND ITS FUNCTIONAL RECONSTITUTION INTO A MEMBRANE ENVIRONMENT By Michael S. Briley' a n d Jean-Pierre Chongeux Neurobiologie Moleculaire lnstitut Pasteur, Paris, France

I. Introduction ........................................................... [ I . Isolation and Purification ... A. The Problem of Identification . . . . ............................ B. Purification of the Subsynaptic Membrane Fragments . . . . . . . . . . . . . . . . . . . C. Solubilization of the Receptor Protein ................................. D. Purification of the Solubilized Receptor Protein . E. Chemical and Structural Properties of the Purifie Receptor Protein ........ 111. Reconstitution ... ...... ...

31 32 32 34 39

44

A. Reconstitution Measured by Na+ Flux.................. 49 B. Reconstitution Measured by Bilayer Membrane Conductance . . . . . . . . . . . . 52 IV. Conclusion ............................................................. 58 R e f e r e n c e s . . . . . . . . . . . . . . . . . . . . ....................................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 59 9

1. Introduction

Synaptic transmission at the neuromuscular junction is mediated through nicotinic cholinergic receptors situated in the postsynaptic membrane. The binding of acetylcholine results in an increased cation permeability leading to a partial membrane depolarization. The nicotinic receptor has been studied in greatest detail using the electric organs of two electric fish, E l e c t r o p h m , a freshwater electric eel and Torpedo, a marine electric ray. The electric organs of these fish represent rich sources of large quantities of a single type of cholinergic synapse amenable to both electroph ysiological and biochemical analysis. The morphology and electrophysiology of these systems have received considerable attention and several recent reviews are available (Bennett, 1970; Rang, 1974; Changeux, 1975; Magazanik, 1976). In this chapter we will concentrate on the isolation and purification of the receptor pro-

' Present address: Synthelabo, 5X Rue de la Glacikre, Paris, France 31

32

MICHAEL S. BRILEY A N D JEAN-PIERRE CHANGEUX

tein, its physical and chemical properties, and attempts at the reconstitution of a functional receptor-membrane complex. Various functional aspects of the receptor such as changes in the binding affinity for agonists and the relationship between these changes and the structural and functional states of the receptor have been reviewed recently in considerable detail (Changeux et al., 1976) and will not be covered here. 11. Isolation and Purification

A. THEPROBLEM OF IDENTIFICATION Important to the purification of any biological material is its easy and unambiguous identification at all stages of purification. In the case of the cholinergic receptor this problem has been largely overcome by the use of covalent affinity labels (Singer et al., 1973; Karlin et al., 1973) and specific snake venom a-toxins (Lee, 1972).

1 . A@nity Labels The first reagent used to covalently label the receptor was the diazo derivative of phenyltrimethylammonium (PTA) (Changeux et al., 1967). This reagent acted as an irreversible antagonist and its action was delayed by the presence of reversible antagonists. After prior reduction with dithiothreitol (DTT)the receptor may be labeled with 4-(N-maleimido)-benzyltrimethylammoniumiodide (MBTA), a quaternary maleimido derivative (Karlin and Winnick, 1968). This irreversibly blocks the response to agonists. Its high degree of specificity has enabled the labeling of the receptor protein in vivo, on membrane fragments and in solubilized preparations (Reiter et al., 1972). A variety of other alkylating and acylating reagents have been tested (for review, see Karlin et al., 1973) but only MBTA and 4(N-maleimido)-phenyl-trimethylammonium iodide (MPTA) have been widely used. Recently Hucho et al. (1976) have used the photoaffinity reagent, 4-azido-2-nitrobenzyltrimethylammoniumfluoroborate, to label the receptor. Unlike the maleimido derivatives, this product labels all of the subunits. Labeling is inhibited by cholinergic agonists and antagonists. The specificity of this reagent has, however, been questioned (Ruoho et al., 1973) and thus its value as a specific receptor label has to be investigated further. 2. Snuke Venom a-Toxins Small basic proteins (molecular weight about 7000)*isolatedfrom the venom of certain -snakes block neuromuscular transmission and the

ISOLATION AND PURIFICATION OF NICOTINIC RECEPTOR

33

synapse of the electroplaque by acting at the level of the acetylcholine receptor (Lee, 1972). Although lacking any structural resemblance to cholinergic effectors, they bind with very high specificity and high affinity and act as very slowly reversible antagonists (Changeux et al., 1970, 1971). They probably bind to a site overlapping the cholinergic ligand binding site (Prives et al., 1972). These a-toxins have been radioactively labeled by a variety of methods (Lee and Tseng, 1966; Menezet al., 1971; Barnard et al., 1971; Cooper and Reich, 1972) and are now used in most laboratories for routine identification and assay of the receptor. The details of the assays used vary considerably, but in all cases involve separation of the receptor-toxin complex from the free toxin. With membrane fragments this is usually achieved by ultracentrifugation or the use of Millipore filters (Weber and Changeux, 1974). In the case of the detergent-solubilized receptor, the receptor-toxin complex has been separated from the free toxin by ammonium sulfate precipitation (Meunier et al., 1972a), filtration through Sephadex (Biesecker, 1973),or Bio-gel (McNamee et al., 1975b) columns. Since the toxin is a small positively charged molecule it can be readily separated from the receptor-toxin complex by passage through diethylaminoethyl (DEAE) resin or filter disk (Fulpius et aZ., 1972; Schmidt and Raftery, 1973). Although too small to be retained on a Millipore filter (0.45 p m diameter pore), the detergent-solubilized receptor forms large aggregates when the detergent is diluted to a concentration below the critical micelle concentration (cmc) in the presence of other detergent-soluble proteins and phospholipids. The aggregates may then be separated from the free toxin by Millipore filtration (Olsen et al., 1972; Meunieret al., 1974). All of these methods allow detection of picomole amounts of bound toxin and in some cases as low as 50 femtomoles. There are several contradictory reports concerning the relationship between the number of toxin binding sites and those binding small ligands or affinity labels. Kasai and Changeux (1971) found that in membrane fragments from Electrophorus the number of toxin binding sites were somewhat greater than those binding decamethonium. The purified receptor from the same source had a similar ratio of 1.6: 1 (Meunier and Changeux, 1973). Both membranes and purified receptor from Torpedo were found to have twice as many toxin binding sites as sites for acetylcholine, d-tubocurarine, or decamethonium (Moody et al., 1973). Chang (1974) found a ratio of 2 : 1, toxin : acetylcholine binding sites with the purified receptor. McNamee et al. (1975a) have found that purified receptor from Torpedo has twice the specific activity when measured by toxin binding than by affinity labeling with MBTA. In a detailed investigation, however, Weber and Changeux (1974) found the relationship between

34

MICHAEL S . BRILEY A N D JEAN-PIERRE

CHANGEUX

the number of toxin and decamethonium binding sites to be very close to unity. This has been confirmed by Sugiyama and Changeux (1975) who found it to be true for the purified receptor as well. Although the use of toxins and affinity labels has largely overcome the problem of identification of the ligand binding site of the receptor, the problem remains of determining whether, after detergent solubilization, the protein carrying the ligand binding site includes all of the apparatus necessary for receptor function (see Section 111). B. PURIFICATION

OF T H E

SUBSYNAPTIC MEMBRANEFRAGMENTS

The isolation of the receptor in the form of receptor-rich subsynaptic membrane fragments many of which exist as closed vesicles or “microsacs” represents an important stage in the total purification of the receptor. This material is “functional” in that the membranes are sensitive to permeability control by cholinergic effectors in a manner closely analogous to that found in vivo (Hazelbauer and Changeux, 1974; Popot et al., 1974, 1976). It is, at the same time, a greatly simplified system, comprising in its purest form only four protein bands by sodium dodecylsulfate (SDS)-gel electrophoresis (Sobel and Changeux, 1977; Hucho et al., 1976). Subcellular fractionation of electric tissue from either Electrophurus or Torpedo results in the separation of membrane fragments derived from the innervated face of the cells (rich in the acetylcholine receptor and acetylcholinesterase) from those derived from the noninnervated face (rich in NafKt ATPase) (Bauman et al., 1969; Duguid and Raftery, 1973a,b). The membrane fragments containing the receptor may then be separated from those containing the esterase (Cohen et al., 1972). Recent modifications of the original procedure (Sobel and Changeux, 1977) have enabled the separation of receptor-rich membrane fragments from the electric organ of Torpedo containing more than 4 nmoles of toxin binding sites per mg/protein with the acetylcholinesterase at least 100 times less. On the basis of the molecular weight of the receptor, this suggests that more than 50% of the protein of these membranes is present as the receptor. In addition to centrifugation, the method of affinity partition has been applied to the purification of these membrane fragments (Flanagan et al., 1976).This method is based on the principles of affinity chromatography (Cuatrecasas, 1970) and phase partition (Albertsson, 1960). It involves partitioning membrane fragments between two immiscible aqueous polymer solutions, one of which carries a bound affinity ligand. The results achieved are comparable to those described above but poten-

ISOLATION A N D PURIFICATION OF NICOTINIC RECEPTOR

35

tially suffer from the same disadvantage as those from affinity chromatography (see Section 11, D). 1. Functional Aspects The receptor-rich membranes form closed vesicles or "microsacs" which are capable of retaining ions such as Na+ and K+. Kasai and Changeux (197 1) using membranes from Electrophorus showed that these microsacs were chemically excitable, i.e., they responded to cholinergic agonists by an increase in their permeability to "Na'. This effect was blocked reversibly by d-rubocurarine or flaxedil (gallamine) and irreversibly by a-bungarotoxin. TheK,,, for agonists and antagonists agree well with those determined electrophysiologically on the intact electroplaque (Table I ) . Recently using a method which maximizes the proportion of agonist-inducible Na' flux, Hess et al. (1975, 1976) have analyzed the kinetics of this process. Similar results have recently been obtained using purified membrane fragments from Torpedo (Hazelbauer and Changeux, 1974; Popot et al., 1976). Here a set of seven ligands were tested and found to have the same relative order of affinities in ziztro as in z h o although the absolute I

TABLE I EFFECTORSWITH T H E RECEPTORFROM ELECTROPHORLT in Vir1o A N D in Vitro

INTERACTIONS OF C H O L I N E R C I C

K,,,,, (in 7 J 7 7 w ) " fM)

K,,, (in vitro)"

K , (a-toxin)"

(M)

(M)

-

1x IOP 8 x lor' 4 x lo-,>

Ag"n b h

2 x lo-" 2 x 10-1;

Acetylcholine Decamethonium Carbam ylcholine

3 x lo-.!

Antagonists d-Tirbocurarine Flaxedil Hexamethonium

3 x lo-' 3 x 10-5

2x

10-7

1 x lo-" 4 x lo-%

2 x lo-' 3 x 10-7 6 x lo-;'

2x 4x

10-7 10-7

6 X lo-"

KallP(in rlirro) apparent dissociation constant determined from depolarisation response of monocellular electroplaqiie (Higman et a / . , 1963: Changeux and Podleski, 1968: Weber and Changeux. 1974: Bartels and Nachmansohn, 1965: Mautner et al., 1966: Karlin and Winnik, 1968). " K,,,,, (in 7tilro) apparent dissociation constant determined from the N a + Hux respor.se of membrane vesicles isolated from El~c/rophorus(Kasai and Changeux, 197 1). ' K,, (a-toxin): protection constant determined from the ability of drugs to decrease by 50% the initial rate of binding 01. ['HI a-toxin o t N . nigrirollis to isolated membranes (Weber and Changeux. 1974). 'I

36

MICHAEL S . BRILEY A N D JEAN-PIERRE

CHANGEUX

values of the apparent dissociation constants appeared higher in vitro (Popot et al., 1976). Conductance measurements of the Torpedo electroplaquein vivo have shown that prolonged application of an agonist causes a reduction in the conductance response (Lester et al., 1975).This pharmacological “desensitization,” which resembles that seen at the neuromuscular junction (Katz and Thesleff, 1957), may also be observed in vitro using microsacs from Torpedo (Sugiyama et al., 1976).Preincubation of the microsacs for several minutes with agonists such as acetylcholine or carbamylcholine caused a subsequent decrease in the magnitude of the response to the same (or a different) agonist (Popot et al., 1974).This effect was blocked by antagonists and reversed by dilution. Both local anaesthetics and Ca2+ions, which enhance desensitization at the neuromuscular junction (see reference in Magazanik and Vyskocil, 1973), show a similar effect in vitro with Torpedo microsacs. A similar effect has been observed on the agonist binding affinity (Weber et al., 1975;for review of desensitization in vitro, see Changeux et al., 1976).

2. Physical Aspects Electron microscopy of the rece tor-rich membranes after negative diameter (Fig. 1) (Cartaud et al., staining shows particles of 80-90 1973; Raftery et al., 1975) or 60-70 A diameter (Nickel and Potter, 1973).These particles have been interpreted as rosettes of 5-6 subunits surrounding a central core. The rosettes are densely packed and sometimes seen arranged as a hexagonal lattice with a center-to-center distance of about 90 A. The lattice is seen more clearly with freeze etching (Cartaud et al., 1973). The purified receptors from Electrophms also appears as rosettes of 80-90 A (Meunier et al., 1974) (Fig. 2). X-ray diffraction studies of centrifugally aligned hydrated membrane specimens show a definite repeating unit in the plane of the membrane (Dupont et al., 1974) which, assuming a hexagonal lattice, has a center-to-center distance of about 90 A. Raftery et al. (1975)have confirmed the presence of an array but have calculated a center-to-center distance of 173 A. I n addition they find a repeating unit perpendicular to the plane of the membrane every 74 A with a secondary repeat every

1

37 A.

Brisson et al. (1975a)have recently introduced a potentially valuable probe for the physical environment of the receptor. The spin-labeled amphipathic cholinergic analog, 8(4’,4’-dimethyloxazolidine-N-oxyl) palmitoylcholine, appears to act as a reversible antagonist of the receptor. The choline end is thus anchored to the receptor binding site allowing the spin-labeled lipid chain to report on the immediate lipid envi-


FIG. 1 . Electron micrograph o f a purified fragment of subsynaptic membrane from electric organ o f Torpedo mrmorata. The preparation according to Sobel and Changeux (1977) was negatively stained with uranyl formate (1 R). (Micrograph kindly provided by Dr. J . Cartaud, Universite de Paris VII.)

38


FIG. 2 . Electron micrographs of' highly purified receptor protein solubilized from electric organ of E/~trop/zonrselectricits by negative staining. (Reproduced from Meunier ei nl., 1974.)

I S O L A T I O N AND PURIFICATION OF N I C O T I N I C RECEPTOR

39

ronment of the receptor. Preliminary results suggest that this immediate lipid environment is completely immobilized. OF THE RECEPTOR PROTEIN C. SOLUBILIZATION

1. Detergent Sokrbilimtion The cholinergic receptor protein is not solubilized by aqueous salt solutions of high or low ionic strength (Olsen et al., 1972; Potter, 1973) or by prolonged sonication (Olsen et ul., 1972). Nondenaturing, nonionic detergents and bile salt solutions that grossly disrupt the membrane structure do, however, solubilize the receptor protein (i.e., it remains in solution after centrifugation at 100,000 g for 1 hour). Of the various detergents used, Triton X-100 (Miledi et al., 1971) and cholate or deoxycholate (Changeuxet al., 197 1 ) are those most commonly reported. The receptor protein may therefore be classified as an integral membrane protein (Singer and Nicolson, 1972). 2. Extraction with Organic Solwnts A novel approach to the isolation of the cholinergic receptor was introduced by De Robertis and his co-workers (for review, see De Robertis, 1971). This method involves the isolation of the hydrophobic receptor protein as a proteolipid by extraction with chloroform-methanol. The extract is incubated (still in chloroform-methanol) with a radioactive cholinergic ligand and then the mixture passed through a column of lipophilic Sephadex LH-20. Several protein peaks are eluted with chloroform-methanol mixtures of increasing polarity but only one, the “cholinergic proteolipid,” is associated with the radioactive ligand. A criticism of this technique (Levinson and Keynes, 1972), that the coelution of protein and ligand was artifactual, has since been refuted (Donellan & Cattell, 1975). There remains, however, several serious objections to this work. Potter (1973) found that the toxin-receptor complex is not extracted with chloroform-methanol, although apparently the cholinergic proteolipid, after being transferred to an aqueous medium in Triton X- 100, can then bind the a-toxin (De Plazas and De Robertis, 1972). Similar attempts in other laboratories, however, have found that treatment with chloroform-methanol irreversibly denatures the toxin binding site (Potter, 1973; Barrantes et al., 1975; Heilbronn, 1975). Similarly the covalent receptor-affinity label complex is not extracted with chloroformmethanol (Karlin, 1973: Barrantes et al., 1975). The fact that treatment with DTT (a preliminary to affinity labeling) greatly reduces the amount

40


of total proteolipid extracted (De Robertis et nl., 1976) has been offered as an explanation for this. However, if after affinity labeling the membranes are reoxidized with 5,5’-dithiobis(2-nitrobenzoicacid) (DTNB), the amount of proteolipid extracted is increased but there is no increase in the extraction of affinity label (Barrantes et al., 1976). Using a Torpedo-like electric fish,Narkejaponica, Kametari et al. (1975) found that most chloroform-methanol soluble acetylcholine binding material could be extracted from the membrane fraction rich in acetylcholinesterase and that very little was extracted from the receptor-rich membrane fraction. The values they obtained for the overall binding of acetylcholine and the dissociation constants were similar to those found by De Plazas and De Robertis (1972).Kametari et ul. (1975)were unable to demonstrate characteristic protein properties for the acetylcholine binding material, but it appears that this may have arisen from their lack of experience with proteolipids and the modifications necessary for their protein assay. Rabbit antiserum raised against purified detergent-extracted receptor from Electrophorus blocks the physiological response in vivo by an immune reaction, confirming its identity as the cholinergic receptor (Patrick and Lindstrom, 1973; Sugiyama et aL, 1973; Heilbronn and Mattson, 1974).Such antisera did not react against the cholinergic proteolipid transferred to detergent solution nor did antisera raised against the cholinergic proteolipid react against the purified receptor (Barrantes et al., 1975;Heilbronn, 1975).Finally a comparison of the protein composition of receptor-rich membranes from Torpedo by SDSpolyacrylamide electrophoresis shows that the band at 40,000 (the only one to be labeled with [ 3 HIMPTA) is not significantly diminished by extraction with chloroform-methanol. The cholinergic proteolipid, therefore, appears to differ in many respects from the now wellcharacterized, detergent-solubilized cholinergic receptor protein. D. PURIFICATION OF

THE

SOLUBILIZED RECEPTOR PROTEIN

Once the receptor had been solubilized in bile salts or nonionic detergents the way was clear for its purification. Difficulties arising from the presence of detergents and the hydrophobic nature of the receptor protein have meant that conventional methods of purification have not, in general, been very successful. However, starting from‘ a preparation of Torpedo membranes ( 1000 nmoles a-toxin boundgm protein), Potter (1973)used a purification procedure involving a sequence of sucrosedensity gradient centrifugation, ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and ultracentrifugation to


41

achieve a preparation close to purity (9500 nmoles a-toxidgm protein) but with a very low yield. Using Electrophorus, which has a much lower specific activity in the membrane fraction (about 50 nmoles a-toxin/gm protein), this approach has not been successful. In the case of mammalian muscle (about l nmoles/gm protein in the membrane fraction) the problems are even greater. The technique of affinity chromatography, introduced by Cuatrecasas (1970), was soon used in the purification of the cholinergic receptor. From the beginning, two basic types of affinity columns were developed. In one group various cholinergic toxins have been coupled directly to activated Sepharose beads. The receptor which binds to the immobilized toxin is eventually displaced by high concentrations of cholinergic ligands. In the other group a cholinergic ligand is attached to Sepharose via a spacer “arm.” Again the bound receptor protein is usually eluted with high concentrations of cholinergic ligand. Results obtained with both types of affinity columns are summarized in Table 11. Toxin columns have the advantage of high specificity. The first unambiguous demonstration that acetylcholinesterase and the acetylcholine receptor are different proteins came from the binding of the receptor to Naja nigricollk toxin coupled to Sepharose beads while acetylcholinesterase remained in solution (Meunieret al., 1971).Desorption from toxin columns is slow and requires very high concentrations of cholinergic ligands. With columns using coupled cholinergic ligands separation of the esterase and receptor is usually achieved by selective desorption using ligands with greater affinity for the receptor than for the esterase. Thus carbamylcholine (Karlin and Cowburn, 1973), flaxedil (Olsen et al., 1972; Meunier et al., 1974), and decamethonium (Biesecker, 1973) have all been used successfully. Similarly a salt gradient also gives selective elution (Schmidt and Raftery, 1972). Both methods usually give yields of 20-50%. While this is adequate from a preparative point of view, it leaves open the question of whether a specific subpopulation of receptor molecules is being selected and that the receptor thus purified is not typical of that existing in vivo. As yet no evidence has been published to support or deny this disturbing possibility. Another criticism of the purification of the receptor by affinity chromatography is the possibility of densitization of the receptor (see Changeux et al., 1976) by the cholinergic effectors which are used in high concentrations both coupled to the gel and as eluants. Sugiyama and Changeux (1975), using a crude Triton X-100-solubilized preparation from Torpedo, found that the affinity for acetylcholine of most of the

TABLE I1 SUMMARY OF AFFINITY CHROMATOGRAPHY METHODSUSED FOR PURIFICATION OF THE NICOTINIC ACETYLCHOLINE RECEPTOR AND COMPARISON WITH CONVENTIONAL METHODS

(fi )

Purity achieved specific activity pmole toxin bound/ gm protein"

E. electricus E . electricus

Hexa-50 Deca-10 or Benzo Q-0.1

6.6 4.1

T. munorata

Carb 0-500 gradient

2.2

T. manorata T. califonica T. nubiliana T. ocelhta E . electricus E . electricus E . electricus E. electricus T. mannoruta T. californica T. califmica

Carb- 1000 Carb- 1000 C a r b 100 Carb-1000 Deca-1 Carb-50 Bis Q-0.003 Flax-2.5 Flax-2.5 NaCl gradient Carb-50

Elution ligand and concentration Affinity ligand

Source material

Reference

ELECTRIC ORGAN P

N

a-Toxin, Najn nnjn

PTA

Flaxedil analog Quaternary ammonium PTA

7.8" 10 12.2" 10 4.5 4" 54.5 5.9 6.9 6.9 6

Klett et al. (1973) Lindstrom and Patrick (1974) Patrick et al. (1975) Karlsson et al. (1972) Heilbronn and Mattson (1974) Eldefrawi and Eldefrawi (1973) Eldefrawi et al. (1975a) Ong and Brady (1974) Rubsamen et al. (1976b) Biesecker (1973) Karlin and Cowburn (1973) Chang (1974) Meunier el al. (1974) Sugiyama and Changeux (1975) Schmidt and Raftery (1972,1973) Weill et a/.( 1 974)

MAMMALIAN SOURCES

a-Toxin. Nnjo nrda

PTA NOA'AFFINI TY METHODS

Conventional methods Conventional methods starting from extensively purified receptor-rich membranes

Mammalian sources Rat diaphragm muscle

Rabbit hindlimb muscle Mouse brain Cat denervated leg muscle

T. mnnornta T. mcinnorntcc

Carb-1000 Carb- 1000 Carh-200 or Hexa-200 Flax-2.1

0.19' 0.53' 0.2'

Not reported 3.5-6

9.3 7 .0

Brockes and Hall (1975) Bradley

~t 01.

(1976)

Romine et nl. (1 974) Dolly and Barnard (1975)

Potter (1973) Sobel and C;liange~x( 1977); Clrangeux P / rrl. ( I 977)

Ahh-Pi1icitions: Hexa, hexamethonium: Deca, decanrethoniiim: Benzo Q. benzoqitinacrium: Carh. carhamykho\ine; Flax, flaxedil (gallamine): bromide: PTA, phenyltrimethylanrmonium. Bis Q, 3,3'-his(n-(triniethylammoni~1m)metl~yl)-azohenzene " These values are final purities L I S L involving ~ ~ ~ sucrose-gradient centrifugation and/or ion-exchange chromatography after the affinity chromatography. " Acetylcholine binding sites. ' Protein by amino acid analysis (all others by the Lowry method). " MBTA binding sites which equal approximately 50% of toxin binding sites (McNaniee et 01.. 1975a). "Junctional receptors. ' Extrajunctional receptors.

44

MICHAEL S . BRILEY AND JEAN-PIERRE CHANCEUX

sites was decreased by about two orders of magnitude after passage through an affinity column (a flaxedil derivative coupled to Sepharose 4B with elution by 2.5 mM flaxedil [Meunier et al., 19741). In the light of these possible problems a purification procedure has recently been developed to avoid the use of affinity chromatography. Using a modification of the method of Cohen et al. (1972), receptor-rich membranes from Torpedo have been very highly purified so that their solubilization in Triton X-100, followed by centrifugation in a sucrose gradient, results in a purification at least equivalent to those achieved with affinity chromatography (Sobel and Changeux, 1977; Changeux et al., 1977). After affinity chromatography one or more other steps are required to purify the receptor to homogeneity. Centrifugation in a sucrose gradient (Meunier et al., 1974; Lindstrom and Patrick, 1974), passage through an ion-exchange column (Klett et al., 1973), and electrophoresis (Eldefrawi and Eldefrawi, 1973) are the most commonly used. Many of the preparations of the receptor protein are homogeneous by various criteria. Ultracentrifugation in a sucrose gradient gives a symmetrical peak which coincides with toxin binding (Meunier et al., 1974; Raftery et al., 1975; McNamee et al., 1975a). Polyacrylamide-gel electrophoresis in nondenaturing detergents at different pH gives one band (Dolly and Barnard, 1975; Raftery et d.,1975; Eldefrawi et al., 1975b) which may be labeled with toxin (Klett et al., 1973; Meunier et al., 1974). Cross-linking of the receptor and toxin with suberimidate (Hucho and Changeux, 1973) or glutaraldehyde (Biesecker, 1973) gives a single band on SDS-gel electrophoresis. Isoelectric focusing (Eldefrawi and Eldefrawi, 1973) and column chromatography (Eldefrawi et al., 1975a) have also been used. Electron microscopy of the purified receptor shows a homogeneous distribution of identical particles (Fig. 2) (Meunier et al., 1974). AND STRUCTURAL PROPERTIES OF E. CHEMICAL RECEPTORPROTEIN

THE

PURIFIED

1. Composition The amino acid composition has now been established for the purified receptor from Electrophmus and three species of Torpedo (Table 111). With the exception of one analysis (Klett et aZ., 1973) which failed to detect tryptophan, the receptor has been found to contain all of the commonly occurring amino acids. According to the classification of Capaldi and Vanderkooi (19721, the receptor contains about 46% polar residues, a value typical of globular water-soluble proteins. This would

45


TABLE I11 AMINOACIDC O M P O ~ I T I(mole/l00 ON mole$) O F T H F RELEITOR PROTEIN PURIFIED FROM T H E ELECTRIC O R ~ ~ oAt NElectrophom\ A N D Torpedo Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteic acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Glucosamine Reference

E. electricus 6.3 2.5 4.2 9.8 6.0 8.2 9.0 6.7 4.8 5.4 1.7 6.9 3.4 8.1 10.7 3.8 5.1 2.4

-

4.6 2.2 4.2 11.4 5.6 6.2 10.2 5.7 .5.9 5.8 2.0 8.6 2.0 6.4 10.5 4.0 5.7 0 -

n

b

T.mannorata

T.californzca

5.0 2.5 3.3 12.4 6.2 8.1 8.7 5.6 5.0 5.0

6.1 2.1 3.5 11.8 6.3 7.1 10.7 6.2 6.4 6.0 2.0 5.5 1.7 5.2 9.3 3.6 4.4 2.1 -

6.1 2.7 4.1 11.9 6.3 6.6 10.2 5.9 4.9 5.1 0.9 7.0 1.8 7.5 9.7 3.8 4.6 0.9

d

e

-

7.3 2.5 7.4 10.1 3.5 4.5 C

5.4 2.4 3.9 11.6 6.4 7.9 10.0 5.9 4.6 5.0 1.2 7.1 2.0 8.2 9.5 3.7 4.5 2.4 -

f

T.nobdinna 4.5 2.5 3.7 12.2 6.8 6.4 9.7 7.1 5.0 4.5 2.8 6.2 1.6 6.2 10.2 4.2 4.2 1.5 2.0

R

Kqr to Refwenus: (a) Meunier et al. (1974); (b) Klett et al. (1973); (c) Heilbronn and Mattson (1974); (d) Eldefrawi and Eldefrawi (1973); (e) Michaelson el al. (1974): (f) Eldef rawi ct NI. ( 1 975a): (g) Moore y t NI. ( 1 974).

suggest that the obvious hydrophobic nature of the receptor is derived from an asymetric distribution of the polar amino acids rather than its overall amino acid composition. On the other hand, using the same data, analyses of hydrophobicity by the methods of Barrantes (1975) or Bigelow (1967) suggest a similarity between the receptor and known integral membrane proteins (Raftery el al., 1976). The receptor appears not to possess any covalently bound phospholipid. No lipid phosphorous was detected (down to a limit of 1 mole P/mole toxin sites) in receptor purified from Electrophorus (Klett et al., 1973). The receptor does, however, contain carbohydrates. By its reaction with concavalin A and various other plant lectins, Meunier et al. ( 1974) detected the presence of D-mannose and N-acetyl-D-galactosamine. N-acetyl-D-glucosamine has been detected in receptor preparations from Torpedo (Michaelson et al., 1974; Moore et al., 1974). Preparations from this source also contain about 5% by weight neutral

46

MICHAEL S . BRILEY AND JEAN-PIERRE CHANGEUX

sugars : mannose, galactose, and glucose in the ratio 8 :2 : 1 (Raftery et al., 1975). In a‘similar preparation, Heilbronn (1975) reported mannose, galactose, and glucose in a similar ratio 8 : 1.8 :0.2. Tests for sialic acid were negative. On a cautionary note, contamination from agarose columns used during preparation exists as a strong possibility and may substantially alter the very small amounts of sugars detected. Analysis by atomic absorption revealed that the receptor contained 4.7% by weight of bound Ca”+ which was not removed by extensive dialysis (Eldefrawi et al., 1975~).Receptor prepared with Ca2+-freesolutions containing l mM ethylenediametetraacetate (EDTA) still bound 0.7% (by weight) Ca2+ (15 moles Ca2+/moleacetylcholine binding site). The significance of this bound divalent cation has not yet been established. Using the fluorescent lanthanide, terbium, as a fluorescent probe, Riibsamen et al. (1976a) demonstrated two types of terbium binding sites both withK,,, of 1.8 x M . About 60% of these sites bind Ca2+with a K,,, of about 1 x 10-3M. These sites which are located on the 40,000 dalton subunit (Riibsamen et al., 197613) also interact with cholinergic agonists but not antagonists such as the a-toxin. These results provide some support for a possible mechanism of changes in membrane ion permeability initiated by activator-induced displacement of Caz+ as has been suggested by Nachmansohn and Neumann (Nachmansohn, 1974; Nachmansohn and Neumann, 1975). 2. Size and Subunit Structure Sucrose-density gradients, in detergent, of crude receptor preparations from Torpedo show two distinct bands of receptor, a predominant one of 9 s and the other of 12s (Raftery et al., 1972; Potter, 1973). The corresponding Stokes radii were found to be 7.0 nm and 8.5 nm, respectively. Triton X-100 solubilization in the presence of 10 mM P-mercaptoethanol gives a single band of receptor of 9 s when centrifuged in a gradient containing 10 mM P-mercaptoethanol (Changeux et al., 1977; Sobel and Changeux, 1977). Centrifugation of the purified receptor from Electrophwus in a detergent-containing sucrose gradient gives a single symmetrical peak with a sedimentation coefficient of 9 S (Meunier et al., 1972a,b; Raftery et al., 1971). Gel filtration through Sepharose 6B in the presence of detergent gives a Stokes radius of 7.3 nm (Meunier et al., 1972b; Raftery et al., 1971). This value is considerably larger than that expected from sedimentation data and was explained by the presence of a large amount of bound detergent (greater than 0.1 mg Triton X-lOO/mg protein) increasing the size and bouyancy of the receptor (Meunier et al., 1972a). This data, after correction for the detergent present, gave estimates of


47

320,000-350,000 daltons (Hucho and Changeux, 1973). Sedimentation equilibrium centrifugation of the purified receptor from Torpedo gave two species of 330,000 and 660,000 daltons, the latter reducing to 330,000 daltons when the Triton X- 100 concentration was increased to 0.1% (Edelstein et al., 1975). Centrifugation of crude purified preparations of receptor from three different species of Torpedo each gave t w o major bands of toxin binding material of molecular weights 190,000 and 330,000 (Gibson et al., 1976). Both oligomers showed similar binding properties. Gel electrophoresis in SDS of the purified receptor from Electrophorus after cross-linking with gluteraldehyde (Biesecker, 1973) or suberimidate (Hucho and Changeux, 1973) gave apparent molecular weights of 260,000 and 230,000, respectively, using globular, nonmembrane proteins as standards. After correction for the underestimation often found when using globular proteins as standards for membrane proteins (Spatz & Strittmatter, 1973), a value of 275,000 was determined (Hucho and Changeux, 1973). SDS-gel electrophoresis of homogeneous preparations from Electrophorus under denaturing conditions has, in general, resulted in t w o bands of about 40,000 and 50,000 daltons (Hucho and Changeux, 1973: Biesecker, 1973; Lindstrom and Patrick, 1974). There are also reports of three bands (40,000, 47,000, and 53,000 daltons) (Karlin et al., 1976), four bands (48,000, 54,000, 60,000, and 110,000 daltons) (Patrick et al., 1975), and one band (size not reported) (Klett et al., 1973). I t is well established that only the subunit of 40,000 daltons binds the affinity label MBTA (Karlin and Cowburn, 1973; Meunier et al., 1974) and therefore carries the cholinergic binding site. This is further demonstrated by the finding that the a-toxin cross-linked to the receptor by suberimidate is found associated with only one subunit of about 40,000 daltons (Gordon et al., 1974). Similarly, the affinity label has been found only associated with the band of 40,000 daltons after labeling the receptor in intact electroplaques (Reiter et al., 1972) and in membrane fragments (Karlin and Cowburn, 1973). Patrick et al. (1975) have suggested that components of less than 48,000 molecular weight arise from proteolytic digestion. This, however, would appear to be an oversimplification since the band at 40,000 daltons occurs equally in receptor purified from Twpedo in the presence of- the protease inhibitor phenylmethyl-sulphonylfluoride (PMSF) (Sobel and Changeux, 1977: Hucho et nl., 1976). Partial cross-linking of the purified receptor gives at least six bands on subsequent SDS-gel electrophoresis (Hucho and Changeux, 1973), suggesting that the receptor is composed of at least five subunits. This is compatible with the rosettes of 5-6 subunits observed by electron microscopy both in membrane fragments (Cartaud et al., 1973; Nickel and

48

MICHAEL S. BRILEY A N D JEAN-PIERRE

CHANCEUX

Potter, 1973) and the purified receptor in detergents (Meunier et al., 1974; Eldefrawi et al., 1975b). The situation with the receptor from Torpedo appears to be more complex. Although a single band on SDS-gel electrophoresis has been reported (Potter, 1973), most studies show multiple bands. Gordon et al. (1974) showed the existence of five bands of 37,000, 49,000, 74,000, 93,000, and 148,000 daltons. T h e higher molecular weight bands, however, are almost certainly aggregates since their abundance increased with the age of preparation. Four bands of 40,000, 50,000, 60,000, and 64,000 daltons in a constant ratio of 4 : 2 : 1 : 1 have been reported (Raftery et al., 1976). The amino acid compositions of the 4 subunits did not differ greatly from each other and each had a similar mean hydrophobicity. Similar subunit patterns have been obtained by Karlin et al. (1976) who reported four subunits of 39,000,48,000,58,000, and 64,000 daltons in the ratio of 5 : 1.5 : 1 : 1.5 and by Rubsamen et al. (1976b); 4 subunits of 40,000, 50,000, 61,000, and 81,000 daltons in the ratio of 3.1: 1.4: 1.0: 1.1. Highly purified Torpedo membrane fragments showed four bands on SDS-gel electrophoresis of 40,000, 43,000, 50,000, and 66,000 daltons (Sobel and Changeux, 1977; Changeux et al., 1977) or 40,000, 48,000, 62,000, and 66,000 daltons (Hucho et nl., 1976). After solubilization and further purification only three bands (40,000, 50,000, and 60,000) were detected. Furthermore these bands varied in their relative proportions from one preparation to another suggesting that the two minor bands may be tenacious impurities (Sobel and Changeux, 1977; Hucho et nl., 1976). In the purest preparation these bands were found in a ratio of 6.3 : 1.4 : 1 (Sobel and Changeux, 1977). As with the receptor from Electrophorus, the only protein band from Torpedo to be affinity labeled is that of 40,000 daltons (Karlin et al., 1976; Changeux et nl., 1977; Sobel and Changeux, 1977). All four of the peptides found by Karlin et al. (1975) and by Raftery et al. (1976) react positively with periodic acid-Schiffs reagent (PAS) indicating the presence of carbohydrate moieties. In each band, mannose, glucose, galactose, and a trace of N-acetylglucosamine have been identified (Raftery et al., 1976). T h e purest protein preparations of receptor from fish electric organ have specific activities near or approaching 10 pmoles of a-toxin binding sites/gm protein (Table I I) or 1 mole of binding sitell 00,000 gm protein. In combination with the molecular weight estimations by physicochemical methods (in the region of 200,000-300,000 daltons), this suggests that there are at least two and possibly three cholinergic binding sites per receptor molecule. In the case ofElectrophorus this might be three of each

ISOLATION A N D PURIFICATION

OF N I C O T I N I C RECEPTOR

49

of the two types of subunit (i.e., 3a 3b) giving a hexamer. With Torpedo the picture is less clear and it is too early to suggest any molecular structure.

IV. Reconstitution

The advances made in our understanding of the nature of the receptor binding site, its various affinity states, its chemical and physical structure have not been matched by our knowledge of how agonist binding controls permeability and the nature of the associated ionophore.‘ At a more specific level one would like to know if all of the components of the functional receptor complex exist in the purified receptor protein isolated on the criterion of the toxin binding site. Equally, do the different types of subunits carry different functional components and if so how do they relate to one another? Permeability changes in artificial biological membranes can be measured directly as the flux of radioactive ions through the membrane or as changes in membrane resistance using the “black lipid” membrane or bilayer technique.

A. RECONSTITUTION MEASUREDBY NA+ FLUX Physical reintegration of the receptor into a membrane structure has been achieved by several groups. Receptor-rich membrane vesicles solubilized in ionic detergents such as sodium cholate or deoxycholate have been reconstituted by removal of the detergent by extensive dialysis. This results in the recovery of particulate material containing the toxin binding activity. Under the electron microscope this material appears as closed membrane vesicles similar to the native “microsacs” (Changeux et al., 1972). A similar preparation was reported to retain **Na+and to show a carbamylcholine-sensitive increase in Na+ efflux which was blocked by toxin, in other words a functional reconstitution. Details of this work, however, have never been published (unpublished observations, cited in Potter, 1973). Later Hazelbauer and Changeux (1974) provided the first demonstration that the receptor could, under certain conditions, be reintegrated into a membrane in a functional form. Receptor-rich membrane vesicles from Torpedo solubilized in sodium cholate were dialyzed for 48 The term ionophore is used here in a general sense and refers to any selective ion pathway, channel, or pore.

50


hours at 4OC. During this time most of the protein and toxin binding capacity was retained. There was, however, a significant loss of phospholipid. To compensate for this a sonicated aqueous dispersion of phospholipids extracted from Torpedo membrane fragments was added after dialysis. To reproducibly obtain closed vesicles it was necessary to add divalent cations, Mpf+ and Ca2+.Centrifugation in a sucrose gradient then gave two fractions, the heaviest of which retained 22Na' and under the electron microscope appeared as vesicles 300-1000 A in diameter bordered by a membrane 70 A thick. T h e rate of Na' efflux was accelerated by carbamylcholine; this increase was blocked by the a-toxin (Fig. 3A). TheK,,, for carbamylcholine was found to be in the region of 5 x lo-" M (Fig. 3B) similar to that obtained for native Torpedo microsacs (Popot et al., 1976). Functional reintegration of the purified receptor into phospholipid vesicles has proved to be difficult. Some partial success has been reported by Michaelson and Raftery (1974).The purified receptor from Torpedo in Triton X-100 was retained on a column of DEAE-cellulose and washed extensively and eventually eluted with solutions of sodium cholate in order to exchange the virtually nondialyzable Triton X-100 for the more freely dializable sodium cholate. A mixture, in 2% sodium cholate, of phospholipids and neutral lipids from Torpedo membranes and receptor protein in the ratio of lipid to protein 10: 1 (w/w) was dialyzed against detergent-free buffers at room temperature for 48 hours (dialysis at 4°C gave a physical but nonfunctional reintegration). After dialysis physical

50 A

0

I

10

I

I

20

30

minutes

10-6

10-7 10-6 1 0 - 3

I O - ~ 10-3

[carbamylcholine].M

FIG. 3. Na' emlux from reconstituted receptor vesicles. A. Reconstituted vesicles equilibrated with 2L'NaC were diluted into Turpedo Ringer's solution ( O ) ,Ringer's solution containing carbamylcholine lo-' M (A),or Ringer's solution containing carbamylcholine lo-' M after preincubation with a-toxin 10P M (m). B . Concentration dependence of' excitability in membrane vesicles (0)and reconstituted receptor vesicles ( A ) . Excitability is calculated a$ ( / t / t + )- 1 . (Reproduced from HaLelbauer and Changeux. 1974.)

ISOLATION A N D PURIFICATION OF NICOTlNIC RECEPTOR

I

7-

---i&c

51

10

TIME (rnin)

FIG. 4. Na+ efflux from vesicles reconstituted from purified receptor from Torpedo. Vesicles were equilibrated with "Na' and diluted into 200 mM NaCI, 10 mM Tris/HCI. p H 7.4 (01the , same solution containing carbamylcholine lo-' M ( O ) , or the same solution containing carbamylcholine lo-' M after preincubation with excess toxin ( W j . (Reproduced from Michaelson and Raftery, 1974.)

reassociation of the receptor and lipids was demonstrated by cosedimentation in a sucrose gradient. Intact reconstituted vesicles and those resolubilized with Triton X- 100 showed similar toxin binding capacities, indicating that the toxin binding sites were asymetrically arranged in an "all-facing out" orientation. The vesicles, which showed osmotic sensitivity, retained "Na+ although they were rather leaky (ti efflux varied between 5 and 15 minutes at room temperature). The rate of efflux was increased in the presence of carbamylcholine at 10-.' M . This excitability was blocked by preincubation with a-toxin (Fig. 4). No evidence was presented on the concentration dependence of the excitability and therefore its similarity to the native system. Furthermore many preparations yielded nonexcitable vesicles and there was no apparent correlation between membrane excitability and the binding capacity for the a-toxin. As yet these results have not been confirmed by other workers. McNamee et al. (1975a,b)have used two methods to incorporate receptor protein purified from Electrophomis and from Torpedo into phospholipid vesicles. In one method detergent-depleted receptor was cosonicated with a mixture of egg lecithin and phospholipids from Electrophwzis receptor-rich membranes. Physical reassociation only was reported (McNamee et al., 1975a). The other method was essentially the same as that used by Michaelson and Raftery (1974) but in this case no excitability could be achieved.

52

MICHAEL S . BRILEY AND JEAN-PIERRE CHANGEUX

Receptor in Triton X-100 purified from Torpedo was detergentexchanged for sodium cholate by centrifuging into a sucrose gradient containing cholate or by repeated dilution with cholate and reconcentration by ultrafiltration. T h e receptor in cholate was mixed with egg lecithin and Twpedo phospholipids and neutral lipids in the ratio of lipid to protein 10: 1 (w/w) and dialyzed at 4°C or 25°C for 72 hours against detergent-free buffers. This treatment resulted in no loss of MBTA labeling capacity. Physical reassociation was again demonstrated by cosedimentation of the receptor and lipids. T h e vesicles formed had a low permeability to .“a+ (t+ influx = 2-3 hours at 25°C) but as found by Michaelson and Raftery (1974) showed no difference between those containing the receptor and those corn posed solely of lipids. The rate of sodium influx could be accelerated by the addition of such artificial ionophores as gramicidin D and valinomycin. Carbamylcholine, however, had no effect on the rate of sodium influx (McNamee et al., 1975b). The uncertain “state of the art” is exemplified by various contradictions between different reports. Hazelbauer and Changeux (1974), for example, stress the importance of divalent cations for the formation of closed vesicles. Others, however, have found no such requirement (Michaelson and Raftery, 1974; McNamee et al., 1975b). Michaelson and Raftery (1974) find that the dialysis must be carried out at room temperature to obtain functional reintegration, whereas Hazelbauer and Changeux (1974) achieved a functional reintegration after dialysis at 4°C. Michaelson and Raftery (1974) have found that 100% of the toxin sites were on the outside of their reconstituted vesicles. Using similar reconstitution methods of cholate dialysis, others have found a symmetrical 50% inside, 50% outside arrangement (Potter, 1973; Briley and Changeux, 1976, 1977; Changeux et al., 1977). Thus the results obtained to date suggest that the functional reconstitution of the purified receptor into membrane vesicles is possible but that all of the parameters involved are not yet identified or controlled.

B. RECONSTITUTION MEASUREDBY BILAYERMEMBRANE CONDUCTANCE 1. “Reconstitution” of Nonionophoric Proteins

Very soon after Mueller and Rudin introduced the technique of bilayer membrane (black lipid films) (Mueller et al., 1962) as a model for biological membranes, attempts were made to modify the membrane properties by inserting specific functional proteins. Del Castillo et a!.


53

(1966) found that trypsin (and other enzymes such as chymotrypsin, lactate dehydrogenase, glutamate dehydrogenase, urease, and acetylcholinesterase) added to the bath became associated with the membrane. Subsequent addition of a substrate caused a drop in membrane resistance of about three orders of magnitude in a manner compatible with the activity of the enzyme. Similarly the incorporation of antigens caused a resistance decrease on subsequent addition of the corresponding antibodies. Although the membranes had a relatively high conductivity (- lov6 mho/cm2 compared with lO-'O mho/cm2 for good membranes made currently), the enzymes appeared to be acting only at the membrane surface since the addition of the substrate and enzyme to opposite sides of the membrane caused no resistance change. Leuzinger and Schneider (1972), working under the misapprehension that acetylcholinesterase and the acetylcholine receptor were identical, incorporated purified preparations of the esterase into bilayers of high resistance (mho/cm2)and found resistance decreases of two orders of magnitude on addition of acetylcholine to the opposite side of the membrane. This effect was blocked by eserine (an inhibitor of acetylcholinesterase which has no action on the acetylcholine receptor at the concentration used). Further work with purified esterase (again on the assumption that it was identical with the receptor) incorporated into high resistance (- lo-' mho/cm2) bilayers gain et al., 1973) showed enzyme-mediated conductance changes on addition. of acetylcholine or carbamylcholine3 to the opposite side of the membrane. These effects were blocked by neostigmine,' atropine," a-toxin;j and d-tubocurarine." In the light of current knowledge that acetylcholinesterase and the acetylcholine receptor are different proteins with different binding properties, these results are difficult to interpret. In a later paper Jain (1974) suggested that the receptor was an impurity of the esterase preparation used. Using a commercial preparation of acetylcholinesterase, he went on to show that dose-response curves for various nicotinic agonists were similar to those with the eel electroplaque. In addition a cation selectivity similar to that occurring in the electroplaque (Na : K : C 1, 3 : 3 : 1) was demonstrated. The previous results with neostigmine and atropine, however, were not explained.

-

Carbamylcholine is a cholinergic agonist which is not a substrate for acetylcholinesterase. ' Neostigmine is an inhibitor of the esterase but does not block the nicotinic receptor. Atropine blocks the muscarinic receptor but not acetylcholinesterase or the nicotinic receptor. 0-Toxin and d-tubocurarine block the nicotinic receptor but are without effect on the esterase.

54

MICHAEL S. BRILEY AND JEAN-PIERRE

CHANGEUX

The incorporation of enzymes into bilayer membranes which results in enzyme activity-induced conductivity changes clearly demonstrates the ambiguity of this method and the difficulty of distinguishing between artifacts inherent in the technique and any conductivity changes due to the acetylcholine receptor. Other experiments which, in the light of more recent findings, appear more confusing than enlightening are those of De Robertis and co-workers. Using a system which gave control bilayers of relatively low resistance ( mhokm’), they incorporated the cholinergic proteolipid (see Section 11, C, 2) into the “membrane-forming solution” prior to bilayer formation (Parisi et al., 1971, 1972). Subsequent local addition of acetylcholine to an unstirred bath gave a small ( 5 - to 10-fold) increase in membrane conductance. This increase was transient, presumably due to the dispersion of the concentrated agonist added. The addition of d-tubocurarine also gave an increase in conductance but less than that of acetylcholine. The effect was not seen with a “noncholinergic” proteolipid. Later the work was extended to electron microscopy of the membrane in the presence and absence of acetylcholine (Vasquez et al., 1971). A change in the appearance of the membrane was interpreted as an opening of the ionophoric channels. As already discussed (see Section 11, C) the cholinergic proteolipid probably differs considerably from the acetylcholine receptor and thus these results are more a demonstration of bilayer artifacts than a recovery of receptor function. A possible explanation for these artifacts has come from Parisi et al. (1975). They have shown that the presence of negatively charged lipids in a bilayer membrane can produce an apparent cholinergic excitability which can be prevented by d-tubocurarine! Whether negatively charged lipids could also explain the binding properties of the cholinergic proteolipid (De Robertis, 197 1) was not investigated.

2. Reconstitution of the Acetylcholine Receptor into Bilayers The first attempt to reintegrate the receptor itself into a bilayer used a partially purified preparation of receptor from rat diaphragm muscle (Kemp et al., 1973). The receptor in 0.6% Triton X-100 was added (at a final dilution of 1000-fold) to the bath after the membrane had thinned. Conductance measurements showed a linear increase with time (Fig. 5). In the presence of acetylcholine, at 5 x M, the rate of increase was accelerated about 4-fold. This effect was blocked by both a-toxin and d-tubocurarine. The sensitivity to acetylcholine was lost on storage although the toxin binding capacity was not altered. No dose-response curves were reported but the authors did note an ion specificity of 3 : 1, K’ : Na’ both in the presence and absence of acetylcholine.


55

'Oi

9 1 N

'E

-I

E

l

6 1 u C c

U U

5

0

4'

TIME (Min)

FIG. 5 . Increase of bilayer membrane conductance by incorporation of receptor. The receptor preparation was added to one side at zero time. Bathing solutions (both chambers) contained 0.1 M KCI (lower curve) or 0.1 M KCI plus acetylcholine (upper curve). (Reproduced from Kemp et al., 1973.)

Shamoo and Eldefrawi (1975) recently described experiments using a purified receptor preparation. A purified and well-characterized receptor preparation from Torpedo (Eldefrawi et al., 1975b) was incorporated into high-resistance bilayer (lo-' mhokm'). With Ca'+ in the bath the conductance increased with time, this increase being proportional to the amount of receptor added. In the presence of Na+ instead of Ca2+the conductance increase was 20- to 100-fold less for the same protein concentration. In either case there was no reproducible effect of carbamylcholine at M. In an attempt to facilitate receptor incorporation into the membrane the receptor was partially digested with trypsin and then treated with Sephadex CM 50 to remove small positively charged peptides. This resulted in a 5% loss of protein and a decrease in the molecular weight of most of the protein bands on SDS-gel electrophoresis. This trypsin treatment, however, had no effect on the acetylcholine binding activity of the receptor. In the presence of Ca2+ the trypsin-treated receptor showed little difference to the native receptor. In the presence of Na+, however, at low protein concentrations no conductance increase with M caused a large time was seen. The addition of carbamylcholine at increase in conductance which could be blocked by curare (Fig. 6). The addition of curare after carbamylcholine could not reverse the conductance increase. The ion specificity for the carbamylcholine-induced con-

56


1

50

TIME(MIN)

FIG.6. Excitability of trypsin-treated purified receptor incorporated into a lipid bilayer membrane. Trypsin-treated receptor (0.8 pglml) was present (in 0.1 M NaCI, 5 mM histidine pH 7.3) in both compartments before formation o f the bilayer at zero time. The concentration of carbamylcholine and curare were 10P M . Arrow indicates membrane rupture. (Reproduced from Shamoo and Eldefrawi, 1975.)

ductance was Na' : K+ : C1-, 4.4 : 4.4 : 1. The increased conductance level remained constant in the presence of constant concentration of carbamylcholine. In other words it did not show desensitization as seen in uivo and in vitro (see Section 11, B). No dose-response curves were reported. While these experiments show some of the properties expected from in uivo and in vitro studies, the necessity of tryptic digestion and the lack of reversibility and desensitization make their interpretation less straightforward. The conductance of a single-receptor channel has recently been estimated from statistical analysis of random "noise" fluctuations in muscle end-plates during iontophoretic application of acetylcholine (Katz and Miledi, 1970, 1972a,b) and found to be about lO-'O mho with an average lifetime of about 1 msec. Further studies with Fourier Transform spectra and end-plate current "noise" (Anderson and Stevens, 1973) gave estimates of unit channel conductance of 3.2 X 10"' mho with an openchannel lifetime of 6-1 1 msec. Some workers have recently attempted to detect such unit-channel conductance in lipid bilayers containing the receptor. Goodall et al. (1974) purified the nicotinic receptor from mouse brain by applying a Triton X-100 extract to a toxin affinity column. The fraction eluted with a carbamylcholine gradient was taken to be the receptor. No biochemical determination of its identity or purity was made. The putative receptor was incorporated into bilayers in the presence of carbamylcholine at 5 x lop4M. Two series of discrete conductance steps or quanta were observed, 1.5 X lo-'' mho (Na'), 2.4 x lo-"' mho (Cl-), and 3.7 x lo-'' mho (Na+),5.9 x lo-'' mho (Cl-).


57

The larger quanta, which appear to be four times the smaller, were diminished in the presence of d-tubocurarine or if the carbamylcholine was removed. This study was extended by Romine et al. (1974) who used receptor purified from both hog brain and mouse brain by toxin affinity columns. Again no biochemical analyses of the receptor were made. The receptor from hog brain gave minimal quanta of 3.8 X lo-” mho (Naf) and 4.7 x lo-’’ mho (C1-) (Fig. 7). Again larger steps were also observed which could be diminished by the addition of d-tubocurarine or atropine (sic). The lifetime of these large steps (the order of several seconds) is too great to result from single channel openings which are more probably part of the membrane “noise.” The aggregated steps were suggested to be due either to groups of receptor opening in a cooperative manner or to some phenomenon associated with desensitization. Equally they may reflect an irreversible channel opening. It is also possible that they may result from some experimental artifact such as membrane instability or receptor exchange with the bath medium. Further work with the nicotinic receptor from mammalian skeletal muscle (Bradley et al., 1976) again showed similar quantal conductance changes again of long duration (tens of seconds). Their frequency was enhanced by the addition of carbamylcholine and reduced by curare and a-toxin but not by atropine. Concanavalin A also showed an antagonistic effect suggesting the involvement of glycoproteins in the phenomenon. Preincubation of the receptor with the reducing reagent, DTT, also reduced the frequency of quantal events. No new explanations were offered to account for the long lifetime of the quanta. The authors did,

10-l0

L 10s

24

FIG. 7 . Bilayer response after addition of receptor extract from hog brain tissue following elution with hexamethonium. Note the increase in “noise” as more quantal jumps in conductance occur. Smallest size conductance increase to Na+ is 3.8 X 10-l’mho. (Reproduced from Romine et al., 1974.)

58

MICHAEL S. BRILEY AND JEAN-PIERRE

CHANGEUX

however, note that in similar reconstitution experiments with receptor isolated from fish electric organ they were unable to observe any quantal changes. The study of membrane “noise” seems to be a potentially sensitive and informative method for determining the properties of receptorcontaining bilayers especially if the temporal resolution in the millisecond range is available. As yet, however, the properties of these quantal events have only some of the properties to be expected for the reconstituted receptor system.

IV. Conclusion

The isolation and purification of the receptor is now well advanced largely due to an abundant source of receptor, the electric organ, and the highly specific, high-affinity ligand, the snake a-toxin. Soon one can expect the quaternary structure to be unequivocally assigned and probably the determination of the primary sequence, at least in the region of the binding site. At the membrane level the various functional states of the receptor and their transitions are becoming increasingly available to study through the use of fluorescent probes (see Changeux et al., 1976, 1977). In spite of considerable attention and some partial successes the functional reconstitution of the receptor and hence our understanding of its mode of action has not progressed very far. Physical association of the receptor with both bilayers and vesicles has been inferred by various methods but the integration of the receptor into the lipid bilayer and its orientation across the membrane have yet to be demonstrated. The functional reintegration of the receptor into vesicles has not been reproducibly demonstrated. With bilayers the distinction between artifactual and receptor-mediated conductance changes has complicated the interpretation of this data. Furthermore, in many cases, the so-called receptor is ill defined in biochemical terms. One may pose the question: Why does the reconstitution of the receptor appear to be so difficult in comparison with other apparently analogous systems such as the adenosine triphosphatase (ATPase) Ca2+ pump (MacLennan, 1975; MacLennan and Holland, 1975)? In order to achieve a functionally active reconstituted receptor-membrane complex it is necessary, on the basis of our current ideas, that the receptor span the membrane orientated with its binding site to the source of agonist. (In a random arrangement this would of course be true for 50% of the sites.) T h e receptor must be in the resting state but capable of activation. The ionophore may be present as a pore in the tertiary

ISOLATION AND PURIFICATION OF NICOTINJC RECEPTOR

59

structure of each subunit, as a specific ionophoric subunit, as a pore formed by the quaternary striictiire of the s~tbunitsof’a single receptor oligomer, or as a pore formed by the quinternary str~ctiireof‘ protein and lipids in the membrane. T h e latter may require a specific “annulus” of lipids in the immediate environment of the receptor as postulated for the ATPase (Warren ut d.,1975). Each of these possible striictiire~ implies its own conditions to be fiilfilled for activity. In addition one may suggest that the lateral stabililty of‘ the receptor in the suhsynaptic membrane (ie., the fact that the receptor remains subsynaptic in spite of lateral diffusion which usually occurs in the plane of the membrane) was also required probably involving a rather inu usual local rigidity in the membrane. T h e possibility that residual detergent may act as a local anesthetic also exists. T h e local anesthetic action of Triton X-100 dt micromolar concentrations (>1000 times lower than the concentration used in receptor purification) has been demonstrated (Bi-isson et al., 197517). This exhausting but not exhaustive list shows that it is not so surprising that the relatively crude methods of reconstitution employed so far have not been routinely successful. I t would thus appear that a more analytical approach to reconstitution is required. I t is not sufficient simply to look for the final product, the excitable membrane, but to study and compare as many properties as possible throughout the process of solubilization, purification, and reconstitution. In this way one may hope to be able to gradually fulfill the requirements for functional reconstitution and in the process probably learn a great deal about the functioning of the receptor! ACKNOWLEDGMENTS Michael S. Briley is the recipient of a long-term EMBO (European Molecular Biology Organization) fellowship. We are indebted to Drs. A . Sobel and J. Cartaud for the preparation and electron microscopy, respectively, of the pitrified membranes shown in Fig. 1. T h e original research was supported by grants from the National Institutes of Health, United States Public Health Service, the Centre National d e la Recherche Scientifique, the Delegation Generale a la Recherche Scientifique et Technique, the Fondation pour la Recherche Midicale FranCake, the College d e France, and the Commissariat a I’Energie Atorniqne. REFERENCES

Albertsson, P. A. (1960). “Partition of Cell Particles a n d Macromolecules.” Almqvist & Wiksell, Stockholm. Anderson, C. R., and Stevens, C. F. (1973).J . Physiol. (London) 235,665-691. Barnard, E. A., Wieckowski, J., and Chiu, T. H. (197 1). Nature ( L o d o n ) 234, 207-209. Barrantes, F. J. (1975).Biochem. Biophy.7. Res. Cornmiin. 62, 4 0 7 4 1 4 . Barrantes. F. J., Changeux, J. P., Lunt, G. G., and Sobel, A. (197.5).Nature (London) 256, 325-327.

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MICHAEL S. BRILEY A N D JEAN-PIERRE

CHANGEUX

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B IOCH EM1CAL ASPECTS 0F NE UROTRANSMISSlON IN THE DEVELOPING BRAIN By Joseph T. Coyle

Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences The Johns Hopkins University School of Medicine Baltimore, Maryland

1. Introduction 11. Prenatal Development of Central Catechdaminergic Neurons

A. Background B. Appearance of Catecholaminergic Neurons in Fetal Brain C. THIThymidine Autoradiography D. Octopamine and 8-Phenylethanolamine in Fetal Brain E. Regional Innervation by Catecholaminergic Processes F. Effects of Drugs on Fetal Brain Catecholamines G. Synaptogenesis on Catecholaminergic Neurons H. Catecholaminergic lnriervation in the Immature Neocortex I . Summary and Speculation 111. Postnatal Development of the Nigmstriatal Circuit A. Background B. Dopaminergic lnnervation to Striatum C. Striatal Cholinergic Neurons D. Striatal GABAergic Neurons E. Ontogenesis of the Function of the Nigrostriatal Circuit 1V. Conclusion References

1. Introduction

T w o related issues have long provoked the interest of both basic and clinical investigators whose focus of research is the development of the central nervous system. First, when are specific neuronal pathways formed in the developing brain; and second, when does effective neurotransmission among the components of such neuronal pathways commence during brain maturation? Biochemical analysis of the processes that mediate chemical synaptic transmission can provide information relevant to both these questions. By measuring the biochemical parame65

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ters that are specific for a neuronal type, it should be possible to “map” in a quantitative fashion the development of the neuronal pathway in the brain. Since neuronal communication occurs by means of chemical transmitters (Krijevic, 1974), the relative development of the processes that mediate neurotransmission should reflect the functional influence of a neuronal pathway at a particular stage of brain development. Four parameters play primary and specific roles in mediating neurotransmission for many identified classes of neurons as demonstrated by the fact that interference with any one of these parameters can profoundly affect the efficacy of neurotransmission (Cooper et al., 1974) (Fig. 1 ) . The neuron possesses the enzyme or enzymes necessary to synthesize its neurotransmitter; in most cases, these enzymes are exclusively localized in or, at least, highly concentrated in the particular neuronal population. Effective neurotransmission requires the presence of adequate stores of neurotransmitters available for release at the synapse; and the vesicles that populate terminal boutons concentrate the neurotransmitter for release as “quanta.” On the neuronal membrane, there are high-affinity transport sites specific for the neurotransmitter utilized by the neuron (Kuhar, 1973). The uptake process terminates the action of released neurotransmitter and may keep the synaptic cleft free of neurotransmitter during periods of presynaptic neuronal quiescence. For interneuronal communication to occur, there must be appropriate receptors for the neurotransmitter on the postsynaptic neuron to decode

REUPTAKE

FIG. 1 . Schematic model of a typical synapse indicating major steps involved in regulating chemical synaptic neurotransmission. These include the specific enzymes that synthesize the neurotransmitter (enzymes), the stores o f neurotransmitter in terminal vesicles (transmitter), the interaction of released neurotransmitter with appropriate postsynaptic receptors (receptors), and the inactivation of the neurotransmitter through removal from the synaptic cleft by high-affinity uptake specific for the neurotransmitter (reuptake).

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the chemical message. On one hand, the presynaptic parameters can be used as specific biochemical markers for quantifying neuronal differentiation; and, on the other hand, the relative development of all four parameters in a brain region provides insight into the potential influence of a neuronal pathway. During the last 25 years numerous investigators have examined the ontogenetic development of one or more of these parameters in the brains of a variety of species, and many comprehensive reviews of these studies have appeared (Baker and Quay, 1969; Filogamo and Marchisio, 1971; Coyle, 1973; Haber and Kuriyama, 1973; Lanier et al., 1976). Accordingly, the purpose of the present chapter is not to review again the diverse aspects of neurotransmitters in the developing brain but rather to critically evaluate the possible correlation between the development of the processes that regulate neurotransmission and the morphologic and functional aspects of neuronal differentiation in rat brain. In the first part of the chapter, studies concerning the early development of central catecholaminergic neurons will be examined because the correlation between morphologic and neurochemical differentiation can be assessed. In the second part, the nigrostriatal circuit shall be the focus of discussion because of the opportunity to compare and contrast neurochemical differentiation of three types of neurons involved in defined synaptic relationships. II. Prenatal Development of Central Catecholaminergic Neurons

A. BACKGROUND The central catecholaminergic neurons provide a unique opportunity for studying the neurochemical aspects of neuronal development because of the possibility of correlating the biochemical results with the neuroanatomic characteristics of the neurons. The cell bodies for the catcholaminergic neurons are limited to the brainstem regions whereas they provide innervation to cortical and subcortical regions quite distant from their perikarya (Ungerstedt, 1971); hence, for neurochemical studies brain regions containing exclusively axons and terminals of the catecholaminergic neurons can be anatomically separated from those possessing the cell bodies of these neurons. In addition, the catecholaminergic neurons can be visualized for histologic studies by histofluorescent (Dahlstrom and Fuxe, 1964) and, more recently, immunochemical techniques (Hartman et nl., 1972; Pickel et d.,1976); thus, one can correlate the neurochemical and morphologic aspects of their differentiation,

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B. APPEARANCE OF CATECHOJAMINERGIC NEURONSIN FETALBRAIN

Information about the stage of brain development at which a neuronal pathway appears is important for assessing possible influences of this pathway on brain function during subsequent differentiation. For the rat, an animal that is born at a relatively early stage of brain maturation, it has been generally accepted that the catecholaminergic neurons develop primarily after birth (Agrawal et al., 1966). However, it has become apparent with application of sensitive radiometric-enzymatic assay techniques that the presynaptic markers for the catecholaminergic neurons are present in the fetal rat brain well before birth. The enzymes in the synthesis pathway for catecholamines, tyrosine hydroxylase, Dopa decarboxylase, and dopamine-P-hydroxylase, as well as the neurotransmitters themselves, dopamine and norepinephrine, are detectable in the fetal rat brain as early as 15 days of gestation when the brain weighs 1.5% of that of the adult (Coyle and Axelrod, 1972a,b; Coyle and Henry, 1973;Lamprecht and Coyle, 1972).The specific activities of the enzymes are approximately 5-10% of that of the adult brain, whereas the concentrations of the catecholamines are only 3% of adult levels. During the last week of fetal development, there is a relatively linear and coordinated increase in the levels of the presynaptic markers for the catecholaminergic neurons to achieve approximately 30% of adult levels by birth. Based upon the linear rate of their rise during the late fetal stages, it has been predicted that these catecholaminergic neuronal markers appear in the brain between 13 and 14 days of gestation (Coyle, 1974). The developmental stage of appearance of the major catecholaminergic nuclei including the noradrenergic cell bodies in the locus coeruleus and the dopaminergic cell bodies in the substantia nigra has been examined with the histofluorescent microscopic technique. With this technique, thin sections of tissues are exposed to paraformaldehyde vapor under rigorously defined conditions; the condensation product formed by paraformaldehyde and catecholamine within neurons can be visualized with the fluorescent microscope (Falck et al., 1962). Nascent noradrenergic perikarya in the medulla pons and dopaminergic perikarya in the midbrain region become apparent at approximately 13 to 14 days of gestation (Maeda and Dresse, 1969; Olson and Seiger, 1972).At this stage, the catecholaminergic cells are still in the process of migration to their ultimate site of localization. Thus, there is an excellent correlation between the neurochemical and histofluorescent microscopic techniques with regard to the time of appearance of the major catecholaminergic nuclei in the rat brain.

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c.

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[’H]‘rHYMlDlNE AUTORADIOGRAPHY

Histofluorescent microscopy and the neurochemical assays are, in fact, measuring a similar parameter, i.e., the presence of catecholamines within the neurons. It is possible that the catecholaminergic neurons are formed well before they develop the capacity to synthesize and store catecholamines, and thus, the “birth” of these neurons may antedate their neurochemical differentiation. Therefore, it is essential to know at what stage in development the catecholaminergic neurons cease dividing or are, in effect, “born.” Such information can be obtained by [3H]thymidine authoradiography (Sidman, 1970). With this method, fetuses of various stages are administered a single dose of rH]thymidine; the animals are then allowed to mature to adulthood for histologic examination. Neuroblasts that are undergoing mitosis during the brief exposure to the [3H]thymidine incorporate it into their deoxyribonucleic acid (DNA), which remains “labeled” unless diluted by subsequent cell division or removed by cell death. Neurons that have ceased dividing no longer incorporate the [3H]thymidine into their DNA. The c3H]thymidine incorporated into the DNA of the neuronal nuclei can be demonstrated in adulthood by autoradiographic techniques: the identity of the radiolabeled neurons is determined by light and histofluorescent microscopy of adjacent sections. With this technique, the neurons in the locus coeruleus exhibit heavy incorporation of [3H]thymidine into their nuclear DNA when injected on days 11 and 13 of gestation but no incorporation when injected on day 14 of gestation or thereafter (Lauder and Bloom, 1974). The nuclei of the dopaminergic cell bodies in the substantia nigra exhibit heavy labeling after exposure to rHIthymidine on days 13-15 of gestation, but no incorporation occurs on subsequent days. Thus, cell division of the noradrenergic neurons in the locus coeruleus ceases by 14 days of gestation and for the dopaminergic neurons in the substantia nigra ceases by 16 days of gestation. I n brief, the full complement of catecholaminergic neurons that form these two major nuclei is attained fully a week before birth in the rat. The developmental stage when cell division of the catecholaminergic neurons ceases coincides with the time when they can first be demonstrated by the histofluorescent technique and the time when their neurotransmitters and biosynthetic enzymes appear. The striking agreement among these three different methodologic approaches provides compelling evidence that the catecholaminergic neurons acquire those specialized processes essential for neurotransmission during

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or immediately after the terminal (phase) mitoses of their precursor neuroblasts. Since the total complement of catecholaminergic neurons in the brain is achieved by 15 days of gestation in the rat, increases in the specific neurochemical markers for these neurons after this date represent neuronal differentiation and not neuronal multiplication. Since the central nervous system is a relatively closed system with respect to the catecholaminergic neurons (i.e., their cell bodies and axonal processes are limited to the cerebrum except for a small portion that innervate the spinal cord), an approximate quantitative assessment of the total increase in these parameters on a per neuron basis can be made by multiplying specific activities of the enzymes or concentrations of the neurotransmitters by brain weight (Fig. 2). When the developmental changes in the neurochemical parameters for the catecholaminergic neurons are expressed in this fashion, there is a 500- to 1000-fold increase between 15 days of gestation and adulthood (Coyle, 1974). This value, of course, I

i 6

Y-

O

Conceptual Age ( Days)

FIG. 2. Development of the biosynthetic enzymes for catecholamines and endogenous dopamine and norepinephrine in fetal rat brain. The activities of tyrosine hydroxylase ( X - . - x ) and dopamine-p-hydroxylase ( x ...X ) and levels of dopamine (0---0) and are expressed in terms of percent of whole brain activity or norepinephrine (0-0) content. The rectangles indicate the period of mitosis, and the arrows indicate the date of appearance by histofluorescent microscopy of the noradrenergic neurons in the locus coeruleus (L.C.) and of the dopaminergic neurons in the substantia nigra (S.N.).

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IN THE DEVELOPING BRAIN

provides only a hint of the enormous changes that occur during the process of differentiation from a discrete cell at one week before birth to a neuron with axonal extensions that make several thousand synaptic contacts. D.

OCTOPAMINE AND P-PHENYLETHANOLAMINE IN

FETALBRAIN

P-Phenylethanolamine and octopamine result from the direct decarboxylation of phenylalanine or tyrosine, respectively, with subsequent P-hydroxylation of the amine by dopamine-/3-hydroxylase (MolinofFand Axelrod, 1972). In mammals, it has generally been thought that these two biogenic amines occur as biochemical “mistakes” due to a side reaction in the normal synthetic pathway for norepinephrine in adrenergic neurons (Kopin et al., 1964). Accordingly, under usual conditions, these two amines are found in relatively low concentrations in tissues with noradrenergic innervation; however, the fact that neither entirely disappears after sympathetic denervation suggests that they may have an additional localization outside of noradrenergic neurons (Saavedra and Axelrod, 1973; Coyle et al., 1974). Notably, in certain invertebrates, octopamine and P-phenylethanolamine appear to be neurotransmitters in their own right for which there are specific receptors (Nathanson and Greengard, 1975; Saavedra et al., 1976). In fetal rat brain at 15 or 16 days of gestation, the concentrations of P-phenylethanolamine and octopamine are 5- to 6-fold higher than those that occur in adult brain (Saavedra et al., 1974) (Table I). More importantly, the concentrations of P-phenylethanolamine and ocTABLE I LEVELS OF’

P-PHENYLETHANOLAMINE, OCTOI’AMINE, A N D N O R E P I N E P H R I N E IN

Conceptual age (days) 15

16 17 18 20 22 (Birth) Adult

FETAL RAT BRAIN“

P-I’henylethanolamine Octopamine ( p m o l e h g tissue) 0. I47 0.154 0.19 1 0.044 0.029

0.037 0.044

Norepinephrine

0.085 0.176 0.065 0.020 0.035 0.039 0.033

” Values are obtained from Coyle and Henry ( 1 973) and Saavedra PI are the mean o f five or more separate determinations.

0.062 0.097 0 . I66 0.297 0.645 0.923 2..556

NI.

( 1 974) and

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JOSEPH T. COYLE

topamine exceed that of endogenous norepinephrine by 2.5-fold at this stage of brain development. Treatments of pregnant mothers at 16 days of gestation with a monoamine oxidase inhibitor, parachlorophenylalanine, or the combination of these drugs with phenylalanine considerably increase the ratio of the amines to norepinephrine in the fetal brain. At 18 days of gestation, there is dramatic decrease in the concentration of these two amines to adult levels, and their concentrations remain relatively constant thereafter. The reasons for this transient high concentration of P-phenylethanolamine and octopamine remain unclear. Although in the adult rat inhibition of monoamine oxidase results in a considerable increase in the levels of these two amines, their levels do not surpass that of endogenous norepinephrine (Molinoffand Axelrod, 1972).Thus, the low activity of monoamine oxidase (10% of adult specific activity) in fetal rat brain does not adequately explain the high levels of these amines (Saavedra et al., 1974; Gripois, 1975). In peripheral sympathetic neurons, the levels of tyrosine hydroxylase are regulated by the cholinergic input to the ganglion and corticosteroids (Mueller et al., 1969; Otten and Thoenen, 1976). It is noteworthy that the pituitaryadrenal axis becomes functional and synapses appear on the locus coeruleus (see below) at 18 days of gestation when the levels of P-phenylethanolamine and octopamine decrease precipitously (Roffi, 1968; Lauder and Bloom, 1975). Thus, it is possible that their elevated levels reflect a disparity between the activity of tyrosine hydroxylase and dopamine-P-hydroxylase within the immature central noradrenergic neurons. The possibility that octopaminergic neurons appear transiently during the early stages of brain development cannot be ruled out. Regardless of the explanation, phenomenologically this represents an example of neurotransmitter ontogeny recapitulating phylogeny.

E. REGIONAL INNERVATION BY CATECHOLAMINERGIC PROCESSES During the last week of gestation, these is a marked increase in the specific activities of the biosynthetic enzymes for catecholamines in fetal rat brain with tyrosine hydroxylase, dopa decarboxylase, and dopamine-P-hydroxylase increasing 4- to 6-fold. The concentrations of endogenous dopamine and norepinephrine increase 15-fold during this time frame, dramatizing the marked differentiation that is occurring in this neuronal population (Coyle and Henry, 1973). The specific activities of the biosynthetic enzymes in the sheared off nerve terminals or synaptosomal fractions increase 10-fold between 15 days of gestation and birth (Coyle and Axelrod, 1972a,b). In accordance with this evidence that

NEUROTRANSMISSION IN THE DEVELOPING BRAIN

73

terminals are being formed at this early stage of development, the highaffinity uptake process for norepinephrine in synaptosomes can be demonstrated by 18 days of gestation (Coyle and Axelrod, 1971). Finally, during the late stages of fetal development, there is a translocation in the distribution of the biosynthetic enzymes with a progressive increase in their activity in the forebrain regions which receive innervation but do not possess intrinsic catecholaminergic cell bodies. T h e extensive histofluorescent microscopic study of Olsen and Seiger on the prenatal development of the central monoaminergic neurons in the rat provide fine-structural correlates for the neurochemical observations (Olsen and Seiger, 1972; Seiger and Olsen, 1973). In the rat embryo with a crown-rump length of 13 mm, which corresponds to 15 days 13rnrn embryo

0

CA cell groups

(3 S-HT re11 groups (.% ’

axon bundles

R-HTaxon bundles (‘.%+h-HT axon bundles

FIG.3. Schematic representation of the distribution of monoaminergic cell groups and axonal projections in the 15day gestational rat brain (13 mm embryo). CA, catecholamine: S-HT, 5-hydroxytq ptarnine. Midsagittal section through fetal b n i n demonstrating cell groups and axon bundles: in, mesencephalic flexure: 1). pontine flexure: c, cervical flexure. (Reproduced from Olson and Seiger, 1972, with kind permission of the authors and publisher, Springer-Verlag.)

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JOSEPH T. COYLE

of gestation, a prominent fluorescent axon bundle arising from the rostral noradrenergic cell complex transverses the lateral part of the diencephalon and reaches the preoptic area (Fig. 3). In the prosencephalon, the catecholaminergic processes have a distinct beaded appearance, a structural characteristic that in adult brain is associated with synaptic specialization. Although it has been generally assumed that synaptogenesis occurs later in rat brain development (Aghajanian and Bloom, 1967), especially in the telencephalon, Konig et al. (1975) have demonstrated by electron microscopy unequivocal synaptic contacts in the developing rat neocortex as early as 16 days of gestation. At approximately 18 days of gestation, widespread projections of the catecholaminergic neurons are evident throughout the fetal brain. Processes with terminals are observed in the medulla, tectum, discrete areas of the hypothalamus, and the tuberculum olfactorium. The medial forebrain bundle is well developed with a large number of catecholaminergic axons; the lateral part of the bundle, which contains dopaminergic fibers, projects to the primordial neostriatum. The striatum itself has a weakly fluorescent marginal zone consisting of small densely packed islands of catecholaminergic processes, and the interior aspects of the nucleus exhibit sparsely distributed varicosities. In the more medial parts of the medial forebrain bundle, which contain primarily noradrenergic fibers, there are axonal projections that can be traced to the hippocampus and the cerebral cortex. During the final 3 days of fetal development, the major morphologic change in the catecholaminergic neurons consists of a proliferation of terminal varicosities in all regions of the central nervous system.

F. EFFECTSOF DRUGSON FETAL BRAINCATECHOLAMINES The neurochernical and morphologic studies demonstrate that catecholaminergic axonal projections reach most regions of the fetal brain by a week before birth and commence the formation of terminal varicosities. During this period (15-16 days of gestation), neurons in the caudal region of the brain are in early stages of differentiation, whereas in the rostra1 regions neuronal cell division continues and the major aspects of neuronal organization are in their earliest stages (Schultze et al., 1974: Hine and Das, 1974; Hicks and D’Amato, 1968). The early, widespread projections of the catecholaminergic neurons may influence the neuronal differentiation or function in the areas receiving these fibers. Since such an influence would likely be communicated by the neurotransmitter itself, it is particularly important to assess the presynaptic processes regulating the disposition of catecholamines at this stage of development. Accordingly, the effects of administration of sev-

75

NELJROTRANSMISSION I N T H E DEVELOPING BRAIN

era1 drugs known to interfere with strategic processes involved in the intraneuronal disposition of catecholamines were examined in fetal rats at 18 days of gestation (Coyle and Henry, 1973; Coyle, 1974) (Table 11). Reserpine, a drug that interferes with the vesicular storage process for monoamines (Iversen, 1967), causes a profound depletion of endogenous dopamine and norepinephrine in the fetal brain. Amphetamine, a drug that displaces catecholamines from their storage sites as well as inhibits the presynaptic high-affinity transport process for them (Axelrod, 1970),causes a 70% decrement in the amine levels in the fetal brain. Thus, endogenous catecholamines are primarily sequestered in storage vesicles in the immature neurons. Inhibition of monoamine oxidase, the enzyme that plays a major role in the intraneuronal catabolism of catecholamines (Iversen, 1967), causes a 37% increase in the levels of both dopamine and norepinephrine. The efFects of reserpine, amphetamine, and monoamine oxidase inhibition are qualitatively similar to that which occur in the adult brain. Administration of a large dose of L-dopa, the amino acid precursor to catecholamines, produces a 60% increase in the concentration of norepinephrine and a massive 70-fold increase in the concentration of dopamine in the fetal brain. A particularly interesting aspect of these pharmacologic studies concerns the effects of inhibitors of catecholamine synthesis. Since inhibition of catecholamine synthesis prevents the restitution of catecholamines lost as a result of release, the time-dependent decrement in catecholamine levels after inhibition of their synthesis is proportional to neuronal TABLE I I EFFECTS01.' PHARMACOLOCIC TREATMENTS ON CATECHOLAMINE LEVELS I N ~ X - D A Y GESTATIONAL FETALRAT B R A I N "

Percent of control Treatment Control Reserpine (4 mg/kg) d-Amphetamine (20 nig/kg) a-Methyl-p-tyrosine (400 mg/kg) Diethyldithiocarhamate (500 mg/kgj Phenipra~ine(20 mglkg) L-dopa (300 mg/kg) + MK486 (150 mg/kg)

Duration

Dopamine

Norepinephrine

100 t 3 .5 t 1 31 ? 6 60 t 3 141 t 6 134 5 6

00 -c 4

5 hours 3 hollr$ 3 hours 3 hours 4 hours

4 hours

7460

230

X k 1

27 t 3

55 2 3 60 2 2 33 t 6 160 2 I 1

" Drugs were administered to pregnant rats at 18 days of gestation and fetuses were delivered by caeserian section between 3 to 5 hours after treatment. Results are expressed in terms of the mean percentage of the untreated control 5 S.E.M. Absolute \dues tor controls are 97 pg/mg for dopamine and 5 0 p g h g for norepinephrine (Coyle. 1973).

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JOSEPH T. COYLE

activity (Anden el al., 1967). Inhibition of tyrosine hydroxylase, the initial enzyme in the synthesis pathway for catecholamines, with a-methyltyrosine results in a 40-45% decrement in the levels of dopamine and norepinephrine after 3 hours in the fetal brain. Inhibition of dopamine-P-hydroxylase with diethyldithiocarbamate causes a 40% fall in the levels of norepinephrine and a 40% increase in the levels of dopamine in the fetal brain. The fact that inhibition of the first and last enzymes in the synthesis pathway for norepinephrine results in the same decrements in the levels of the amine indicates that negligible amounts of the intermediates L-dopa and dopamine are present in the noradrenergic neurons; thus, the synthesis pathway for norepinephrine is tightly coupled as is the case in the mature neurons (Goldstein and Nakajima, 1967). More importantly, the decrease in the concentrations of dopamine and norepinephrine after inhibition of their synthesis indicates that they are in a dynamic state. The half-life for the two neurotransmitters is approximately 3.5 hours, which is 50-7’5% of the half-life of the neurotransmitters in the whole brain of adult rats under basal conditions (Korf el al., 1973; Neff et al., 1971). This half-life suggests that fetal catecholaminergic neurons are sustaining an impulse flow near that of the adult.

G. SYNAPTOGENESIS O N CATECHOLAMINERCIC NEURONS These pharmacologic studies which suggest that catecholaminergic neurons are spontaneously active as early as 18 days of gestation raise the question of when synapses are formed on the catecholaminergic soma and dendrites. This issue has been examined by a combination of histofluorescent and electron microscopy (Lauder and Bloom, 1975). For the dopaminergic neurons in the substantia nigra, both neuropil and somatic synapses are evident by 18 days of gestation, the earliest time point examined; however, the density of synapses is extremely low until well after birth when they markedly increase in number between 15 and 30 days postpartum. With regard to the noradrenergic neurons of the locus coeruleus, faintly stained synaptic profiles are first observed in the neuropil at 19 days of gestation; by 20 days of gestation, synaptic contacts are clearly apparent both in the neuropil and on the somata, I n the locus coeruleus, the most rapid phase of synaptogenesis occurs between 5 and 10 days after birth. The combination of the morphologic and pharmacologic results suggest that the noradrenergic and possibly dopaminergic neurons are spontaneously active prior to the development of presynaptic input. That neuronal activity appears spontane-

NEUROTRANSMISSION IN T H E DEVELOPING BRAIN

77

ously prior to development of presynaptic input has been well established in other neuronal systems (Woodward et al., 1971). The role that presynaptic input to the central catecholaminergic neurons may play in terms of regulating their differentiation remains unclear; however, it should be noted that 95% of their total differentiation occurs after the appearance of synapses on their somata and dendrites. In a series of elegant experiments, the cholinergic input to the superior cervical ganglion has been demonstrated to modulate the differentiation of the sympathetic neurons. In this system, a major increase in the activity of tyrosine hydroxylase occurs after the development of cholinergic synapses in the ganglia; decentralization of the ganglia to prevent its cholinergic innervation significantly reduces the developmental increases in tyrosine hydroxylase (Black et al., 1971, 1972). Treatment with drugs that block the acetylcholine nicotinic receptor at this critical time also reduces the subsequent increase in tyrosine hydroxylase activity, thus demonstrating that acetylcholine liberated by the presynaptic terminals is the chemical cue that promotes the differentiation of the postsynaptic neurons (Black and Green, 1973, 1974). Furthermore, this cholinergic input not only regulates enzyme levels in the perikarya but also the formation of target organ innervation (Black and Mytilineou, 1976). Based upon these observations, it is not unreasonable to speculate that the presynaptic input to the central noradrenergic and dopaminergic neurons may play an important but yet undefined role in modulating their differentiation. H. CATECHOLAMINERGIC INNERVATIONI N

THE

IMMATURE NEOCORTEX

Although these histofluorescent and biochemical studies indicate that axonal projections reach the primordial neocortex by a week before birth, the innervation of the cortex at birth appears to be surprisingly sparse. The specific activities of the biosynthetic enzymes for catecholamines are only 10-20% of adult (Porcher and Heller, 1972; Nomura et al., 1976; Coyle and Axelrod, 1972a,b). In agreement, Loizou ( 1972) reports that catecholaminergic terminal varicosities are extremely rare in the rat neocortex at birth. The disparity between the antenatal and postnatal observations suggest that either there is a long delay between the arrival of catecholaminergic axons to the fetal cortex and the subsequent formation of synapses, or that immature catecholaminergic axons innervating the cortex at birth are deficient in their ability to synthesize endogenous catecholamines. To circumvent the possible pitfall of relying upon endogenous catecholamines as the markers for the

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JOSEPH T. COYLE

nerve terminals, neonatal rats have been pretreated with catecholamine congeners and precursors prior to electron microscopic and biochemical analysis in an attempt to uncover previously inapparent terminals. Monoaminergic terminals can be identified at the ultrastructural level by the presence of small (40-50 nm) granular vesicles which are storage sites for the amines (Bloom, 1973; Hokfelt and Ljungdahl, 1972). Although the small granular vesicles are rarely seen in the central nervous system in routine ultramicroscopic preparations, their demonstration can be markedly enhanced by exposure of brain tissue to the catecholamine congener 5-hydroxydopamine (Richards and Tranzer, 1970; Ajika and Hokfelt, 1973). This “false” neurotransmitter is selectively taken up and concentrated in the synaptic vesicles of the monoaminergic nerve terminals, whereupon it forms an electron-dense precipitate after aldehyde fixation. Thus, this indirect method permits the ultrastructural visualization of those synaptic terminals which have an uptake-storage mechanism specific for monoamines. During the first week after birth, the lateral cortex consists of three layers divided into the marginal zone, cortical plate, and subplate layer (Kristt and Molliver, 1975). Synapses are quite sparse throughout the layers of the neonatal cortex; however, there are regions of relatively greater synaptic density in the marginal zone, in the deep third of the cortical plate, and in the subplate layer. In untreated animals, all synaptic terminals contain clear or “empty” vesicles. In neonatal rats treated with 5-hydroxydopamine, approximately 30% of all synaptic terminals are filled with small round vesicles that contain a granular deposit (Molliver and Kristt, 1976) (Fig. 4). By 6 days after birth, the synapses containing small granular vesicles are mostly concentrated in the deep third of the cortical plate wherein 70% of the synaptic boutons contain these granular vesicles. Many of the axons form multiple synapses de passage: notably the same type of granular vesicles is found at every junctional site for a given axon. Pretreatment of the neonatal rat with reserpine, a drug that blocks the monoamine vesicular storage process prior to the administration of 5-hydroxydopamine, abolishes the development of vesicular precipitates. Hence, the vesicular accumulation of 5-hydroxydopamine occurs in a reserpine-sensitive site that is probably restricted to monoaminergic terminals. Since an apparent high density of monoaminergic terminals can be demonstrated in the neonatal cortex only after loading with the catecholamine congener, the effect of pretreatment with the catecholamine precursor L-dopa on the levels of endogenous catecholamines in the cortex of the neonatal rat has also been examined (Coyle and Molliver, 1977) (Table 111). Previous studies in the fetal and

NEUROTRANSMISSlON IN T HE DEVELOPING BRAIN

79

Flc. 4. Aminergic synapses in lateral newortex of the 6-day-old rat. Rats were pretreated with 5-hydroxydopamine: cortical sections were fixed in aldehyde-osmiiim. Llpper left: Synaptic Imuton with many sinall vesicles containing dense granules and two large dense core vesicles. LJpper right: .A termirial containing g n n n l a r vesicles t h i t edi ibits three areas of synaptic contact with asymmetric membrane speckalizations. Middle: Ax011 forms two ,ytup.s;inhibition, . . . . ’ >.

CYCLIC NUCLEOTIDES IN T H E NERVOUS SYSTEM

109

marized in a recent monograph (Daly, 1977). The present chapter will, therefore, attempt only an outline of current research and advances in this field of cyclic nucleotide research. The following areas will be covered: ( 1) the properties of adenylate cyclases, guanylate cyclases, phosphodiesterases, cyclic nucleotide-dependent kinases, and phosphatases as delineated with cell-free preparations and as studied in intact cells of brain slices, ganglia, and cultured brain cells; (2) the possible relationships of cyclic nucleotides to the control of biochemistry and neurophysiology in the intact nervous system; (3) the possible relationships of cyclic nucleotides to the pharmacology of centrally active drugs to behavior and mental dysfunctions. Literature has been surveyed to September, 1976, but the coverage is intended to be selective rather than comprehensive. II. Cyclic AMP

A. LOCALIZATION OF ADENYLATE CYCLASES The high levels of adenylate cyclases in brain, particularly in gray matter of neocortex and cerebellum (Klainer el al., 1962; Sutherland et al., 1962; Weiss and Costa, 1968), and the association of a major portion of the enzyme with synaptosome fractions from brain (De Robertis et al., 1967) strongly suggested important roles for cyclic AMP in the nervous system, presumably related to synaptic transmission. The activation of adenylate cyclases from brain and ganglia by various putative neurotransmitters lent further support to this belief. Synaptosomes, however, consist of both a presynaptic vesicular moiety and an attached postsynaptic membrane, and definitive evidence for presynaptic versus postsynaptic localization of adenylate cyclase has not, as yet, been obtained with brain synaptosome preparations. I n this regard, it should be noted that adenylate cyclase, usually an enzyme of the plasma membrane, has been demonstrated in the membranes from epinephrine-secretory vesicles of the adrenal medulla (Nikodijevic el al., 1976). In contrast to adenylate cyclases from plasma membranes, the cyclase of these secretory vesicles was inhibited by P-adrenergic agonists. Whether adenylate cyclases are associated with presynaptic noradrenergic vesicles and other neurotransmitter vesicles of central neurons is unknown. Recent studies have employed sucrose gradients to assess to what extent adenylate cyclases are associated with the synaptosome fractions which exhibit active uptake mechanisms for catecholamines. Active uptake mechanisms are, of course, thought to serve as a marker for the

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JOHN W. DALY

presynaptic entities. With synaptosomes from rat cerebral cortex, a significant portion of the adenylate cyclase activity was not associated with fractions exhibiting marked uptake of radioactive norepinephrine (Davis and Lefkowitz, 1976). High levels of binding sites for the /I-adrenergic antagonist dihydroalprenolol were associated almost entirely with the fractions which accumulated norepinephrine. It is interesting that these ‘‘&receptors,” normally thought to be associated with adenylate cyclase, were not associated with the major portion of adenylate cyclase in synaptosome fractions. An activation of adenylate cyclase by catecholamines was stated to be undetectable in these synaptosome preparations. With synaptosomes from rat striatum, the distribution of cyclase activity did not exactly correspond to the fractions which accumulated radioactive dopamine (Sieghart et al., 1976). Basal adenylate cyclase appeared on these sucrose gradients as one broad peak, while dopamine-stimulated cyclase activity appeared to represent two broad peaks. These studies, while not definitive, are at least consonant with significant postsynaptic rather than only presynaptic localization of adenylate cyclase in synaptosome preparations. Accumulations of cyclic AMP elicited by norepinephrine in synaptosomes appeared in one study to be derived from endogenous ATP within the synaptosome (Harris, 1976), a result consonant with a presynaptic generation of cyclic AMP. Intracellular ATP of brain slices was labeled by incubation with radioactive adenine, followed by homogenization, isolation of synaptosomes, and measurement of catecholamineelicited generation of cyclic AMP from “intrasynaptosomal” ATP. Norepinephrine appeared to be activating a /3-adrenergic receptorlinked cyclase. Other studies on adenylate cyclases associated with synaptosomes have been based on assays with exogenous ATP, a technique which will measure only the cyclases which have catalytic sites accessible to the medium. Synaptosomes are normally isolated after homogenization of brain tissue in isotonic sucrose. If, instead, brain tissue is homogenized in physiological medium, vesicular entities were formed whose adenylate cyclases appeared to accept as substrate not exogenous ATP, but instead the intravesicular ATP (Chasin et al., 1974; Shimizu et al., 1975a; Horn and Phillipson, 1976). The major morphological components of these homogenates were 100-800 nm vesicular membrane fragments, but in addition larger synaptosomelike entities were present whose postsynaptic elements when present had formed a vesicular structure. The responses of the cyclic AMP-generating systems in these cell-free preparations to neurotransmitters were remarkably similar to responses of cyclic AMP systems in brain slices. Responses of adenylate cyclases in synaptosomes isolated from sucrose homogenates have in contrast often


111

lost or greatly reduced responsiveness to neurotransmitters (cf. Daly, 1977). The morphological localization of adenylate cyclase and of cyclic AMP in tissue slices from brain has been studied to a limited extent. In rat cerebral cortex, a histochemical assay demonstrated aden ylate cyclase activity at a limited number of synapses on plasma membranes of astrocytes and associated with capillaries goo et al., 1975).The validity of such assays for adenylate cyclase has been questioned (Lemay and Jarett, 1975). Lead ions used in many such assays are potent inhibitors of adenylate cyclase (Nathanson and Bloom, 1975, 1976). Catecholaminesensitive adenylate cyclase was stated to have been detected histochemically at presynaptic sites in cerebral cortex and caudate nucleus (cited in Hervonen and Rechardt, 1976). In rat cerebellum, high levels of cyclic AMP were detected by immunofluorescent assay in Purkinje neurons and granule neurons (Bloom et al., 1972; Siggins et al., 1973). The weak fluorescence in the cerebellar molecular layer was associated with the Purkinje cell dendrites. Norepinephrine greatly increased the fluorescence of Purkinje cells. In view of these results, it is perhaps surprising that the basal levels of cyclic AMP in cerebellum were not lower in a mutant strain of mice, the nervous mouse, in which Purkinje cells are virtually absent (Ma0 et al., 1975). Levels of cyclic AMP were slightly higher in the granular layer than in the molecular layer of Swiss-Webster mice (Rubin and Ferrendelli, 1976). The generation of cyclic AMP has been studied extensively in incubated slices of brain tissue, but little has been learned from these studies as to the morphological sites at which cyclic AMP is formed (cf. Daly, 1977). Basal levels of cyclic AMP were similar in incubated cerebellar slices from control and X-irradiated rats (Hoffer et al., 1976). Thus, elimination of neurons of the granular layer by neonatal X-irradiation had little effect on basal levels of cyclic AMP. However, the accumulation of cyclic AMP elicited by norepinephrine was markedly reduced in slices from X-irradiated rats. The data were indicative of the presence of cyclic AMP systems controlled by P-adrenergic receptors both in Purkinje cells and in neurons of the granular layer of cerebellum. Another aspect of brain slice studies is relevant to the localization of cyclic AMP systems. Thus, adenine phosphoribosyltransferase, the enzyme responsible for incorporation of adenine into intracellular adenine nucleotides, was closely associated with cyclic AMP-generating systems in brain slices (cf. Daly, 1977). Adenosine incorporation into adenine nucleotides was much less closely associated with the cyclic AMP compartments. Labeling of synaptosomes, presumably the presynaptic elements, occurred more rapidly with adenosine than with adenine (Kuroda and Mcllwain, 1974). Such data provide further evidence for a postsynaptic

112

JOHN W. DALY

rather than presynaptic localization of cyclic AMP systems in brain tissue. Destruction of central noradrenergic presynaptic terminals with 6-hydroxydopamine had either no effect or resulted in an apparent adaptive increase in responses of norepinephrine-sensitive cyclic AMPgenerating systems in brain slices (Huang et al., 1973b; Kalisker et al., 1973; Dismukes and Daly, 1975b; Dismukes et al., 1976b; Skolnick and Daly, 1976a, 1977). Similar results pertain after destruction of ascending noradrenergic fibers by lesions of the medial forebrain bundle (Dismukes et al., 1975, 1976b). Levels of adenylate cyclase in homogenates of rat cerebral cortex were only marginally decreased by the B-hydroxydopamine treatment (Kalisker et al., 1973). Electrolytic or 6-hydroxydopamine lesions of the nigrostriatal dopaminergic pathway had no effect or apparently resulted in an increase in dopamine-sensitive adenylate cyclases in striatum (Krueger et al., 1976; Mishra et al., 1974; Von Voightlander et al., 1973). The lack of change or, in some instances, an apparent adaptive increase in catecholamine-sensitive cyclases after destruction of presynaptic terminals, is at least consonant with postsynaptic loci for the cyclic AMP systems. In a region of the substantia nigra containing dendrites of the dopaminergic nigrostriatal neurons, the dopamine-sensitive cyclase activity was unaffected by 6-hydroxydopamine-induced destruction of the dopaminergic neurons (Kebabian and Saavedra, 1976). Thus, the dopamine-sensitive cyclases must be associated with some other cell type in this brain area. One further aspect of sites of generation of cyclic AMP in the nervous system needs to be considered. Brain consists mainly of neurons and glia. The latter cells, the astrocytes and the oligodendroglia are more numerous than neurons and comprise about one-half of the total brain tissue. A variety of data with cultured glioma (astrocytoma) and neuroblastoma cells suggested that catecholamine-sensitive adenylate cyclases might, in brain, be associated primarily with astrocytes, not neurons. Indeed norepinephrine elicited a profound accumulation of cyclic AMP in cultures of fetal brain cells in which extensive proliferation of cells, presumably glial cells, had occurred (Gilman and Schrier, 1972). Norepinephrine elicited a much lower accumulation of cyclic AMP in reaggregation cultures in which proportions of neurons and glia are probably still similar to those of fetal brain (Seeds and Gilman, 1971). Studies with brain tissue have not resolved the neuron-glia question. It would appear likely that neurotransmitter-sensitive adenylate cyclase will be found to have roles in physiological regulation in both neurons and glia. Adenylate cyclase has been detected in homogenates of both neuron and gliaenriched fractions from rat brain (Palmer, 1973). Cyclase activity was lower in the homogenates from glia-enriched fractions. Norepinephrine,

CYCLIC NUCLEOTIDES I N THE NERVOUS SYSTEM

113

dopamine, and histamine-sensitive cyclases were reported from the neuron and glia-enriched preparations (Palmer, 1973, 1976; Palmer and Manian, 1976; Spiker et al., 1976). In cultured rat superior cervical ganglia, basal levels and responses of cyclic AMP-generating systems to isoproterenol were found to decrease markedly during culture (Cramer et al., 1973). In view of survival of postganglionic noradrenergic cell bodies and degeneration of interstitial cells and presynaptic terminals during culture, it was proposed that the P-adrenergic cyclases were associated in part with ganglionic glial cells. However, in other studies on superior cervical ganglion, the in vivo increase in cyclic AMP elicited by isoproterenol was found to be virtually lost when the noradrenergic cell bodies had been destroyed by prior administration of nerve growth factor or 6-hydroxydopamine (Otten et al., 1974). These treatments had little effect on basal levels of cyclic AMP. Dopamine-elicited accumulations of cyclic AMP in bovine superior cervical ganglion were subsesquently shown by immunofluorescent assay to occur primarily in the dendrites and cell bodies of the noradrenergic neurons (Kebabian et al., 1975a). Norepinephrine-elicited accumulations of cyclic AMP occurred in the postganglionic noradrenergic neurons, but also occurred in fibroblast and blood vessellike cells. In the superior cervical ganglion as in brain slices, radioactive adenine selectively labeled ATP compartments associated with cyclic AMP-generating systems (Lindl et al., 1975). In lumbar sympathetic ganglia from chick, norepinephrine (0.1 mM)- and dopamine (3 mM)-sensitive adenylate cyclase activity appeared by histochemical assay to be localized at both postsynaptic dendrites of the sympathetic neurons and at presynaptic “aminergic” nerve terminals (Hervonen and Rechardt, 1976). Isoproterenol has been reported to increase levels of cyclic AMP in desheathed frog sciatic nerves (Horn and McAfee, 1976). Accumulation of particulate adenylate cyclases, proximal to a constriction of chicken sciatic nerve, was indicative of significant axonal transport of the enzyme to distal cholinergic terminals (Bray et al., 1971). In summary, the majority of data on localization of cyclic AMPgenerating systems in nervous tissue is consonant with a postsynaptic localization, probably primarily in neurons but perhaps to some extent in glia. Presynaptic cyclic AMP-generating systems are probably present in certain types of neurons.

B. REGULATION OF ADENYLATE CYCLASES Cyclic AMP-generating systems in brain tissue appear important to central homeostatic mechanisms as evident in the adaptive changes in

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responsiveness of these systems which occur as a result of alterations in transsynaptic input of specific neurotransmitters (cf. Dismukes and Daly, 1976b, 1977). Such adaptation could involve changes in adenylate cyclases, phosphodiesterases, and cyclic AMP-dependent protein kinases, but such alterations are studied with difficulty in heterogeneous brain tissue. In pineal gland, during the day when noradrenergic input is low, levels of /3-adrenergic-sensitive adenylate cyclases were found to have undergone a compensatory increase, while during the night when noradrenergic input is elevated, P-adrenergic-sensitive cyclases had undergone a compensatory decrease (cf. Romero and Axelrod, 1975; Kebabian et al., 1975~).Isoproterenol-elicited decreases in pineal gland adenylate cyclase did not appear to involve protein synthesis. Adaptive changes in phosphodiesterases and protein kinases also occurred in pineal gland. The adaptive changes in adenylate cyclase activity which occurs in cultured neuroma cells on exposure to neurotransmitters which elevate cyclic AMP levels appeared in certain cell lines to involve cyclic AMP-elicited synthesis of a protein which inhibits adenylate cyclase (De Vellis and Brooker, 1974). In another cell line cyclic AMP-dependent reductions of cyclase activity did not require protein synthesis (Browning et al., 1976). Stimulation of cyclic AMP-generating systems of glioma cells by norepinephrine or prostaglandin resulted in specific rather than general reductions in responsiveness of adenylate cyclases (Perkins et al., 1975). 1. Norepinephrine

T h e effect of norepinephrine on formation of cyclic AMP has been studied extensively with brain slice preparations. Unfortunately, adenylate cyclases assayed with exogenous ATP in homogenates of brain retains little of the norepinephrine-mediated regulation of activity seen in brain slice preparations (cf. Daly, 1977). Certain investigators, most notably Von Hungen, Roberts, and co-workers, have been able to obtain small but reproducible amine-elicited stimulations of adenylate cyclase in brain homogenates through the use of EGTA-inhibited preparations (cf. Von Hungen and Roberts, 1974). Recently, the stimulation of asolubilired adenylate cyclase from bovine brain by norepinephrine and dopamine, but not by epinephrine or serotonin, was reported (Stellwagen and Baker, 1976). Norepinephrine-sensitive cyclic AMP-generating systems have been studied in cell-free vesicular preparations from guinea pig cerebral cortex and cerebellum (Chasin et al., 1974; Shimizu et al., 1975a) and from rat limbic forebrain (Horn and Phillipson, 1976). In the cortical preparations, the response to epinephrine was blocked by an a-adrenergic antagonist, while in the cerebellar preparations it was


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blocked by a P-adrenergic antagonist. In rat limbic forebrain, the response to norepinephrine was blocked by &antagonists and partially blocked by various phenothiazines and other antipsychotics such as clozapine and haloperidol. Thus, the nature of the adrenergic receptors modulating cyclic AMP generation in these cell-free preparations was quite consonant with data from brain slice preparations (see below). With slices from various brain regions, (nor) epinephrine reproducibly elicits a marked activation of cyclic AMP-generating systems. The nature of the noradrenergic receptors regulating formation of cyclic AMP differs greatly among brain regions and between species. Thus, in rat cerebral cortex, the response has been characterized as due to a mix of a- and P-adrenergic receptors (Perkins and Moore, 1973; Skolnick and Daly, 1976b), while in rat cerebellum only P-adrenergic receptors pertain (Skolnick et al., 1976; Schwabe and Daly, 1977). In guinea pig cerebral cortex, in contrast to rat, the activation of cyclic AMPgenerating systems by norepinephrine involved virtually only a-adrenergic receptors (Chasin et al., 1971, 1973; Sattin et al., 1975). In guinea pig cerebellum only P-adrenergic receptors pertained. In mice the nature of responses again differed, involving primarily P-adrenergic receptors in cerebral cortex (Schultz and Daly, 1973d) and a mix of aand /3-adrenergic receptors in cerebellum (Ferrendelli et al., 1975). Thus, in different brain regions and different species, the interaction of adrenergic agonists and antagonists with the function of norepinephrineresponsive cyclic AMP systems will differ markedly. I t follows that in z~ivo differences in the effects of such drugs might provide insights into the functional role of norepinephrine-sensitive cyclic AMP systems. The marked synergism between norepinephrine and adenosine with respect to activation of cyclic AMP-generating systems in brain slices appeared to involve mainly a-adrenergic receptors (Perkins and Moore, 1973; Schultz and Daly, 1973c,d; Perkins et al., 1975; Sattin et al., 1975; Skolnick and Daly, 1975b). The synergism was particularly striking in guinea pig cerebral cortical slices where a-adrenergic receptors predominate and where norepinephrine has virtually no effect on cyclic AMP levels except in the presence of adenosine. In rat cortical slices, a-adrenergic receptors appeared involved to a greater extent than were P-adrenergic receptors in the synergism between norepinephrine and adenosine, while in mouse cerebral cortical slices where P-adrenergic receptors predominate, there was no clear synergism between norepinephrine and adenosine. In rat caudate slices, 2-chloroadenosine and isoproterenol had greater than additive effects on cyclic AMP levels (Wilkening and Makman, 1975). Synergisms between norepinephrine and histamine were quite pronounced in guinea pig cerebral cortical

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slices (Huang et al., 1971, 1973a; Chasin et al., 1973; Schultz and Daly, 1973a) but were minimal in rabbit (Kakiuchi and Rall, 1968a) and rat (Huang et al., 1971; Palmer et al., 1973; Schultz and Daly, 1973d; French et al., 1975) cerebral cortical slices. It appeared that a histamine-elicited release of adenosine might be partially responsible for the synergism in guinea pig cortical slices. The physiological significance of the synergisms between adenosine and biogenic amines and between norepinephrine and histamine is unknown. Norepinephrine, based on studies with brain slices, can be expected to elicit accumulations of cyclic AMP in brain via interaction with both aand P-adrenergic receptors, methoxamine via interaction with a-receptors (Skolnick and Daly, 1975b), and isoproterenol via interaction with P-receptors. The a-component of norepinephrine responses is blocked by a-antagonists such as phentolamine, phenoxybenzamine, ergot alkaloids, and clonidine (Skolnick and Daly, 1975c, 1976a), while the ,&component is blocked by P-antagonists such as alprenolol, propranolol, dichlorisoproterenol, and sotalol (Skolnick and Daly, 197613). Fluphenazine appeared capable of blocking the P-adrenergic receptors associated with cerebellar Purkinje cells (Skolnick et al., 1976; Hoffer et al., 1976). Antipsychotic drufs such as clozapine, chlorpromazine, and haloper idol partially blocked the response of P-adrenergic-controlled cyclic AMP systems to norepinephrine in slices from limbic forebrain (Blumberg et al., 1976). In brain slices, dopamine-elicited accumulations of cyclic AMP were minimal so that it appeared unlikely that a significant portion of the norepinephrine response was due to activation of dopaminergic receptors. In bovine superior cervical ganglia, norepinephrine elicited accumulations of cyclic AMP via interaction with both a P-adrenergic receptor and via interaction with what appeared to be a dopaminergic receptor (Kebabian and Greengard, 1971; Kalix et al., 1974). In rat superior cervical ganglia, dopamine was relatively ineffective and norepinephrine appeared to elicit accumulations of cyclic AMP via interaction with aand P-adrenergic receptors (Lindl and Cramer, 1975).

2 . Dopamine In contrast to norepinephrine, the effects of dopamine on formation of cyclic AMP are studied most satisfactorily with brain homogenates rather than in brain slices. In homogenates or with synaptosomes, dopamine elicited a small but reproducible activation of adenylate cyclases which was unaffected by P-adrenergic antagonists and was blocked by antipsychotics such as ffuphenazine and haloperidol (cf, Iversen, 1975; Kebabian et al., 1975d). Dopamine-sensitive adenylate cyclases


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have been studied primarily in homogenates of striatum (caudate nucleus), but have also been studied in homogenates from substantia nigra (Kebabian and Saavedra, 1976; Phillipson and Horn, 1976), amygdala (Racagni and Carenzi, 1976; Weinryb and Michel, 1976), olfactory tubercle, and nucleus accumbens (Kebabian et al., 1972, 1975d; Clement-Cormier et al., 1974; Horn et al., 1974; Miller et al., 1974; Carenzi et al., 1975; Mishra et al., 1975; Weinryb and Michel, 1976). No pronounced differences in the potencies of dopamine antagonists in different brain regions have emerged from these studies. Thus, these studies have not clarified the factors involved in antipsychotic, extrapyramidal, and endocrinological effects of dopamine antagonists. Antipsychotic effects have been proposed to relate to antagonism of dopaminergic receptors in the mesolimbic system (olfactory tubercle, nucleus accumbens), extrapyramidal side effects to antagonism of receptors in striatum (caudate nucleus), and endocrinological side effects to antagonism of receptors in the median eminence of the hypothalamus. The relatively low potency of haloperidol and other butyrophenones as antagonists of dopamine-sensitive cyclases is also puzzling in view of their high potency as antipsychotics (cf. Laduron, 1976). In preparations from brain regions other than the limbic system, dopamine-elicited activations of adenylate cyclases have not been well characterized. Based on studies with brain homogenates dopamine, apomorphine, 2-amino-l,2,3,4-tetrahydronaphthalene, and probably lysergic acid diethylamide (Spano et al., 1975a; Von Hungen et al., 1975) can be expected to elicit accumulations of cyclic AMP in brain via interaction with dopaminergic receptors. Phenothiazines such as fluphenazine, trifluoperazine, chlorpromazine, and thioridazine, thioxanthenes such as flupenthixol and chlorprothixene, butyrophenones such as droperidol and haloperidol, and certain other antipsychotic agents such as butaclamol, pimozide, and clozapine block these dopaminergic receptors. The reason underlying the lack of responsiveness of dopaminesensitive cyclic AMP-generating systems in brain slices is not apparent. Dopamine did elicit small accumulations of cyclic AMP in slices of rat caudate nucleus (Fornetal., 1974; Kruegeretal., 1976) and in slices from cerebral cortex (Dismukes and Daly, 1974; Harris, 1976), but the presence of high concentrations of a phosphodiesterase inhibitor was required. In caudate slices, the dopamine response was blocked by fluphenazine, while the response in cortical slices has not been evaluated and might represent a partial activation of adrenergic receptors by dopamine. Adenosine did not appear to potentiate dopamine responses in rat (Schwabe, unpublished results) or guinea pig (Shimizu et al., 1970; Sattin et al., 1975) cerebral cortical slices. Dopamine in recent studies has

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been reported to elicit small accumulations of cyclic AMP in rat caudate (Wilkening and Makman, 1975) and mouse and rat cortical (Martres et al., 1975; Schwabe and Daly, 1977) slices in the absence of a phosphodiesterase inhibitor. I n adenine-labeled striatal synaptosomes where endogenous rather than exogenous ATP is serving as substrate for adenylate cyclase, dopamine was ineffective and formation of cyclic AMP appeared activated by interaction of catecholamines with a P-adrenergic receptor (Harris, 1976). In a preliminary report on dopamine and p-adrenergic-sensitive cyclic AMP-generating systems in homogenates from rat caudate nucleus, it was proposed that the dopamine-sensitive compartment lacked endogenous ATP and required lysis by hypotonic media to permit exogenous ATP access to catalytic sites of adenylate cyclase (Sheppard and Burghardt, 1976). The p-adrenergic-sensitive compartment was proposed to contain sufficient endogenous ATP which was lost on lysis and could not be effectively replaced by exogenous ATP. Whether similar lack of ATP in intact cells is responsible for minimal responses of dopamine-sensitive cyclic AMP-generating systems in caudate slices appears somewhat unlikely. However, in an intracellular cyclic AMP system with high levels of phosphodiesterases and low ATPgenerating capacity, high turnover of cyclic AMP after activation of cyclases by dopamine might result in depletion of ATP. The lack of responses to dopamine in terms of generation of radioactive cyclic AMP in adenine-labeled slices of rat striatum suggests that adenine did not significantly label the dopamine-sensitive compartment (Harris, i976). P-Adrenergic agonists did elicit generation of radioactive cyclic AMP in striatal slices. In bovine and rabbit superior cervical ganglion, dopamine activated cyclic AMP-generating systems via interaction with receptors antagonized both by a-adrenergic antagonists, and less effectively by central dopaminergic antagonists such as chlorpromazine and haloperidol (Kebabian and Greengard, 1971; Kalix et al., 1974; Roch and Kalix, 1975a,b),while in rat superior cervical ganglion, convincing evidence for the presence of dopamine-sensitive cyclases has not been obtained (cf. Lindl and Cramer, 1975). 3. Serotonin

Stimulations of cyclic AMP-generating systems by serotonin in homogenates from brain tissue and in brain slices have usually been marginal. Responses of adenylate cyclases to serotonin were reported in EGTA-inhibited homogenate preparations from rat hippocampus, anterior and posterior colliculi, midbrain, and hypothalamus (Von Hungen

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and Koberts, 1974). Methysergide antagonized the serotonin response. Serotonin has been reported to elicit small, often marginal accumulations of cyclic AMP in brain slices from rabbit cerebral cortex and cerebellum (Kakiuchi and Rall, 1968a,b), guinea pig cerebral cortex (Chasin et al., 197l), hippocampus, amygdala, diencephalon, and brain stem (Chasin et al., 1973), mouse cerebral cortex (Martres et al., 1975), monkey polysensory cortex (Skolnick et al., 1973), and human cerebral cortex (Shimizu el al., 1971; Kodama et al., 1973). In rat cerebral cortical slices serotonin had only marginal effects on cyclic AMP levels even in the presence of phosphodiesterase inhibitors (Dismukes and Daly, 1974: Dismukes et al., 1975; French et ad., 1975). Many of these studies have been with adenine-labeled slices, and it is possible that adenine does not effectively label serotonin-sensitive compartments. It is also possible that high phosphodiesterase activity or low activity of ATP-regenerating systems associated with serotonin-sensitive compartments make difficult the detection of serotonin responses in brain slices. Serotonin, although having no effect in cerebral cortical slices from control rats, did elicit a small response in slices from rats during withdrawal from chronic ethanol treatment (French et al., 1975). The response to serotonin was, however, blocked by methysergide, phenoxybenzamine, or propranolol. The apparent nonspecific nature of the response casts doubts on whether it really represents a specific activation of a serotonin-sensitive cyclic AMP system. Other marginal responses to serotonin may also represent nonspecific or indirect activation of cyclic AMP systems. Further studies on serotonin responses in brain tissue are clearly required before any speculations as to central function of serotonin-sensitive cyclic AMP systems are warranted. In guinea pig cerebral cortical slices, serotonin had, at best, marginal effects on cyclic AMP-generating systems except in the presence of exogenous adenosine (Shimizu et al., 1970; Huang et al., 1971; Huang and Daly, 1972; Schultz and Daly, 1973a,c; Dismukes et al., 197613). The synergism between adenosine and serotonin was antagonized in guinea pig cortical slices by methysergide. No synergism between serotonin and low concentrations of adenosine was reported in one study with guinea pig cerebral cortical slices (Sattin et al., 1975). In rat and mouse cortical slices, combinations of serotonin and adenosine had effects on accumulations of cyclic AMP no greater than that elicited by adenosine alone (Huang et al., 1973b: Schultz and Daly, 1973d; Skolnick and Daly, 1974b). Serotonin had no effect on cyclic AMP levels in cultured cells from fetal rat brain (Gilman and Schrier, 1972). Effects of serotonin on cyclic AMP levels in sympathetic ganglia of mammals have not apparently been investigated. Serotonin did increase cyclic AMP levels

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in the abdominal ganglion of the mollusc Aplysia califomica (Cedar and Schwartz, 1972; Cedar et al., 1972; Levitan et al., 1974) and in cockroach thoracic ganglion (Nathanson and Greengard, 1973, 1974). 4. Histamine T h e stimulation of cyclic AMP generation in brain tissue by histamine has been studied extensively in brain slices and more recently in homogenates. Histamine activated adenylate cyclases in homogenates from guinea pig neocortex, hippocampus, and striatum (Hegstrand et al., 1976). The 2-fold stimulation in hippocampal preparations was antagonized by an Hz-antagonist, metiamide, but not by an HI-antagonist, pyrilamine. Histamine elicited a small increase in adenylate cyclase activity in preparations from rabbit cortex and hippocampus (Palmer, 1973; Spiker et al., 1976). Histamine has no, or only marginal effects, on cyclases from rat brain (Burkard and Gey, 1968; Von Hungen and Roberts, 1973a,b; De Belleroche et al., 1974; Izumi et al., 1975a; Hegstrand et al., 1976) or on cyclases from monkey hippocampus (Weinryb and Michel, 1976). I n brain slices, the magnitude of responses to histamine had been largely predictive of the recent results with brain homogenates. Thus, histamine elicited large accumulations of cyclic AMP in slices from rabbit cerebral cortex (Kakiuchi and Rall, 1968a) and in slices from guinea pig cerebral cortex and hippocampus (Chasin et al., 1973; Rogers et al., 1975), while in slices from rat and mouse cerebral cortex or hippocampus minimal responses pertained (Krishna et al., 1970; Schultz and Daly, 1973d; Skolnick and Daly, 1974b, 1975a; Dismukeset al., 1975).Characterization of the nature of the histaminergic receptors regulating cyclic AMP generation in brain tissue is far from complete. However, studies with H1-agonists such as 2-methylhistamine and 2-aminoethylthiazole, with the He-agonist 4-methylhistamine, with HI-antagonists such as brompheniramine, pyrilamine, and diphenhydramine, and with H2antagonists such as metiamide provided evidence that both HI- and H2histaminergic receptors are involved in activation of cyclic AMP systems in brain tissue. In guinea pig cerebral cortical and hippocampal slices a mix of HI- and H2-receptors appeared involved in activation of cyclic AMP systems (Baudry et al., 1975; Rogers et al., 1975; Dismukes et al., 1976a). In rat cortical slices, the results were less conclusive because of the magnitude of the small response. H2-receptors, however, appeared to be primarily involved (Dismukes et al., 1975). Histamine responses in chick cortical slices involved mainly Hz-receptors (Nahorski, et al., 1974). Responses to histamine in other species or brain regions have not been well characterized. Histamine had no effect on cyclic AMP levels in cultured cells from fetal rat brain (Gilman and Schrier, 1972). The syner-


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gism between histamine and adenosine in guinea pig cortical slices appeared to involve mainly HI-receptors (Dismukes et al., 1976a). The apparent proportion of HI- and Hz-receptor contributions to histamine responses will, therefore, be strongly dependent on endogenous levels of adenosine in the brain slice preparations. Recently, clonidine (EC,,, 50 1.1M ) has been proposed to elicit accumulations of cyclic AMP in guinea pig hippocampal slices via interaction with an Hz-receptor (Audiger et al., 1976). In chick cortical slices histamine elicits a large accumulation of cyclic AMP via interaction with an Hz-receptor, but clonidine had no effect (Nahorski et al., 1975b). In rat superior cervical ganglion, histamine activated cyclic AMPgenerating systems via interaction with a receptor that was antagonized by both HI- and Hz-antagonists (Lindl and Cramer, 1974). In bovine superior cervical ganglion, histamine-elicited accumulations of cyclic were antagonized by an Hz-antagonist (Kebabian et al., 1975b; Roch and Kalix, 1975a). 5. Adenosine

Activation of cyclic AMP-generating systems by adenosine occurs, apparently at an extracellular site, in brain slices from a variety of species and brain regions (cf. Mah and Daly, 1976; Daly, 1977). Theophylline, isobutylmethylxanthine, caffeine, and certain adenosine analogs such as 2'-deoxyadenosine, act as adenosine antagonists in brain slices. Other adenosine analogs such as N"-phenylisopropyladenosineand 2-chloroadenosine are agonists. In addition to stimulatory effects on cyclic AMP-generating systems, adenosine has striking effects on the responsiveness of amine-sensitive systems. Synergistic interactions of adenosine with norepinephrine, serotonin, histamine (see above), and glutamate (Shimizu et al., 1974, 1975b) have been reported. In addition, adenosine has been reported to prevent refractoriness of amine-sensitive cyclic AMP-generating systems in guinea pig cerebral cortical slices (Schultz and Daly, 1973b; Schultz, 1975a,b). Adenosine appeared to be involved as an intermediary in the activation of cyclic AMP-generating systems in brain slices by depolarizing agents and certain metabolic inhibitors (cf. Daly, 1977). Although firmly established as an important factor in the regulation of cyclic AMP generation in brain slices, activation of adenylate cyclases by adenosine has as yet not been demonstrated in homogenates of brain tissue prepared in isotonic sucrose (Sattin and Rall, 1970; McKenzie and Bar, 1973). It should be noted that exogenous ATP employed for assay of adenylate cyclase in such preparations will undergo enzymatic conversion to adenosine. Basal levels of adenylate cyclase in homogenates of rat

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caudate nucleus were indeed lower when assayed in the presence of the adenosine antagonists theophylline and isobutylmethylxanthine, than when assayed in the presence of other phosphodiesterase inhibitors which are not adenosine antagonists such as papaverine or dipyridamole (Fredholm et al., 1976). Theophylline reduced levels of cyclic AMP in rat cerebral cortical synaptosornes (De Belleroche et al., 1974). It should be noted that adenosine is converted to an inactive metabolite inosine by the action of the enzyme adenosine deaminase. This enzyme occurs in various brain regions (Sun et al., 1976) and has been used for the study of adenosine-dependent activation of cyclic AMP systems in brain slices (Huang et al., 1973a; Schwabe et al., 1977). Adenosine has been demonstrated to activate adenylate cyclases in homogenates of blood platelets ( Haslarn and Lynham, 1972), cultured neuroblastoma (Blume and Foster, 1975, 1976a,b; Penit et al., 1976), and glioma (Clark et al., 1975; Perkins et al., 1975; Clark and Seney, 1976) cells, and undoubtedly conditions will be found under which activation of adenylate cyclases of synaptosome preparations by adenosine can be demonstrated. Indeed, in the vesicular preparations obtained after homogenization of guinea pig cerebral cortex in physiological medium, adenosine (ECoo 10 p M ) elicited a marked stimulation of cyclic AMP-generating systems (Chasin et al., 1974; Shimizu etal., 1975a). In cultured cells from fetal rat brain, adenosine elicited accumulations of cyclic AMP (Gilman and Schrier, 1972; Sturgill et al., 1975). Unlike results with brain slices, combinations of catecholamines and adenosine did not have synergistic effects on cyclic AMP generation in cultured cells. In rat superior cervical ganglia, effects of adenosine on cyclic AMP levels were not detected (Roch and Kalix, 1975b). Furthermore, unlike results with brain slices, theophylline did not antagonize, but was instead required in order for depolarizing agents to elicit accumulations of cyclic AMP (Kalix and Roch, 1975; Roch and Kalix, 1975b; Webbetal., 1975). 6. Acetylcholine Acetylcholine had no effect or inhibited the activity of adenylate cyclases from rat brain (Von Hungen and Roberts, 1973a,b; Duffy and Powell, 1975) and rat striatum (Walker and Walker, 1973). In brain slices, acetylcholine or cholinergic agonists had virtually no effect on levels of cyclic AMP (cf. Daly, 1977), although in some instances a slight reduction pertained in the presence of acetylcholine (Kuo et al., 1972). In bovine superior cervical ganglion, acetylcholine and cholinergic agonists enhanced the levels of cyclic AMP (McAfee et al., 1971; Greengard et al., 1972; Kalix et ad., 1974). The mechanism appeared to involve an acetylcholine-induced release of doparnine from ganglionic

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interneurons and a resultant stimulation of cyclic AMP-generating systems in postganglionic noradrenergic cell bodies by the released dopamine. Acetylcholine, however, slightly inhibited the accumulation of cyclic AMP elicited by exogenous dopamine (Kebabian et al., 1975b). In rat superior cervical ganglion acetylcholine had no significant effect on levels of cyclic AMP (Cramer and Lindl, 1974). Acetylcholine had no effect on levels of cyclic AMP in peripheral neurons (Kebabian et nf., 1975b). 7. Prostaglandins Prostaglandin-sensitive cyclic AMP-generating systems have been demonstrated in homogenates and slices of brain tissue, but quite high concentrations have been required to elicit significant responses. Early studies with the low concentrations of prostaglandins, which are effective in other tissues, had no effect in brain preparations (Schmidt et al., 1970; Robison et al., 1970; Zanella and Rall, 1973). In recent years stimulation of adenylate cyclases in rat brain homogenates by prostaglandins of the E series has been reported (Collier and Roy, 1974a,b; D ~ f f yand Powell, 1975). Concentrations of prostaglandin from 2.5 to 150 CLMwere employed. Morphineand other narcotic analgesics were effective antagonists of prostaglandin-elicited activation of brain adenylate cyclases. Other groups, however, have been unable under a variety of conditions to demonstrate significant activation of adenylate cyclases by prostaglandins in homogenates of rat brain, cerebral cortex, or caudate nucleus (Van Inwegen et al., 1975; Tell et nl., 1975). Similarly, in brain slices reports on prostaglandin-sensitive cyclic AMP systems have not been definitive. Very high concentrations of prostaglandins of the E series (EC,, 20 P M ) did elicit significant accumulations of cyclic AMP in slices from rat cerebral cortex (Berti et al., 1972; Kuehl et al., 1972; Dismukes and Daly, 1975a). Prostaglandin antagonists such as 7-oxa- 13-prostynoic acid and the dibenzooxazepine hydrazine, SC 19220, had no effect of responses to prostaglandin E, in rat cortical slices. Morphine slightly potatiated the prostaglandin response, in contrast to its inhibitory effects in brain homogenates (Collier and Roy, 1974a,b) and cultured cells (cf. Sharma et al., 1975). Tentative evidence for partial antagonism of’ norepinephrine and isoproterenol-elicited accumulations of cyclic AMP by prostaglandin El in rat cortical slices has been reported (Dismukes and Daly, 1975a). However, in guinea pig cerebellar slices, prostaglandin E, did not antagonize the norepinephrine response (Ohga and Daly, 1977a). In view of the lack of potency of prostaglandins in brain preparations, the relevance of these efTects to physiological roles of prostaglandin in the central nervous system must be subjected to further investigation. Even at high concentrations, prostaglandin El had no apparent

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effect on cyclic AMP levels in cerebral cortical slices from species other than rat (Berti et al., 1972). Prostaglandin El ( E G O< 3 p M ) elicited large accumulations of cyclic AMP in cultured cells from fetal rat cerebral cortex (Gilman and Schrier, 1972). Effects of prostaglandin on cyclic AMP-generating systems in ganglia have apparently not been studied. 8. Amino Acids

The status of putative amino acid neurotransmitters, particularly glutamate and aspartate, with respect to function of cyclic AMPgenerating systems in brain tissue, is as yet not completely resolved. Neither glutamate (Shimizu et al., 1974) nor y-aminobutyrate (Von Hungen and Roberts, 1973a,b) had any effect on adenylate cyclase activity in brain homogenates. Both glutamate and aspartate at very high concentrations (EGO1.5 mM) stimulated cyclic AMP generation in slices from cerebral cortex and cerebellum of various species (Ferrendelli et al., 1974, 1975; Shimizu et al., 1974, 1975b,c; Mah and Daly, 1976; Schmidt et aE., 1976). y-Aminobutyrate either had no effect or slightly reduced levels of cyclic AMP in brain slices. y-Aminobutyrate has been reported to antagonize responses to norepinephrine in rat cortical slices (French et al., 1975) and mouse cerebellar slices (Ferrendelli et al., 1975). Glycine had only marginal effects on cyclic AMP levels in brain slices. It would appear possible that enhanced formation and release of adenosine are responsible in incubated brain slices for the responses to glutamate and aspartate. The excitatory amino acid, glutamate, might, through stimulation of neuronal activity o r through ATP-dependent uptake in brain slices, deplete ATP and enhance adenosine release (cf. Pull and McIlwain, 1975). The inhibitory amino acid y-aminobutyrate might, by reduction in neuronal activity, tend to reduce adenosine release. Theophylline, an adenosine antagonist, did block glutamate-elicited accumulations of cyclic AMP in brain slices. However, another adenosine antagonist, 2’-deoxyadenosine, had no effect on glutamate responses. In addition, the response to combinations of glutamate and adenosine was greater than additive. Glutamate (EC,, 20 p M ) elicited significant accumulations of cyclic AMP in vesicular preparations obtained after homogenization of guinea pig cerebral cortical tissue in physiological medium (Shimizu et al., 1957a). Clearly, further studies are needed to resolve the mechanism and significance of glutamate-elicited accumulations of cyclic AMP in brain slices. At least two interpretations of the results are possible: one, that glutamate directly activates cyclic AMP systems and that theophylline is not a specific adenosine antagonist. It would appear more likely that glutamate elicits “release” of adenosine in brain slices and vesicular brain preparations and that “released”


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adenosine is responsible for stimulation of cyclic AMP-generating systems, hence the blockade by theophylline. I n order to explain the lack of antagonism of glutamate responses with 2'-deoxyadenosine and at least additive responses to combinations of glutamate and adenosine, it must be proposed that exogenous adenosine and 2'-deoxyadenosine d o not readily reach the sites at which glutamate elicits release of adenosine. It should be noted that if glutamate does directly stimulate cyclic AMPgenerating systems in brain slices then it, along with substance P, will represent the only excitatory putative neurotransmitters which appear to have such activity. All other substances-norepinephrine, dopamine, serotonin, histamine, adenosine, and prostaglandin-which stimulate cyclic AMP systems have pronounced inhibitory effects on electrical activity of certain central neurons. 2,3-Diaminopropionate has been reported as a specific antagonist of glutamate-elicited accumulations of cyclic AMP (Shimizu et al., 1975~).Cysteine sulfinate and kainic acid represent glutamate analogs which stimulate cyclic AMP accumulation in brain slices (Shimizu et al., 1974, 1975b,c; Schmidt et al., 1976). At least in the case of kainic acid the mechanism of stimulation of cyclic AMP-generating systems appeared different from that of glutamate. 9. Peptides

Substance P, an extremely potent excitatory peptide, has been reported to stimulate adenylate cyclase activity in homogenates from rat brain (Duffy and Powell, 1975). Enkephalins in the presence of a peptidase inhibitor caused a small reduction in cyclic AMP levels in slices from rat cerebral cortex (Minneman and Iversen, 1976b). In rat brain homogenates, enkephalins inhibited formation of cyclic AMP (H. 0. J. Collier and A. C. Roy, cited in Goldstein, 1976). The relationship of the central analgesic and behavioral activity of enkephalins and of other larger endorphins to cyclic AMP mechanisms is at present under active investigation. In rat superior cervical ganglia, nerve growth factor elicited a transient increase in levels of cyclic AMP (NikodiJevicet al., 1975). Angiotensin, a ganglionic depolarizing agent, had no effect on cyclic AMP levels in bovine superior cervical ganglia (Kebabian et al., 1975b). Inhibitory peptides from neurosecretory cells of the mollusc Aplysia cal$mica increased levels of cyclic AMP in the neuropil of Aplysia abdominal ganglia (Treistman and Levitan, 1976). 10. Macromolecular Factors Heat-stable factors from brain have been reported to activate adenylate cyclases (Kauffmanet al., 1972; Izumietal., 1975a, 1976). The heat-

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stable calcium-binding protein which is required for activation of calcium-dependent phosphodiesterases has recently been established as an activator of adenylate cyclase in brain homogenates (Brostrom et al., 1975; Cheunget al., 1975a; Lynch et al., 1976). This activator protein(s) was present in brain homogenates nearly equally distributed between soluble and membrane fractions (Cheung et al., 1975a; Gnegy et al., 1976a). The activator protein was released from membrane fractions of rat brain or striatum after cyclic AMP-dependent phosphorylation of a membrane protein (Gnegy et al., 1976a,b). It is tempting to speculate that in the membrane the activator protein is associated with adenylate cyclases, thus rendering enzymatic activity sensitive to regulation by calcium, and that as a result of cyclic AMP generation, a feedback control mechanism elicits a release of the activator protein into the cytosol where it and calcium ion can activate hydrolysis of cyclic AMP by soluble phosphodiesterases. In a preliminary communication, the activation of adenylate cyclase in membrane fractions from caudate nucleus by dopamine was reported to be greatly reduced after cyclic AMP-dependent release of the activator protein from the membrane (Gnegy et al., 1 9 7 6 ~ )Levels . of activator protein in caudate membranes were increased after treatments of animals with agents such as a-methyltyrosine, reserpine, clozapine, and haloperidol, which cause in vivo supersensitivity to dopaminergic agonists. Further studies on the role of macromolecular activators of adenylate cyclase, for example ubiquitin, an ubiquitous activator of P-adrenergic receptor-controlled cyclases (Goldstein et al., 1975) and on inhibitory factors (Levey et al., 1975; Izumi et al., 1975b), should provide valuable insights into the complex intracellular control of cyclic AMP systems in the nervous system. 11. Calcium Ions The activity of adenylate cyclases in homogenates was inhibited by high concentrations of calcium ions, but was activated by low concentrations of calcium, apparently through interaction with a high-affinity calcium-binding site (cf. Johnson and Sutherland, 1973; MacDonald, 1975). EGTA, by chelation of calcium ions associated with adenylate cyclases, markedly reduced enzymatic activity. The relationship of this calcium dependency to the presence of calcium-dependent activator protein has not been established. In brain slices, responses of cyclic AMP-generating systems to biogenic amines and adenosine were significantly influenced by removal of extracellular calcium with EGTA (cf. Schwabe and Daly, 1977; Schwabe et al., 1977). Responses to amines were reduced in the absence of extracellular calcium, while responses to adenosine appeared

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somewhat enhanced. The a-adrenergic component of responses to norepinephrine in rat brain slices appeared to be completely dependent on the presence of extracellular calcium ions. 12. G T P .

The regulation of activity and hormone responsiveness of adenylate cyclases by GTP has been extensively studied with cell-free preparations. In such preparations GTP or a stable analog, guanylylimidodiphosphate (Gpp(NH)p),activates adenylate cyclases and often potentiates responses to biogenic amines and other activators. In recent studies with adenosine-sensitive and prostaglandin-sensitive adenylate cyclases from neuroblastoma cells, a model was proposed in which activation of adenylate cyclase was dependent upon a relatively irreversible binding of GTP to a guanine nucleotide site (Blume and Foster, 1976a). Hydrolysis of GTP to GDP on the enzyme was proposed to yield an inactive cyclaseGDP complex. Dissociation of GDP was proposed to be facilitated by “hormones” (adenosine or prostaglandin) to yield adenylate cyclases with the guanine nucleotide binding site again available for activation by GTP. In this model GTP would be the physiological activator of adenylate cyclase with hormones merely facilitating dissociation of inhibitory GDP from the enzyme from the cyclase. In unstimulated systems the adenylate cyclase would be present mainly in the inactive GDPcomplexed form. Such a model is consonant with GTP control of adenylate cyclase even in intact cells where levels of GTP (100-200 p M ) are far greater than the concentrations required for activation of adenylate cyclase in cell-free preparations. It would appear possible that under conditions in which GTP levels are reduced in a morphological compartment by activation of guanylate cyclase, associated adcnylate cyclases would become refractory to hormonal activation. Such inhibitory interrelationships of cyclic AMP and cyclic GMP-generating systems have been proposed (Goldberg et al., 1973, 1975), but have not been clearly demonstrated in either brain slices (Ohga and Daly, 1977a) or in ganglia (Kebabian et al., 1975b). Methods for the study of regulation of cyclic AMP-generating systems by GTP in intact cells are, unfortunately, not available.

C. LOCALIZATION OF PHOSPHODIESTERASES High levels of phosphodiesterases were associated in homogenates of brain tissue with both soluble and membrane fractions, including soluble and membrane fractions from lysed synaptosomes (De Robertis et al., 1967; Weiss and Costa, 1968; Beavo et nl., 1970; Gaballah and Popoff,

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1971a). The presence of variety of phosphodiesterase isozymes some of which hydrolyze both cyclic AMP and cyclic GMP and some of which are more or less specific for either cyclic AMP or cyclic GMP complicates studies of this enzyme in both brain (Uzunov and Weiss, 1972a; Fertel and Weiss, 1974; Weiss et al., 1974; Kakiuchiet al., 1975a,b; Pledgeret al., 1975) and ganglia (Boudreau and Drummond, 1975; Lindlet al., 1976). Calcium-dependent activator protein for calcium-dependent phosphodiesterases occurred nearly equally distributed between soluble and membrane fractions from brain (Cheung et al., 1975a,b; Gnegy et al., 1976a) and underwent a cyclic AMP-dependent release from synaptosome membranes (Gnegy et al., 1976a,b). The morphological localization of phosphodiesterase in tissue slices from brain has been studied to a limited extent. Cyclic AMP phosphodiesterases assayed histochemically with high concentrations of cyclic AMP appeared localized at postsynaptic dendritic sites of neurons in rat cerebral cortical slices (Florendo et al., 1971) and at postsynaptic dendritic sites in the molecular layer of developing mouse brain (Adinolfi and Schmidt,. 1974). In earlier histochemical studies with rabbit, phosphodiesterase activity was found associated with glial cells, synaptic areas of neurons, in the neuropile of the cerebral cortical plexiform layer, and in the cerebellar molecular layer which contains Purkinje cell dendrites (Shanta et al., 1966). Phosphodiesterase activity assayed in homogenates from gray matter of rabbit cerebral cortex or olfactory bulb did not appear uniquely associated with particular layers (Breckenridge and Johnston, 1969). Phosphodiesterase activity in cerebral cortex was not decreased after lesions of the ascending noradrenergic, serotoninergic, and histaminergic nerve fibers of the medial forebrain bundle or after denervation of the superior cervical ganglia (Breckenridge and Johnston, 1969). Phosphodiesterase activity in rat cerebral cortex was only marginally decreased after destruction of presynaptic noradrenergic terminals with 6-hydroxydopamine (Kalisker et al., 1973). A soluble phosphodiesterase increased proximal to a constriction of the chicken sciatic nerve, suggesting axonal transport of phosphodiesterases to distal cholinergic terminals (Bray et al., 1971). In summary, phosphodiesterases appear to be localized to a significant extent at postsynaptic neuronal sites. The enzyme is probably also associated with glia and presynaptic terminals. D. REGULATION OF PHOSPHODIESTERASES

A role for a calcium-dependent activator protein in the control of calcium-dependent cyclic AMP and cyclic GMP phosphodiesterases has


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been apparent since the discovery of this activator in 1970 (Kakiuchi and Yamazaki, 1970; Cheung, 1970). The activator has been studied extensively (cf. Teshima and Kakiuchi, 1974; Wickson et al., 1975; Brostrom and Wolff, 1976; Liu and Cheung, 1976; Uzunov et al., 1976) and has been recently shown to be capable of activating both adenylate cyclases and phosphodiesterases (Brostrom et al., 1975; Cheung et al., 1975a). Translocation of the activator provides an attractive basis for feedback control of cyclic AMP formation. An activator isolated from porcine brain appeared to be a phosphoprotein (Wolff and Brostrom, 1974), while activator isolated by other groups from rat, porcine, or bovine brain did not contain phosphate (Liu and Cheung, 1976; Watterson et al., 1976). A factor from rat brain activated phosphodiesterases of synaptosomes but not soluble phosphodiesterases (Izumi et al., 1976). Certain phospholipids have been reported to activate the calcium-dependent phosphodiesterases (Wolff and Brostrom, 1976). A protein isolated from bovine rod outer segments inhibited phosphodiesterases from rat brain (Dumler and Etingof, 1976). Similar heat-stable inhibitory proteins were present in brain homogenates (T. Kanamori, C. R. Creveling, and J. W. Daly, unpublished results). Inhibition of phosphodiesterases by calcium ions has been reported (cf. Cheung, 1971; Boudreau and Drummond, 1975). Inhibitions of cyclic AMP phosphodiesterases by cyclic GMP and vice versa represent a possible interrelationship between metabolism of the two cyclic nucleotides (Goldberg et al., 1970; Roberts and Simonsen, 1970; Weiss et al., 1974; Weiss and Greenberg, 1975). Stimulations of cyclic AMP phosphodiesterases by cyclic GMP have been reported (Beavoetal., 1971; Boudreau and Drummond, 1975; Hidakaetal., 1975). Regulation of hydrolysis of cyclic AMP in intact cells is obviously quite complex. In addition, adaptive changes in apparent levels of phosphodiesterase appear responsible in part for alterations in responsiveness of cyclic AMP-generating systems to changes in neurotransmitter input. Such adaptation in responsiveness is difficult to study in heterogeneous tissue such as brain (cf. Dismukes and Daly, 1976b; Daly, 1977). I n pineal gland, refractoriness of postsynaptic cyclic AMP-generating systems to isoproterenol was due in part to an adaptive increase in phosphodiesterases (Oleshansky and Neff, 1975). During the night when noradrenergic input to the pineal gland is elevated, levels of phosphodiesterases have apparently undergone a compensatory adaptive increase (Minneman and Iversen, 1976a). The mechanisms involved in adaptive changes in phosphodiesterase activity appear to involve cyclic AMP-dependent mechanisms and, at least in cultured neuroma cells, to involve protein synthesis (Schwartz and Passonneau, 1974; Browning et al., 1976). In certain cell lines, exposure to stimulants of cyclic AMP-

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dependent generating systems did not result in adaptive changes in phosphodiesterases (De Vellis and Brooker, 1974; Perkins et al., 1975).

E. INHIBITORS A N D ACTIVATORS OF PHOSPHODIESTERASES Drugs which specifically inhibit or active phosphodiesterases would provide extremely valuable tools for the study of functional roles of cyclic AMP and cyclic GMP in the central and peripheral nervous system. Unfortunately, most known inhibitors of phosphodiesterases have side effects and few appear to be specific with respect to inhibition of specific phosphodiesterase isozymes. The situation is even less satisfactory with respect to agents which activate phosphodiesterases. Thus, imidazoles, generally accepted as phosphodiesterase activators, are maximally effective with brain phosphodiesterases only at 10-20 mM concentrations (cf. Cheung, 1971). Inhibition of phosphodiesterases by imidazole has also been reported (Goldberg et al., 1970). 1 . MethylxanthineJ Theophylline and caffeine have low potencies as inhibitors of phosphodiesterases from brain (Weinryb et al., 1972; Fredholm et al., 1976; Levin and Weiss, 1976). Thus, it remains rather doubtful that the central pharmacological activities of these two compounds are primarily due to inhibition of phosphodiesterases. Isobutylmethylxanthine was manyfold more potent than theophylline or caffeine as a phosphodiesterase inhibitor in brain preparations (cf. Fredholm et aL, 1976). Isobutylmethylxanthine appeared to be more potent as an inhibitor of calciumdependent phosphodiesterases than of crude phosphodiesterases from rat cerebral cortex (DuMoulin and Schultz, 1975). Isobutylmethylxanthine had similar potency with regard to inhibition of hydrolysis of cyclic AMP and cyclic GMP (Fredholm et al., 1976). Methylxanthines, in addition to their activity as phosphodiesterase inhibitors, are active antagonists of adenosine-sensitive cyclic AMP systems in brain slices (cf. Mah and Daly, 1976).

2 . Benzodiazepines The centrally active benzodiazepines, which include diazepam, chlordiazepoxide, and medazepam, were potent inhibitors of phosphodiesterases from brain (Weinryb et al., 1972; Dalton et al., 1974; DuMoulin and Schultz, 1975; Levin and Weiss, 1976). N o striking selectivity, with respect to inhibition of phosphodiesterases from different brain regions, was noted for this class of inhibitor. Benzodiazpeines inhibited hydrolysis of cyclic AMP and cyclic GMP by brain phosphodies-


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terases equally effectively. Pharmacological activities of benzodiazepines may not be, in all cases, linked to inhibition of phosphodiesterases. Diazepam and chlordiazepoxide have, for example, been demonstrated to be potent, presumably directly acting antagonists of y-aminobutyrate-elicited inhibition of Purkinje cells in explants of rat cerebellum (Gahwiler, 1976). 3. Phenotliiazines

Although relatively potent as phosphodiesterase inhibitors, compounds of this class such as fluphenazine, trifluoperazine, and chlorpromazine have too many other activities, for example as dopaminergic antagonists, inhibitors of adenylate cyclases (Uzunov and Weiss, 1972b; Palmer and Manian, 1974a,b),and inhibitors of norepinephrine uptake, to be useful as specific tools for in situ investigation of phosphodiesterases. The phenothiazines were very potent inhibitors of calciumdependent phosphodiesterases (Uzunov et al., 1974; Weiss et al., 1974; Weiss and Greenberg, 1975; Levin and Weiss, 1976). I t would appear that trifluoperazine, chlorpromazine, and other antipsychotics such as pimozide inhibited calcium-dependent phosphodiesterases primarily through competitive antagonism of the activation by the calciumdependent activator protein. Chlorpromazine was much more potent in inhibiting hydrolysis of cyclic AMP than in inhibiting hydrolysis of cyclic GMP by calcium-dependent phosphodiesterases. Trifluoperazine inhibited phosphodiesterases from rat cerebrum and brain stem more effectively than phosphodiesterases from cerebellum (Uzunov and Weiss, 1971).

4. Papaverine The alkaloid papaverine is a relatively potent inhibitor of brain, phosphodiesterases, but in view of other pharmacological activities, such as inhibition of ATP-generating systems and inhibition of uptake of adenosine, it is not a particularly selective tool for the in situ study of phosphodiesterases. Inhibition of cyclic AMP phosphodiesterases by papaverine was competitive at low concentrations of the alkaloid and noncompetitive at high concentrations (Weiss, 1975). Papaverine was more potent as an inhibitor of certain calcium-independent enzymes than as an inhibitor of the calcium-dependent enzymes. Particulate enzymes appeared to be inhibited more readily than soluble enzymes by papaverine (Furlanut et al., 1973; Fredholm et al., 1976). Papaverine was a potent inhibitor of hydrolysis of both cyclic AMP and cyclic GMP by calcium-dependent phosphodiesterase (Kakiuchi et al., 1975a; Weiss, 1975; Fredholm et al., 1976.)

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5. 1-H-Pyrazolo[3,4b]pyridines This class of compounds, exemplified by SQ 20009, represents a group of extremely potent inhibitors of brain phosphodiesterases (Weinryb et al., 1972; Hess et al., 1975; Kakiuchi et al., 1975a). S Q 20009 was a potent inhibitor of hydrolysis of cyclic GMP by calcium-dependent phosphodiesterases (Kakiuchi et al., 1975a). I n studies with brain slices SQ 20009 had, unlike other potent phosphodiesterase inhibitors, little effect on accumulations of cyclic AMP elicited by biogenic amines, adenosine, or glutamate (Schultz, 1974a,b; Mah and Daly, 1976). Potentiations of amine responses in brain slices by relatively high concentrations of SQ 20009 have been reported by other groups (Forn et al., 1974; Hess et al., 1975; Wilkening and Makman, 1975). The potency of SQ 20009 in intact brain cells would appear much lower than would have been predicted based on its potency with cell-free preparations. 6. Dipyridamole This polar compound probably penetrates intact cells to a limited extent, thus circumscribing its usefulness as a tool for the in situ study of phosphodiesterases. With brain phosphodiesterases, dipyridamole was a relatively potent inhibitor (Weinryb et al., 1972; Fredholm et al., 1976). Dipyridamole was ineffective with cyclic GMP phosphodiesterases. Potentiation of amine responses by dipyridamole in brain slices appeared mainly due to inhibition of adenosine uptake rather than inhibition of phosphodiesterases (Huang and Daly, 1974). 7. Dialkoxybenzyl-2-imidazolzdinones This widely used class of phosphodiesterase inhibitors is exemplified by RO 20-1724. In brain homogenates RO 20-1724 was not nearly as potent an inhibitor of phosphodiesterases as isobutylmethylxanthine, diazepam, papaverine, or SQ 20009 (Sheppard et al., 1972; DuMoulin and Schultz, 1975). RO 20-1724 had little or no effect on cyclic GMP phosphodiesterases (Sheppard et al., 1972; Schwabe et al., 1976). RO 20- 1724 was quite effective in potentiating amine and adenosine-elicited accumulations of cyclic AMP in brain slices (Schultz, 1974a,b; Mah and Daly, 1976; Schwabe el al., 1976). In part, potentiation of amine responses by RO 20-1724 in brain slices appeared due to inhibition of uptake of endogenous adenosine resulting in synergistic amineadenosine interactions (Mah and Daly, 1976; Schwabe et al., 1977). 8. Dialkoxyphenyl-2-pyrlidones

Recently, a dialkoxyphenyl-2-pyrrolidone,ZK 627 11, has been proposed as the phosphodiesterase inhibitor of choice for study of the enzyme in situ in the nervous system (Schwabe et al., 1976b). ZK 6271 1 was


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many-fold more potent as an inhibitor of calcium-dependent phosphodiesterases from brain than RO 20-1724 and was much more potent than RO 20- 1724 in potentiating norepinephrine and adenosine-elicited accumulations of cyclic AMP in brain slices. Adenosine mechanisms appeared to have only a minor role in potentiation of amine responses by ZK 6271 1 in brain slices. ZK 6271 1 had little effect on cyclic GMP phosphodiesterases. F. CYCLICAMP-DEPENDENT PROTEINKINASES

Cyclic AMP-dependent protein kinases in homogenates from brain tissue were present both in soluble fractions, and associated with particulate fractions including synaptosomes (Gaballah et al., 1971; Maeno et al., 1971; Gaballah and Popoff, 1971b; Uno et al., 1976) and microtubules (cf. Sloboda et al., 1975; Rappaport et al., 1976). Synaptic membranes from synaptosomes contained high levels of cyclic AMP-dependent protein kinases. Only low levels of protein kinase activity were associated with nuclei, although in other tissues such as liver, protein kinases from nuclei appeared involved in phosphorylation of histone and nonhistone chromatin proteins (cf. Kish and Kleinsmith, 1974). Low- and highaffinity binding sites for cyclic AMP were present in synaptic membranes (Weller and Rodnight, 1975). Presumably, the high-affinity binding sites represent, at least in part, sites on the regulatory unit of cyclic AMPdependent protein kinases. A number of different protein kinases were present in brain (cf. Miyamoto et al., 197 1 ; Inoue et al., 1973; Kuo, 1974; Takahashi et al., 1975; Uno et al., 1976). Soluble cyclic AMP-dependent protein kinases obtained from membrane and cytosol fractions appeared distinct in terms of physical properties and substrate specificities (Uno et al., 1976). Cyclic AMPdependent protein kinases have been divided into two types (Corbin et al., 1975). Type I enzymes were only slowly dissociated into regulatory and catalytic subunits by histunes and reassociated quite rapidly after cyclic AMP-elicited dissociation. Type I1 enzymes were dissociated relatively rapidly by histone and reassociated relatively slowly after cyclic AMP. The major enzyme in rat brain was of type 11. The type I kinase and another kinase were minor constituents. The physiological substrates for cyclic AMP-dependent protein kinases in brain tissue will be dependent on both the substrate specificity of the kinase and the accessibility of various potential substrates. Thus, while histones are active substrates for most cyclic AMP-dependent protein kinases, their nuclear localization renders them inaccessible to, for example, protein kinases associated with synaptic membranes. Translocation of the catalytic unit of cyclic AMP-dependent protein kinase from cytosol to nucleus, however, occurred in glioma cells (Salem

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and DeVellis, 1976) and in adrenal medulla (Costa et al., 1976) after activation of cyclic AMP-generating systems. Synaptic membranes (Johnson etal., 1971) and ribosomes (Schmidt and Sokoloff, 1973) represent two particulate fractions from brain which contain substrates for cyclic AMP-dependent protein kinases. An “inhibitory” modulator protein has been reported from various tissues including brain (cf. Kuo, 1975; Kuo et al., 1976a,b). This inhibitory modulator protein inhibited cyclic AMP-dependent protein kinase activity assayed with histone as substrate, but actually stimulated activity when assayed with protamine as substrate. In brain homogenates, the inhibitory modulator protein was associated with soluble proteins and with synaptosome fractions (Roskoski et al., 1976). Virtually all of the modulator in lysed synaptosomes was associated with soluble proteins. Other endogenous factors would appear capable of regulating cyclic AMP-dependent protein kinases. These include calcium ions, adenosine, AMP, and ADP which inhibit kinase activity (Kuo and Greengard, 1969; Miyamoto et al., 1969; Kuoet al., 1970). ADP was the most active inhibitor. Formation of cyclic AMP in various tissues including brain has apparently been accompanied by the formation of a compound similar in properties to cyclic AMP which inhibited activation of kinases by cyclic AMP (Murad et al., 1969; Wasner, 1975). Activation of cyclic AMP-dependent protein kinases by cyclic GMP does not appear, in view of the specificity of binding sites on the regulatory subunits of the kinase, to be of significance under physiological conditions. Cyclic GMP has, however, been reported to antagonize cyclic AMP-dependent phosphorylation of microtubular protein (Sandoval and Cuatrecasas, 1976b). Cyclic AMP-dependent protein kinases catalyzed phosphorylation of their own regulatory subunits (Maeno et al., 1974). The significance of phosphorylation of the regulatory subunit is unclear. In heart preparations phosphorylation facilitated cyclic AMP dissociation of regulatory and catalytic subunits (Erlichman et al., 1974; Rangel-Aldao and Rosen, 1976). In summary, it is clear that regulation of phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinases is complex. Steady-state levels of cyclic AMP-dependent protein phosphorylation will reflect not only the activity of kinases, but also the rates of hydrolysis by phosphatases.

G. PHOSPHOPROTEIN PHOSPHATASES These enzymes which presumably serve to terminate the physiological responses to cyclic nucleotides in intact cells were present in homogenates of brain tissue in both soluble and membrane fractions (Weller and Rodnight, 1971; Maeno and Greengard, 1972; Maeno et al., 1975;

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Miyamoto and Kakiuchi, 1975). A significant proportion of the phosphoprotein phosphatases was associated with synaptosome fractions. Lysed synaptosome fractions afforded both soluble and membrane phosphatase activity. Histochemically, phosphoprotein phosphatases have been cited as appearing to be associated with postsynaptic dendritic sites of neurons in rat cerebral cortex (Greengard ct al., 1972). At least three isozymes with differing substrate profiles have been detected in brain preparations (Maeno and Greengard, 1972). Magnesium and calcium ions had little effect on phosphatase activity; manganese was sornewhat stimulatory and zinc was inhibitory. A compound which had properties similar to cyclic AMP and which was apparently formed along with cyclic AMP in various tissues has been reported to stimulate a phosphoprotein phosphatase from beef muscle (Wasner, 1975). Cyclic AMP inhibited the phosphatase. Dephosphorylation of the regulatory unit of cyclic AMP-dependent protein kinase has been reported to be stimulated by cyclic AMP (Maeno ef nl., 1975). In membranes, the localization of kinases, phosphatases, and their protein substrates are probably interrelated, perhaps as functional complexes (cf. Ueda et d,, 1975). H. CYCLICAMP-DEPENDENT AUTOPHOSPHORYLATION OF PROTEINS

The activity of cyclic AMP-dependent protein kinases can be studied either with exogenous substrates or with endogenous substrates present in soluble, synaptosome, or microtubule preparations from brain tissue or in brain slices. The latter technique, which will be referred to as autophosphorylation, provides data relevant to the normal physiological substrates whose phosphorylation is regulated by cyclic AMP. Incorporation of radioactive phosphate from [:"P]ATP into such endogenous proteins with soluble or membrane preparations will be dependent on accessibility of ATP to the catalytic site of protein kinases and on the activity of protein kinases and phosphoprotein phosphatases. Rates of turnover of phosphorylated proteins are conveniently assessed by addition of EDTA to inhibit the kinases. The rate of dephosphorylation by phosphatases can then be measured. In tissue slices, steady-state incorporation of phosphate into protein will, in addition, be dependent on the rate of incorporation of radioactive phosphate into ATP and the degree to which alternate pathways for ATP utilization compete with the kinase for labeled ATP. 1. Soluble Fractions

Autophosphorylation of proteins in soluble fractions from brain might be expected to provide data on possible physiological substrates for cyclic AMP-dependent kinases in the cytosol of brain cells. However,

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interpretation of the data must be tempered by a realization that many morphological relationships in the cytosol have been disrupted on homogenization, and that nonphysiological substrates can now compete with physiological substrates for the kinase. Nonetheless, in soluble fractions from brain homogenates, cyclic AMP clearly stimulated phosphorylation of one protein, apparently identical with the regulatory subunit of cyclic AMP-dependent protein kinase (Malkinson, 1975). Presumably, the phosphorylation of other cytosol proteins, such as phosphorylase 6 kinase, glycogen synthetase I, and tyrosine hydroxylase-activator protein was also stimulated by cyclic AMP but was undetectable in heterogeneous soluble fractions from brain.

2 , Synaptosomes Cyclic AMP stimulated phosphorylation of a t least three proteins in synaptic membranes from rat brain (Johnson et al., 1972; Ueda et al., 1973; Ehrlich and Routtenberg, 1974; Krueger et al., 1975; Maenoet al., 1975; Malkinson et al., 1975; Routtenberg and Ehrlich, 1975; Weller and Morgan, 1976). A protein with a molecular weight of about 49,000 appeared to correspond to the regulatory unit of cyclic AMP-dependent protein kinase and was phosphorylated in soluble fractions, synaptic membrane fractions, and other membrane fractions suggesting that it has no special o r unique role in synaptic events. However, two higher molecular weight proteins (80,000 and 86,000) appeared to be unique to synaptic membranes (Weller and Morgan, 1976).Levels of these proteins increased markedly in rat cerebrum 2 to 3 weeks after birth, a time at which synapses are rapidly being formed in the rat central nervous system (cited in Greengard, 1976). Calcium ions have been shown to stimulate phosphorylation of a number of proteins in synaptic membranes, in particular proteins with molecular weights of about 62,000 and 49,000 (DeLorenzo, 1976). The relationship of these proteins to those phosphorylated by cyclic AMP-dependent mechanisms has not been investigated.

3. Microtubules The autophosphorylation of protein constituents of microtubules has been studied extensively since an initial report on phosphorylation of microtubular protein in 1970 (Goodman et al., 1970). It now appears that a high molecular weight cyclic AMP-dependent protein kinase is associated with microtubular protein and phosphorylates certain other trace, high molecular weight proteins rather than tubulin itself (cf. Sloboda et al., 1975; Rappaport et al., 1976; Sandoval and Cuatrecasas, 1976a,b). The significance of the phosphorylation of microtubular pro-


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teins is unclear, although it has been proposed that phosphorylation may facilitate polymerization of tubulin (Sandoval and Cuatrecasas, 1976a). However, neither the rate nor extent of polymerization of microtubular proteins was significantly affected by inhibition of protein phosphorylation or by cyclic AMP-elicited phosphorylation (Rappaport et al., 1976). Others have proposed the stabilization of microtubules by cyclic AMP (Gillespie, 1971; cf. review by Daly, 1977).

4. Brain Slices and Ganglia Phosphorylation of proteins in brain slices has been studied extensively during incubations with agents or under conditions expected to stimulate cyclic AMP accumulations (Reddington et al., 1973; Weller and Rodnight, 1973a,b; Williams et al., 1974a,b; Williams and Rodnight, 1975, 1976). Norepinephrine, histamine, serotonin, and electrical pulsation increased incorporation of radioactive phosphate into proteins of guinea pig cortical slices. Adenosine, another stimulant of cyclic AMPgenerating systems, but in addition an inhibitor of protein kinase, had little effect alone, and selectively blocked the increase in protein phosphorylation elicited by histamine. T h e increase in protein phosphorylation elicited by norepinephrine was blocked by P-adrenergic but not by a-adrenergic antagonists. The increase in protein phosphorylation elicited by norepinephrine and electrical pulsation appeared to have been associated with neuronal elements, while the increase elicited by histamine and serotonin appeared to have been associated with glial elements in guinea pig cortical slices. The stimulations of protein phosphorylation elicited by norepinephrine and electrical pulsation were not additive. The data are difficult to completely rationalize in terms of a-adrenergic receptor-elicited accumulations of cyclic AMP in guinea pig cortical slices, and synergistic responses of cyclic AMP-generating systems to the combination of electrical pulsation with norepinephrine and to combinations of adenosine with histamine, serotonin, or norepinephrine. In slices of rat striatum, all of the biogenic amines-norepinephrine, histamine, serotonin, and dopamine-nhanced protein phosphorylation (Williams, 1976). y-Aminobutyrate and acetylcholine also stimulated protein phosphorylation. Adenosine reduced protein phosphorylation. T h e stimulatory effect of dopamine was antagonized by fluphenazine and haloperidol. 8-Bromo cyclic AMP or the phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced phosphorylation of three proteins in slices of rat caudate nucleus (Krueger et al., 1975). The two higher molecular weight proteins (80,000 and 85,000) appeared associated with synaptosome membranes, while the lower molecular weight protein (49,000) appeared to be the regulatory subunit of cyclic AMP-dependent protein kinase.

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In the abdominal ganglion of the mollusc, Aplysiu culijimica, dibutyryl cyclic AMP enhanced the phosphorylation of a specific high molecular weight protein ( 1 18,000) probably associated with synaptic entities of the ganglia (Levitan and Barondes, 1974; Levitan et al., 1974). Octopamine and serotonin also enhanced phosphorylation of this protein. 111. Cyclic GMP

A. LOCALIZATION OF GUANYLATE CYCLASES Guanylate cyclase, unlike adenylate cyclase, was associated with both soluble and particulate fractions in brain homogenates (Goridis and Morgan, 1973; Bensinger et al., 1974; Nakazawa and Sano, 1974; Kimura and Murad, 1974, 1976a,b; Nakazawa et al., 1976; Troyer and Ferrendelli, 1976). High levels of guanylate cyclase were found in synaptosome fractions. Lysis of synaptosomes, however, afforded mainly soluble guanylate cyclase, a result strongly indicative of a presynaptic 10calization of this enzyme. Recent data indicated that soluble and particulate guanylate cyclases from cerebellum were distinct entities (Troyer and Ferrendelli, 1976). The soluble enzyme did not appear merely to represent readily solubilized membrane-bound enzyme. In cultures of neonatal or fetal brain cells from rat or chicken, guanylate cyclase was mainly associated with neurons rather than with glia (Goridis and Morgan, 1973; Goridis et al., 1974; Zwiller etal., 1976). Guanylate cyclase was present in most regions of rat brain with lowest levels in pons medulla and spinal cord (Nakazawa and Sano, 1974; Nakazawa et al., 1976). T h e high levels of cyclic GMP and cyclic AMP in cerebellum (Schmidt et al., 1972; Kuo et al., 1972; Steiner et al., 1972) probably primarily reflect not high levels of cyclases, but instead the low levels of cerebellar phosphodiesterases. Cyclic GMP levels in mouse cerebellum were 2-fold higher in the molecular layer containing the dendrites of Purkinje cells than in the granular layer (Rubin and Ferrendelli, 1976). Cyclic GMP levels were markedly reduced in cerebellum from “nervous” mutant mice in which Purkinje cells are nearly completely absent (Ma0 et al., 1975). No interrelationships between levels of cyclic AMP and cyclic GMP were apparent in studies with cerebellar slices from rat, mouse, guinea pig, and rabbit, suggesting that cyclic AMP and cyclic AMPgenerating systems are present in different morphological loci (Ohga and Daly, 1977a). Other data have been indicative of an association of both cyclic AMP and cyclic GMP systems with Purkinje cells. The effect of various putative neurotransmitters and depolarizing conditions on


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accumulations of cyclic GMP has been studied extensively in incubated slices of brain tissue, particularly in slices from cerebellum where responses of cyclic GMP-generating systems are quite large (cf. Ferrendelli, 1975). The data have not provided definitive insights into the site of generation of cyclic GMP in brain tissue. In bovine superior cervical ganglion, an immunofluorescent assay indicated that acetylcholine-elicited accumulations of cyclic GMP occurred primarily in postganglionic neurons (Kebabian et al., 1975a). In rat pineal glands, however, denervation greatly decreased the norepinephrine-elicited accumulation of cyclic GMP, a result indicative of a presynaptic site for cyclic GMP formation in this sympathetically innervated tissue (O’Dea et al., 1976). Denervation, however, was reported by another group to result in an increase of guanylate cyclase in pineal gland (Strada el al., 1976). In summary, definitive statements as to the localization of cyclic GMP-generating systems in nervous tissue are not, as yet, warranted. Clearly, a significant portion of cyclic GMP systems would appear to be associated with neurons at presynaptic loci. However, the presence of cyclic GMP-generating systems at post-synaptic loci in neurons and in glia cannot be excluded. I t would appear that a significant portion of the enzyme is associated with cytosol, while the remainder is a membrane enzyme.

B. REGULATION OF GUANYLATE CYCLASES T h e activity of guanylate cyclases in the nervous system appears to be under a complex set of controls, including exogenous factors such as excitatory neurotransmitters and calcium ions, intracellular macromolecules, and levels of GTP and ATP. Guanylate cyclase from brain tissue required manganese for optimal activity, and has been reported to be inhibited by ATP (White and Aurbach, 1969; Boehme, 1970; Goridis and Morgan, 1973; Nakazawa and Sano, 1974; Olson et al., 1976: Troyer and Ferrendelli, 1976). Magnesium ions could partially activate the enzyme. Calcium ions usually inhibited membrane-bound guanylate cyclases and stimulated soluble guanylate cyclases. However, although guanylate cyclase activity from a neuroblastoma cell line was predominantly particulate, a stimulation of total enzyme activity by calcium pertained (Zwiller et al., 1976), The calcium-dependent activator protein had no effect on activity of guanylate cyclases (Olson et al., 1976). Other macromolecular factors may, however, be important to the activity of guanylate cyclases. Thus, the presence of a macromolecular factor was required in order for azide to activate soluble guanylate cyclases from

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brain (Mittal et al., 1975). Azide alone was able to activate membranebound guanylate cyclases from brain and to activate cyclic GMPgenerating systems in brain slices (Kimura et al., 1975). Activation of soluble or membrane-bound guanylate cyclase by putative neurotransmitters or neuromodulators has proven difficult to demonstrate in brain and other tissue. Thus, activation of cyclic GMP-generating systems in brain slices or ganglia by cholinergic and noradrenergic agonists, by glutamate, by histamine, by adenosine, and by depolarizing agents or electrical stimulation may represent not a direct activation of the cyclase by a neurotransmitter, but instead may reflect only neurotransmitter or depolarization-elicited increases in influx of calcium and an intracellular activation of the guanylate cyclase by calcium. Indeed, virtually all instances of neurotransmitter or depolarization-elicited increases in cyclic GMP in brain slices or ganglia have been dependent on extracellular calcium (cf. Ferrendelli et al., 1976; Ohga and Daly, 1977b). Feedback control and adaptive changes in cyclic GMP-generating systems have not really been investigated. In vivo treatment with harmaline elevated cyclic GMP levels in brain and led to an increase in soluble but not particulate guanylate cyclase in rat cerebellum (Spano et al., 197513).A protein factor inhibitory to cyclic GMP-dependent protein kinases was decreased in cerebellum by harmaline treatment (Szmigielski and Guidotti, 1976). Denervation of the pineal gland and hence cessation of noradrenergic input led to an increase in guanylate cyclase activity (Strada et al., 1976). A decrease in responses of cyclic GMP-generating systems to norepinephrine occurred after denervation (ODea et al., 1976). It was proposed that the decrease was due to loss of presynaptic cyclic GMP systems. A brief survey of the effects of different neurotransmitters on cyclic GMP generation in brain and ganglia tissue is relevant to the role of cyclic GMP in the nervous system. 1. Norepinephrine

The effects of norepinephrine on levels of cyclic GMP in cerebral cortical slices from various species have been inconsistent. Either no effect or only a marginal stimulation by norepinephrine has been reported (Kinscherf et al., 1976; Ohga and Daly, 1977a; Schwabe et al., 1977). In cerebellar slices from various species norepinephrine did elicit a significant accumulation of cyclic GMP (Ferrendelli, 1975; Ferrendelli et al., 1975; Kinscherf et al., 1976; Ohga and Daly, 1977a,b; Schmidt et al., 1976). In mouse cerebellar slices, the response to norepinephrine was blocked by both a- and P-adrenergic antagonists, while in guinea pig cerebellar slices only the P-adrenergic antagonist was effective. In pineal


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gland, norepinephrine appeared to elicit accumulations of cyclic GMP at presynaptic sites via interaction with an a-adrenergic receptor (O’Dea et al., 1976). 2. Other Biogenic Amines Dopamine and serotonin have not been reported to elicit significant accumulations of cyclic GMP in brain tissue (Ferrendelli et al., 1975). Dopamine did appear to slightly antagonize acetylcholine-elicited accumulations of cyclic GMP in bovine superior cervical ganglion (Kebabian et al., 1975b). Histamine has been reported to elicit accumulations of cyclic GMP in cerebral cortical slices but not in cerebellar slices of rabbit, rat, and guinea pig (Kuo et al., 1972; Lee et al., 1972; Ohga and Daly, 1977a,b; Schwabe et al., 1977). Histamine increased cyclic GMP levels in bovine superior cervical ganglion (Kebabian et al., 1975b).

3. Adenosine Adenosine has been reported to have either no effect (Ferrendelli et al., 1973, 1975) or a small stimulatory effect (Ohga and Daly, 1977a,b; Schwabe et al., 1977a) on cyclic GMP levels in brain slices. Further studies are clearly required to establish adenosine as a valid modulator of cyclic GMP-generating systems in brain tissue. The response in guinea pig cerebellar slices was antagonized by theophylline. 4. Acetylcholine Acetylcholine and cholinergic agonists elicited accumulations of cyclic GMP in brain slices from cerebral cortex and cerebellum of rabbit and rat (Kuo et al., 1972; Lee et al., 1972; Palmer and Duszynski, 1975; Palmer et al., 1976) and in bovine superior cervical ganglion (Kebabian et al., 197513) via interaction with muscarinic receptors. In other studies with cortical and cerebellar slices from various species, acetylcholine and cholinergic agonists had no effect on cyclic GMP levels (Ferrendelli et al., 1973; Kinscherf et al., 1976; Ohga and Daly, 1977a). Carbamylcholine had no effect on cyclic GMP levels in rat superior cervical ganglion (Hanbauer et al., 1975a). Acetylcholine and cholinergic agonists have been reported to elicit accumulations of cyclic GMP in peripheral neurons (Kebabian et al., 1975b; Horn and McAfee, 1976). 5 . Prostaglandins Prostaglandins did not, in preliminary experiments, have effects on levels of cyclic GMP in brain slices (Ferrendelli, 1975; Ohga and Daly, 1977a).

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6. Amino Acids Glutamate, at quite high concentrations, elicited accumulations of cyclic GMP in cerebral cortical slices from rabbit, guinea pig, cat, and mouse, but not in cerebral cortical slices from rat (Kinscherf et al., 1976). Glutamate elicited accumulations of cyclic GMP in cerebellar slices from guinea pig, rat, and mouse, but not in rabbit, Other groups have reported no effect of glutamate in guinea pig (Ohga and Daly, 1977a) or rat cerebellar slices (Schmidt et al., 1976). T h e glutamate response has been studied in some detail in mouse cerebellar slices (Ferrendelli et al., 1974, 19’75; Ferrendelli, 1975). The mechanism remains unclear, although it appears likely that glutamate-elicited activation of cyclic GMP-generating systems is related to its function as a n excitatory neurotransmitter in the nervous system. Kainic acid, an excitatory analog of glutamate, was a potent activator of cyclic GMP generation in rat cerebellar slices but not in slices from cerebral cortex, hippocampus, midbrain, hypothalamus, or brain stem (Schmidt et al., 1976). Glutamate had no effect on activity of cerebellar guanylate cyclase (Biggio and Guidotti, 1976b). The inhibitory neurotransmitters glycine and y-aminobutyrate had no effect on cyclic GMP levels in slices from cerebral cortex or cerebellum of various species (Kinscherfet al., 1976; Ohga and Daly, 1977a; Schmidt et al., 1976), except in cerebellar slices from mouse where both glycine and 7-aminobutyrate at quite high concentrations elicited modest accumulations of cyclic GMP (Ferrendelli et al., 1974, 1975; Ferrendelli, 1975). It is unlikely that the stirnulatory effects of y-aminobutyrate at such high concentrations on cyclic GMP systems bear any relationship to its physiological functions in brain, where a large body of evidence implicates y-aminobutyrate as an inhibitor of cyclic GMP generation and glutamate as an activator of cyclic GMP generation (cf. Costa et al., 1975a,b). 7. Peptides Enkephalins, in the present of a peptidase inhibitor, increased cyclic GMP levels in rat striatal slices (Minneman and Iversen, 1976b). The analgesic antagonist, naloxone, blocked the response. In bovine superior cervical ganglion, angiotensin, a ganglionic depolarizing agent, had no effect on cyclic GMP levels (Kebabian et al., 1975b). C. CYCLICGMP PHOSPHODIESTERASES It has proven difficult to establish which of the various phosphodiesterases in nervous tissue are concerned with hydrolysis of cyclic AMP and


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which are concerned with hydrolysis of cyclic GMP. Certain phosphodiesterases are relatively selective while others appear to hydrolyze both cyclic AMP and cyclic GMP (cf. Thompson and Appleman, 1971a,b; Pledger et al., 1974, 1975). Regional distribution of cyclic GMP phosphodiesterases has been investigated in rat (Nakazawa and Sano, 1974) and cat (Dalton et al., 1974) brain. Cyclic GMP phosphodiesterases like cyclic AMP phosphodiesterases were found in both particulate and soluble fractions from rat brain (Nakazawa and Sano, 1974). It has been suggested that a high molecular weight phosphodiesterase isozyme might be concerned primarily with cyclic GMP hydrolysis (Thompson and Appleman, 1971b), and more recently that the calciiim-dependent phosphodiesterase of brain might be concerned primarily with cyclic GMP hydrolysis (Kakiuchi et al., 1973). No definitive evidence has, however, been obtained to support such hypotheses. In part this reflects the fact that selective activators or inhibitors of cyclic AMP or cyclic GMP phosphodiesterases for studies in intact cells have not as yet been defined. Dipyridamole, RO 20-1724, and ZK 62771 are, however, relatively ineffective inhibitors of cyclic GMP phosphodiesterase activity (see above). In brain slices RO 20-1724 and ZK 62771 were relatively ineffective with regard to elevating cyclic GMP levels (Schwabe et al., 1976). Isobutylmethylxanthine proved to be the most effective inhibitor with regard to elevation of cyclic GMP levels in guinea pig cerebellar slices (Ohga and Daly, 1977b). Theophylline and SQ 20009 were very ineffective, while RO 20-1724, diazepam, and papaverine had intermediate effects. Until really selective inhibitors of cyclic AMP and cyclic GMP phosphodiesterases are developed, the role of the different phosphodiesterases in brain cyclic nucleotide metabolism will probably remain rather poorly defined. Cyclic AMP has, with certain phosphodiesterases, been shown to competitively inhibit hydrolysis of cyclic GMP (Goldberg et al., 1970; Williams et al., 1971; Weiss et al., 1974: Uzunov et al., 1976). D. CYCLICGMP-DEPENDENT PROTEINKINASES The properties and function of the relatively unstable cyclic GMPdependent protein kinases in nervous tissue are as yet poorly understood. Levels of cyclic GMPdependent protein kinase were higher in cerebellum than in cerebral cortex (Sold and Hofmann, 1974). The enzyme occurred in both soluble and membrane fractions in brain homogenates, required magnesium ions for activity, and was inhibited by calcium ions (Greengard and Kuo, 1970; Hofmann and Sold, 1972; Kuo, 1974). At least two isozymes were present in brain (Takai et nl., 1975). Binding sites for cyclic GMP in brain homognates, presumably at

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least in part, reflect sites of a regulatory subunit of cyclic GMPdependent protein kinases (Sold and Hofmann, 1974; Gill and Kanstein, 1975; Takai et al., 1975). Indeed, in cerebellar homogenates the major portion of binding sites have been recently shown to be associated with cyclic GMP-dependent protein kinase (Lincoln et al., 1976). Cyclic GMP-elicited dissociation of regulatory and catalytic subunits of the enzyme was not readily demonstrable, and it may therefore be premature to conclude that the activation of cyclic GMP-dependent kinase is similar in this respect to the activation of cyclic AMP-dependent kinases. Histones are excellent substrates for both cyclic AMP and cyclic GMPdependent protein kinases, but different serine residues in histone were phosphorylated by the two classes of protein kinases (Takai et al., 1975). Cyclic GMP-dependent kinases did not activate phosphorylase 6 kinase. A “stimulatory” modulator protein present in mammalian tissues alters the substrate specificity of cyclic GMP-dependent protein kinases (Donnelly et al., 1973; Kuo et al., 1976a,b; Kuo and Kuo, 1976). With arginine-rich histones or protamine as substrate, kinase activity was stimulated by the modulator protein, while with a histone mixture the kinase activity was somewhat inhibited. An inhibitory modulator protein for cyclic GMP-dependent protein kinase has recently been reported in rat cerebellum (Szmigielski and Guidotti, 1976). T h e physiological substrates for cyclic GMP-dependent protein kinases in brain tissue are unknown. In smooth muscle, but not apparently in other tissues, cyclic GMP stimulated phosphorylation of two membrane proteins (Casnellie and Greengard, 1974; Greengard, 1976).

IV. Cyclic Nucleotides and the Function of the Central and Peripheral Nervous System

Neurochemists have now provided a wealth of data on the various parameters related to function of cyclic AMP and cyclic GMP systems in the nervous system. It has, however, in brain, as in other even less complex tissues, proven immensely difficult to correlate changes in cyclic nucleotide levels with changes in physiological function. In nervous tissue, such correlations have been attempted at the level of biochemical changes in homogenates, intact cells, or even brain itself, at the level of neurophysiological changes in neurons or glia, and at the levels of brain pharmacology and behavior (cf. Daly, 1977). In view of the complexity and difficulties involved in establishing such correlations with cyclic nucleotide systems, only a brief overview of current areas of research will be attempted in the present review.


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A. BIOCHEMISTRY Cyclic AMP in isolated systems clearly has effects on at least three biochemical parameters: ( 1) intermediary metabolism through activation of phosphorylase b kinase and inactivation of glycogen synthetase I, thereby increasing glycolysis and glycogenolysis; (2) neurotransmitter pathways through activation of biosynthetic enzymes such as tyrosine hydroxylase; (3) cyclic AMP systems through feedback control of adenylate cyclase, phosphodiesterase, and protein kinases. Certain of these cyclic AMP-dependent effects appear to involve alterations in RNA and protein synthesis, but such alterations have not been defined in brain. Ribosomes did serve as substrates of cyclic AMP-dependent protein kinase (Schmidt and Sokoloff, 1973). Dibutyryl cyclic AMP, administered intraventricularly, did alter labeling patterns of brain RNA in goldfish (Shashoua, 1971). Stabilization of microtubules by cyclic AMPdependent mechanisms has been proposed (see Section I1,H). Activation of Na+-K+-ATPaseby cyclic AMP-dependent mechanisms has been proposed (cf. Phillis, 1976) and would be relevant to inhibitory effects of amines and cyclic AMP on neurons and perhaps to proposed roles for cyclic AMP in the control of transmitter release (see below). 1. Intermediary Metabolism One of the problems inherent in attempts to correlate changes in glycolysis or glycogenolysis with changes in cyclic AMP levels is that small increases in cyclic AMP often appear to evoke full activation of the phosphorylase kinase and inactivation of glycogen synthetase. This problem has rendered metabolic correlations in brain tissue particularly difficult, since sacrifice of animals results in large increases in brain cyclic AMP. In hypothermic mice, postdecapitation increases in cyclic AMP and phosphorylase a and decreases in glycogen synthetase have been shown to follow similar time courses (Lust and Passonneau, 1976). Simi-larly, the increases in brain cyclic AMP elicited by convulsions, stab wound trauma, and ischemia were accompanied by the expected changes in metabolic parameters (Watanabe and Passonneau, 1974; Folbergrova, 1975; Lust and Passonneau, 1976; Mrsulja et al., 1976; Watanabe and Ishii, 1976). Elevations of brain cyclic AMP under such conditions probably are due in large part of adenosine-dependent activation of cyclic AMP-generating systems. In slices of rat caudate nucleus both isoproterenol and 2-chloroadenosine increased glycogenolysis (Wilkening and Makman, 1976). In neonatal chicks elevations of cyclic AMP in brain elicited by intravenous P-adrenergic agonists were correlated with increased .glycogenolysis (Edwards et al., 1974; Nahorski et al.,

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1975a). In contrast, elevations of cyclic AMP elicited in chick brain by intravenous histamine were blocked completely by an Hi?-antagonist,but the increases in glycogenolysis elicited by histamine were only partially blocked. In sum, the results indicate that amine and adenosine-sensitive cyclic AMP mechanisms have significant roles in the regulation of central glucose metabolism. The sites-neurone or glia-at which such roles pertain are as yet unknown. 2. Neurotransmitter Metabolism Activation of tyrosine hydroxylase by cyclic AMP-dependent mechanisms has been extensively investigated. The mechanism involved appears to involve a cyclic AMP-dependent phosphorylation of a protein, which then activates tyrosine hydroxylase by increasing the enzyme’s affinity for tetrahydropteridine cofactor and reducing its affinity for inhibitory catecholamines (cf. Lloyd and Kaufman, 1975; Lovenberg et al., 1975). Activation of tyrosine hydroxylase by this mechanism has been studied primarily with synaptosome preparations, but also appears to pertain in vivo in brain (cf. Lovenberg and Bruckwick, 1975; Roth et al., 1975; Zivkovic et al., 1975, 1976). Activation of phenylalanine hydroxylase by cyclic AMP was, in contrast, due to direct phosphorylation of the enzyme (Milstien et al., 1976). In superior cervical ganglia, cyclic AMP-dependent activation of tyrosine hydroxylase required protein synthesis (Mackay and Iversen, 1972). In vivo P-adrenergic agonists induced tyrosine hydroxylase activity in ganglia apparently through a low, sustained increase in cyclic AMP, followed by enhanced synthesis of the enzyme (Hanbauer et al., 1975a,b). Effects of cyclic AMP on metabolism of other neurotransmitters or neuromodulators are much more poorly defined (cf. Daly, 1977).

3 . Adaptation of Cyclic AMP Systems T h e adaptive changes in cyclic AMP systems which occur as a result of changes in synaptic input to receptors controlling cyclic AMP generation in brain have been recently reviewed (Dismukes and Daly, 1976b; Daly, 1977). It is clear that as a result of reductions in neurotransmitter input to a cyclic AMP systems, the system in many cases attempts an adaptation by becoming “supersensitive” to the particular neurotransmitter. Conversely, increases in synaptic input to cyclic AMP systems in brain have often been followed by a compensatory “subsensitivity” of the system. “Supersensitivity” to the neurotransmitter may be due to increases in neurotransmitter-sensitive adenylate cyclase activity, reductions in associated phosphodiesterase activity, or increases in cyclic AMP-dependent protein kinase activity. Reductions in neurotransmitter input to cyclic


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AMP systems in brain can be evoked by various manipulations including (1) lesions, either physical or chemical, of neuronal pathways: (2) inhibition of neurotransmitter synthesis: (3) blockade of neurotransmitter receptors: and (4) treatment with drugs or manipulations which reduce levels or turnover of a neurotransmitter. A few recent examples are illustrative. Pretreatment of rats with 6-hydroxydopamine to destroy noradrenergic terminals resulted in enhanced responsiveness of norepinephrine-sensitive cyclic AMP-generating systems in limbic forebrain (Blumberg et al., 1976; Vetulani el nl., 1976). Similar adaptation of norepinephrine-sensitive cyclic AMP-generating systems after 6-hydroxydopamine treatment has been found in cerebral cortex of Sprague-Dawley rats but not in cerebral cortex of Fisher 344 rats (Skolnick and Daly, 1977; see also Section 11, A) or in cerebral cortex of guinea pig (Dismukes et al., 1976b). Furthermore, although central dopaminergic mechanisms clearly become supersensitive after lesions of the nigrostriatal dopaminergic pathways, after chronic administration of dopamine antagonists, after inhibition of dopamine synthesis with a-methyltyrosine, and after reduction of dopamine levels with reserpine, levels or responses of dopamine-sensitive cyclases of‘ striatum did not appear to be increased after such treatments (Von Voightlander et al., 1973, 1975; Rotrosen et al., 1975; Biggio et al., 1976; Krueger et nl., 1976). Two other groups, however, reported the expected increases in dopamine-sensitive cyclases after lesions (Mishra et al., 1974: Gardner el al., 1976) or treatment with a dopamine antagonist (Iwatsubo and Clouet, 1975). The reason for lack of apparent adaptation of cyclic AMP-generating systems in certain species, strains, or brain regions requires further investigation. It is possible that adaptation may occur at the level of protein kinase or its physiological substrates (cf. Romero and Axelrod, 1975; Routtenberg et al., 19’75). Subsensitivity of cyclic AMP systems to a neurotransmitter may be due to decreases in neurotransmitter-sensitive adenylate cyclase activity, increases in associated phosphodiesterase activity, or decreases in cyclic AMP-dependent protein kinase activity. Increases in neurotransmitter input to cyclic AMP systems can be evoked by various means including: (1) electrical stimulation of nerwonal pathways; (2) augmentation of neurotransmitter synthesis; (3) stimulation of receptors by “false transmitters”; (4) treatment with drugs or environmental manipulations which cause augmented release of the neurotransmitter; and ( 5 ) treatment with drugs which prevent the inactivation of the transmitter by either reuptake or metabolism. For example, recent studies have demonstrated that chronic treatment of rats with compounds such as imipramine, desipramine, or chlorpromazine, which prevent inactivation of’ norepinephrine by blocking reuptake into presynaptic terminals, re-

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sulted in a significant reduction in the responses of cyclic AMPgenerating systems to norepinephrine in slices of cerebral cortex (Frazer et al., 1974; Schultz, 1976) or limbic forebrain (Vetulani and Sulser, 1975; Vetulani et al., 1976). Chronic treatment of mice with amphetamine, a norepinephrine-releasing agent, led to a similar reduction in responsiveness of cerebral cortical norepinephrine-sensitive cyclic AMP-generating systems (Martres et al., 1975). Such adaptive changes in cyclic AMP systems after drug or environmental manipulations can be expected to provide valuable insights into the mechanism of action of the drug or to the effect of environmental manipulations on turnover of central neurotransmitters. For example, chronic electroconvulsive shock treatments, known to increase norepinephrine turnover, resulted in rat limbic forebrain in a reduction in responsiveness of norepinephrine-sensitive cyclic AMP-generating systems (Vetulani and Sulser, 1975; Vetulani et al., 1976). Surprisingly, electroshock treatment reduced the responsiveness to norepinephrine even in 6-hydroxydopamine-treated rats. In such animals presynaptic noradrenergic terminals have been destroyed so that shock treatment could not be causing enhanced release of nonexistent norepinephrine. The effects of drug treatment of animals on the responsiveness of brain cyclic AMP-generating systems have been studied in detail for ethanol and morphine. For ethanol, the adaptive changes in responsiveness of cyclic AMP-generating systems were consonant with enhanced turnover of norepinephrine during chronic treatment with ethanol and with reduced turnover of norepinephrine during withdrawal (French and Palmer, 1973; French et al., 1974, 1975). For morphine the results have been rather inconsistent (cf. Daly, 1977).Recent studies have not clarified the situation with one report of an enhanced level of striatal adenylate cyclase during chronic morphine treatment (Merali et al., 1976), and another report that chronic morphine treatment had no effect on basal or dopamine-sensitive adenylate cyclase in striatal homogenates (Bosse and Kuschinsky, 1976). Supersensitivity of prostaglandin-sensitive cyclic AMP-generating systems has been demonstrated in cultured neuroma cells after morphine treatment (Sharma et al., 1975; Traber et al., 1975). The responsiveness of prostaglandin-sensitive cyclic AMP-generating systems in cerebral slices was increased after rearing rats under conditions of environmental impoverishment (Dismukes and Daly, 1976a).

B. NEUROPHYSIOLOGY The effects of neurotransmitters known to increase cyclic AMP levels and of cyclic AMP analogs on ( 1 ) spontaneous and evoked firing of


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central neurons; (2) transmission or spontaneous firing in ganglion preparations; and (3) evoked or spontaneous transmitter release in peripheral neurons have been studied extensively. Effects of neurotransmitters known to increase cyclic GMP levels and of cyclic GMP analogs have been studied to a lesser extent. 1 . Cyclic A M P and Central Neurons I n cerebellar Purkinje cells and in a variety of other central neurons, norepinephrine-elicited accumulations of cyclic AMP have been linked to inhibitory effects on spontaneous firing of the neurons. These extensive studies have been recently reviewed (Bloom, 1975; Daly, 1977). Norepinephrine and cyclic AMP analogs caused an increase in membrane resistance and a hyperpolarization of central neurons, probably via a phosphorylation of a membrane protein associated with ionic channels. Such a phosphorylation might have caused hyperpolarization and an increase in membrane resistance by either activating Na’-K+-ATPase or by reducing passive membrane conductances for sodium ions or calcium ions. Catecholamines have been reported to activate Na+-K+-ATPase (Yoshimura, 1973; Gilbert et al., 1975; Logan and O’Donovan, 1976). Ouabain and other inhibitors of the ATPase have been reported to antagonize catecholamine-elicited inhibitions of central neurons (Phillis, 1976; Yarbrough, 1976). Although a variety of evidence implicates cyclic AMP in the inhibition of central neurons by biogenic amines, it should be noted that calcium ions are inhibitory to central neurons and that the norepinephrine-elicited inhibition of central neurons can be antagonized by “calcium antagonists,” leading to the postulate that the inhibitory effects of norepinephrine are dependent on extracellular calcium ions (cf. Phillis, 1976). Studies by another group (Freedman et al., 1975; Nathanson et al., 1976) have, however, provided evidence indicating that “calcium antagonists” either d o not block the inhibitory effects of norepinephrine on Purkinje cells, or in the case of lanthanum do so by direct inhibition of norepinephrine-sensitive adenylate cyclases. T h e inhibition of firing of caudate neurons by dopamine would appear, like effects of norepinephrine on central neurons, to involve a cyclic AMPdependent mechanism (Siggins et al., 1974; Bloom, 1975).Other amines, such as histamine and serotonin, inhibit firing of certain central neurons, but the relationship of these inhibitions to cyclic AMP mechanisms has not been established. Adenosine, 5’-AMP, and certain other adenine nucleotides have recently been found to be potent inhibitors of central neurons (Phillis et al., 1974, 1975; Phillis and Kostopoulos, 1975). Adenosine and 5’-AMP are, of course, effective stimulants of cyclic AMP generation in brain slices. In a slice of guinea pig olfactory cortex,

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adenosine and adenine nucleotide stimulated cyclic AMP generation and inhibited postsynaptic potentials evoked by stimulation of the olfactory tract (Kuroda and Kobayashi, 1975; Okada and Kuroda, 1975; Kuroda et al., 1976a,b). Thus, the adenosine-elicited accumulations of cyclic AMP, presumably at postsynaptic sites, appeared to inhibit synaptic transmission in this brain region. Adenosine had no effect on synaptic transmission in a slice of superior colliculus. Neurotransmitters, in particular norepinephrine, have been proposed to have effects in brain on both neurons and glia. It should, therefore, be noted that norepinephrine and other putative neurotransmitters have no effect on membrane potentials of glial cells (Wardell, 1966; Krnjevic and Schwartz, 1967; Hosli et al., 1976). Thus, if norepinephrine does elicit accumulations of cyclic AMP in glial cells, cyclic AMP does not appear to affect glial membrane potentials as it does neuronal potentials.

2 . Cyclic GMP in Central Neurons Recent data suggested that the activation of firing of rat cerebral cortical neurons by acetylcholine occurred via interaction with a muscarinic receptor and was mediated by cyclic GMP (Stone et al., 1975). Cyclic GMP excited many of the cortical neurons. Interestingly the same neuron was often excited by cyclic GMP or acetylcholine and inhibited by dibutyryl cyclic AMP or norepinephrine. In studies by another group, both cyclic AMP and cyclic GMP inhibited firing of cortical neurons (Phillis et al., 1974). A role for cyclic GMP in the effects of acetylcholine on spinal motor neurons has been questioned (Krnjevic et al., 1976).

3. Cyclic A M P in Ganglia In rabbit and bovine superior cervical ganglia release of dopamine from interneurons and dopamine-elicited accumulations of cyclic AMP on postganglionic neurons appeared to be responsible for the slow inhibitory postsynaptic potentials (cf. Greengard and Kebabian, 1974). It appears likely that norepinephrine is the inhibitory neurotransmitter in rat superior cervical ganglion (see Section 11, B). In cat, dopamine appeared likely to be the inhibitory neurotransmitter (Machova and Kristofova, 1973). In the ganglia of molluscs, peptides which elicit accumulations of cyclic AMP and cyclic AMP analogs caused marked hyperpolarizations in ganglion neurons during silent periods between bursts of electrical activity (Treistman and Levitan, 1976). Intracellular injection of cyclic AMP into neurons of the mollusc, Helix pomatia, has been reported to cause depolarization and enhanced spontaneous firing (Liberman et al., 1975).

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4. Cyclic GMP in Ganglia In the superior cervical ganglion, acetylcholine via interaction with muscarinic receptors on postganglionic neurons appears to elicit an accumulation of cyclic GMP which is responsible for the slow excitatory postsynaptic potentials (cf. Greengard and Kebabian, 1974). A prior treatment of rabbit superior cervical ganglion with dopamine or dibutyryl cyclic AMP had prolonged facilitative effects on the slow excitatory postsynaptic potentials (Libet and Tosaka, 1970; Libet et al., 1976). Thus, activation of synaptic cyclic AMP-dependent mechanisms appeared to have long-term facilitative effects on cyclic GMP-dependent neurotransmission. In bullfrog sympathetic ganglia, cyclic GMP has been proposed to have a role in the inhibition of post-tetanic potentiation of postsynaptic potentials by the phosphodiesterase inhibitors isobutylmethylxanthine and diazepam (Suria, 1976).

5 . Cyclic Nucleotides in Peripheral N m e s As yet, conclusive effects of cyclic nucleotides on axonal conduction or on membrane properties of axons have not been demonstrated. In one report, cyclic AMP was found to be inhibitory to generation and transmission of action potentials in frog sciatic nerve (Van d e Berg, 1974), while in another report neither cyclic AMP, cyclic GMP, nor stimulation of cyclic nucleotide-generating systems had any effect on membrane properties or action potentials in desheated frog sciatic nerve (Horn and McAfee, 1976). 6. Cyclic A M P and Transmitter Release

A possible facilitative role for cyclic AMP in the release of acetylcholine or norepinephrine from peripheral neurons has been extensively investigated during the ten years since its initial proposal (Breckenridge et al., 1967; cf. review by Daly, 1977). Neither norepinephrine nor cyclic AMP analogs had any effect on spontaneous release of acetylcholine from the rat phrenic nerve diaphragm preparation (Miyamoto and Breckenridge, 1974). However, acetylcholine release in depolarized preparations was enhanced by a cyclic AMP analog, and it was proposed that cyclic AMP-dependent mechanisms might increase availability of calcium in stimulated preparations, thereby enhancing transmitter release. Recent data on facilitative effects of dibutyryl cyclic AMP on neuromuscular transmission in vizjo were consonant with this interpretation (Standaert et al., 1976). Another phosphodiesterase inhibitor, SQ 20009, has recently been reported to facilitate neuromuscular transmission (McNiece and Jacobs, 1976). The inhibition of neuromuscular

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transmission by adenosine (Ginsborg and Hirst, 1972; Miyamoto and Breckenridge, 1974) has been interpreted as due to inhibition of cyclic AMP-dependent protein kinase (Dretchen et al., 1976). Adenosine has recently been reported to inhibit release of norepinephrine from sympathetic neurons (Hedqvist and Fredholm, 1976). Stimulus-evoked release of norepinephrine in spleen and vas deferens was enhanced by cyclic AMP analogs and by phosphodiesterase inhibitors (Cubeddu et al., 1974, 1975; Wooten et al., 1973). However, in another report neither dibutyryl cyclic AMP nor theophylline appeared to have any effect on stimulus-evoked release of norepinephrine in vas deferens (Stjarne, 1976). Activation of Na+-K+-ATPase has been proposed to play an important role in control of transmitter release via interaction of norepinephrine with a presynaptic a-adrenergic receptor (Gilbert et al., 1975). The activation of ATPase by norepinephrine has been considered as involving cyclic AMP mechanisms (see above), but as yet no evidence linking presynaptic a-adrenergic receptors with cyclic AMP-generating systems has been provided (cf. Skolnick and Daly, 1975c, 1976a). C. CENTRAL PHARMACOLOGY, BEHAVIOR, AND CYCLICNUCLEOTIDES The effects of various centrally active drugs on levels of cyclic nucleotides in brain have been studied extensively with the hope that correlations of central activity and alterations in levels of cyclic AMP and cyclic GMP would become apparent. Levels of cyclic nucleotides have been measured in specific brain regions, in cerebrospinal fluid, and in urine after drug treatments, after environmental manipulations, in different strains of rats or mice, and in patients with mental disorders. I n addition, the effects of intraventricular or intracerebral administration of cyclic nucleotides on behavior, on pharmacological responses to drugs, and on central vegetative functions have been investigated. These attempts to relate cyclic nucleotide mechanisms to the pharmacology of centrally active drugs and to behavior have been comprehensively reviewed (Daly, 1977), and only a few salient results will be mentioned in the present review. 1. Neurotransmitters The various neurotransmitters known to stimulate cyclic AMP or cyclic GMP formation in brain have quite different gross effects on behavior. Catecholamines are central stimulants (Laverty, 1975). Intraventricular norepinephrine was more effective in eliciting spontaneous motor activity in a rat strain with a highly responsive norepinephrinesensitive subocortical cyclic AMP-generating system than in a strain with a less responsive cyclic AMP-generating system (Segal at al., 1975).


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Dopamine administered to the nucleus accumbens elicited increases in locomotor activity as did activation of adenylate cyclases in this brain region by injection of cholera toxin (Miller and Kelly, 1975; Iversen et al., 1975). The behavioral effects of histamine (Monnier and Hall, 1969; Asakawa and Yoshida, 1971; Schwartz et al., 1974),serotonin (Chase and Murphy, 1973), and cholinergic agonists (Pradhan and Dutta, 1971) are complex and difficult to categorize in specific terms. Histamine and serotonin have inhibitory effects on many central neurons, while acetylcholine usually has excitatory effects. Prostaglandin is a central depressant (Horton, 1964). Transient increases in brain levels of cyclic AMP elicited by prostaglandin El were not, however, correlated with the long-lasting sedation (Wellmann and Schwabe, 1973). Adenosine, administered parenterally or centrally, has profound sedative effects (Marley and Nistico, 1972; Haulica et al., 1973; Maitre et al., 1974). Glutamate, an excitatory neurotransmitter, elevated cerebellar levels of cyclic GMP and is a potent convulsant (Guidotti et al., 1975; Biggio and Guidotti, 1976b). The inhibitory neurotransmitter, glycine, elevated cerebellar levels of cyclic GMP, while another inhibitory neurotransmitter, y-aminobutyrate, reduced cerebellar levels of cyclic GMP and increased levels of cyclic AMP (Ma0 et al., 1974; Guidotti et al., 1975). Antagonists of glycine and y-aminobutyrate and drugs which deplete y-aminobu ty rate have convu lsant activity . 2. Phosphodiesterase Inhibitors Except for the central stimulants, theophylline and caffeine, which are, of course, very weak inhibitors of brain phosphodiesterases, the various phosphodiesterase inhibitors such as papaverine, diazepam, chlorpromazine, RO 20-1724, ZK 6271 1, and SQ 20009 are central depressants (cf. Beer et al., 1972; Schwabe et al., 1976). The effect of a variety of phosphodiesterase inhibitors on turning behavior in rats with unilateral lesions of the substantia nigra has been investigated (Fredholm et al., 1976). This turning behavior is thought to be dependent on activation of striatal dopamine-sensitive adenylate cyclase. Isobutymethylxanthine and a phenylazapurinone greatly potentiated dopa-elicited turning, while dipyridamole and theophylline were less effective and papaverine had no effect. In an earlier study, another group reported that diazepam, chlordiazepoxide, and a triazolopyrimidine did not potentiate apomorphine-induced turning (Arbuthnott et al., 1974). 3. Cyclic Nucleotide Analogs I n most studies intraventricular or intracerebral administration of cyclic AMP or of cyclic AMP analogs resulted in increases in locomotor activity, gross excitation, and often convulsions (cf. Daly, 1977). Di-

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bu tyryl cyclic AMP shortened amobarbital-induced narcosis (Kraynack et al., 1976 and references therein) and increased the rate of development of tolerance to ethanol (Wahlstrom, 1975) and to morphine (Ho et al., 1973, 1975). Dibutyryl cyclic AMP antagonized the analgesia elicited by morphine. It is noteworthy that peripheral administration of a cyclic GMP analog, 8-bromo cyclic GMP, caused a 2-fold increase in levels of cyclic AMP in brain (Fernandez-Pol and Hays, 1976). Such effects on cyclic AMP levels could confound any interpretation of the pharmacology of cyclic GMP analogs. 4. Central Stimulants Various central stimulants such as apomorphine, dopa, amphetamine, and tricyclic antidepressants, whose activity is linked to enhanced stimulation of catecholamine receptors, have been reported to elevate cyclic AMP and often cyclic GMP in brain or in specific brain regions (cf. Daly, 197’7). The apomorphine-elicited increases in cyclic GMP levels in rat cerebellum were proposed to be due to enhanced excitatory input to the cerebellum due to activation of striatal dopaminergic receptors (Burkard et al., 1976). The increase in levels of cyclic AMP in rat cerebrospinal fluid elicited by dopa appeared primarily mediated by activation of central /3-adrenergic rather than dopaminergic receptors (Cramer and Kiessling, 1976). Convulsants such as isoniazid, picrotoxin, pentylenetetrazole, glutamate, and tremorigenic agents such as oxotremorine and harmaline elevated cyclic GMP levels, particularly in cerebellum. Oxotremorine and harmaline apparently increase excitatory input into the cerebellum, thereby elevating cyclic GMP levels (cf. Ferrendelli et al., 1970, 19’72; Guidotti et al., 1975; Biggio and Guidotti, 1976b; Opmeer et al., 1976). I t would appear the y-aminobutyrate, an inhibitory neurotransmitter, is inhibitory to cyclic GMPgenerating systems, perhaps via an inhibition of release or action of excitatory neurotransmitters (cf. Suria, 1976). 5. Central Depressants Ethanol had minimal effects on central levels of cyclic AMP, but caused marked reductions in levels of cyclic GMP (Redos et al., 1976a,b). Other central depressants such as barbiturates, papaverine, reserpine, and chlorpromazine reduced central levels, particularly cerebellar levels, of cyclic GMP (Ferrendelli et al., 1972: Kimura et al., 1974; Lust et al., 1976; Opmeer et al., 1976). Morphine reduced levels of cyclic GMP in cerebellum (Lust et al., 1976), apparently due to decreased mossy fiber input to the cerebellum (Biggio and Guidotti, 1976a). Morphine had, in various studies, somewhat inconsistent effects on levels of cyclic AMP in brain. However, morphine has been reported by a number of groups to

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elicit increases of cyclic AMP in striatum (cf. Bonnet, 1975: Clouet et al., 1975; Merali et al., 1975). Such morphine-elicited increases in cyclic AMP levels contrast with inhibitory effects of morphine on cyclic AMP generation in homogenates or cultured cells (see Section 11, A). 6. Behavioral Correlates It should be obvious that many drugs affect such a variety of cyclic AMP-dependent inhibitory pathways andlor cyclic GMP-dependent pathways in the central nervous system so as to make impossible the interpretation of the behavioral results. Certainly, this will be is the case for cyclic AMP analogs and for phosphodiesterase inhibitors. Activation of adenylate cyclase by injection of cholera toxin into specific brain regions would appear to be a somewhat more selective approach. Another type of approach has been to attempt to correlate behavioral parameters with (1) responses of cyclic AMP-generating systems in specific brain regions of different rat or mouse strains (Skolnick and Daly, 1974a, 1975b; Williams and Pirch, 1974; Sattin, 1975; Stalvey et al., 1976); ( 2 ) levels of cyclic AMP in brains of mouse strains (Barchas et al., 1974; Orenberg et al., 1975); and (3) cyclic AMP-dependent phosphorylation of synaptosome proteins from rat strains (Ehrlich and Brunngraber, 1976). In the last study, autophosphorylation of a synaptosome protein with a molecular weight of 49,000, presumably the regulatory unit cor cyclic AMP-dependent protein kinase, was found to be high in striatal synaptosomes from a strain of rat with high spontaneous behavioral activity and low in a less active strain of rat. Autophosphorylation of this protein in cortical synaptosomes was low in the behaviorally active strain of rat and high in the less active strain of rat. These results in conjunction with data on responsiveness of norepinephrine-sensitive cyclic AMP-generating systems from the two rat strains (Skolnick and Daly, 1974a, 1975b) suggest that at least in these two rat strains, brain regions with highly responsive norepinephrine-sensitive cyclic AMP-generating systems have associated a high capacity for autophosphorylation of a synaptosome protein. Although the various studies cited above have provided a number of interesting examples of apparent correlations between particular behaviors and functions of the cyclic AMP systems, much more detailed studies of this type will be required if significant insights into the specific roles of cyclic nucleotide systems in complex behaviors are to be established. 7. Clinical Correlates During the past six years many groups have reported correlations or lack of correlations between clinical state of patients with mental disorders, in particular manic-depressive syndrome, and cyclic AMP levels in

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urine or cerebrospinal fluid (cf. Daly, 1977). Recent studies provide further data which show no correlation between mental dysfunction and cyclic AMP levels in cerebrospinal fluid (Smith et al., 1976) or urine (Moyes and Moyes, 1976). V. Summary

Biochemical studies have provided evidence that cyclic AMP systems are associated in brain and ganglia with postsynaptic sites on neurons. Neurophysiological studies indicate that such cyclic AMP systems are involved in inhibitory neurotransmission for noradrenergic, dopaminergic, serotoninergic, and histaminergic pathways. Adenosine appears to serve as an inhibitory neuromodulator, probably via interaction with the postsynaptic cyclic AMP-generating systems. Behavioral studies suggest that cyclic AMP mechanisms may be involved in regulation of certain complex behaviors. The results do not preclude significant roles for cyclic AMP at presynaptic sites regulating neurotransmitter formation and/or release or at glial sites regulating perhaps metabolic events. Biochemical studies have provided evidence that cyclic GMP systems are associated in brain and ganglia with both presynaptic and postsynaptic sites. Neurophysiological studies indicate that the postsynaptic cyclic GMP systems are involved in responses to excitatory neurotransmitters such as acetycholine and glutamate. Roles for cyclic GMP systems at presynaptic sites are less well defined but might involve effects on release of transmitters. Cyclic GMP-generating systems appear to be associated with neurones, but their presence in glia cannot be excluded. Behavioral studies on the role of cyclic GMP have been hindered by lack of specific agents for activation or inhibition of the cyclic GMP-generating systems. The results obtained during the past decade and a half thus provide a solid basis for further studies on the precise nature and role of cyclic AMP and cyclic GMP as regulatory messengers in the central and peripheral nervous systems. REFERENCES Adinolfi, A. M., and Schmidt, S. Y . (1974). Brain Res. 76, 21-31. Arbuthnott, G . S., Attree, T.J., Eccleston, D., Loose, R. W., and Martin, M. J. (1974).Med. Biol. 52, 350-353. Asakawa, T., and Yoshida, H. (1971).Jap.J. Pharmatol. 21,569-583. Audiger, Y . , Virion, A., and Schwartz, J.-C. (1976).Nature (London) 262,307-308. Barchas,J. D., Ciaranello, R. D., Dominic, J. A., Deguchi, T.,Orenberg, E., Renson,J., and Kessler, S. (1974).Adu. Biochem. Psychophannacol. 12, 195-215. Baudry, M., Martres, M. P., and Schwartz,J.-C. (1975).Nature (London) 253,362-363. Beavo, J. A., Hardrnan,J. G., and Sutherland, E. W. ( 1 970).J . Baol. Chem. 245,5649-5655.


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