INTERNATIONAL REVIEW OF
Neu robiology VOLUME 22
Editorial Board W. Ross ADEY JULIUS
AXELROD
SEYMOUR KETY KEITHKILL...
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INTERNATIONAL REVIEW OF
Neu robiology VOLUME 22
Editorial Board W. Ross ADEY JULIUS
AXELROD
SEYMOUR KETY KEITHKILLAM
Ross BALDESSARINI
CONAN KORNETSKY
SIRROGERBANNISTER
ABELLAJTHA
FLOYD BLOOM
BORISLEBEDEV
DANIEL BOVET
PAULMANDELL
PHILLIP BRADLEY
HUMPHRY OSMOND
JOSE
DELGADO
SIRJOHN ECCLES JOEL
ELKES
RODOLFO PAOLETTI SOLOMON SNYDER STEPHEN SZARA
H. J. EYSENCK
JOHN
KJELLFUXE
MARATVARTANIAN
Bo HOLMSTEDT
RICHARD WYATT
PAULJANSSEN
OLIVER ZANGWILL
VANE
INTERNATIONAL REVIEW OF
Neurobioloav -
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Edited by JOHN R. SMYTHIES Department of Psychiatry ond the Neurosciences Program University of Alabama Medical Center Birminghorn, Alabama
RONALD J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birmingham, Alabama
VOLUME 22
1981
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York
London
Toronto
Sydney
San Francisco
COPYRIGHT @ 1981, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R 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.
111 Fifth Avenue, New York. New York 10003
United Kingdom Edition published b y ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NWI 7DX
LIBRARY OF CONGRESS CATALOG CARDNUMBER:59- 13822 ISBN 0- 12-366822-0 PRINTED IN THE UNITED STATES OF AMERICA 81 82 83 84
9 8 7 6 5 4 3 2 1
CONTENTS
CONTRIBUTORS ......................................................
ix
Transport and Metabolism of Glutamate and GABA in Neurons and Glial Cells
ARNESCHOUSBOE
............................
11. Transport of Glutamate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 3
................
16
V. Metabolism of GABA . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . VI. Concluding Remarks . . . . References
....................................................
34
Brain Intermediary Metabolism In Vivo: Changes with Carbon Dioxide, Development, and Seizures
ALEXANDER L. MILLER
I. 11. 111. IV. V.
General Introduction to Brain Intermediary Metabolism . . . . . . . . . . . . . . Methods of Studying Brain Intermediary Metabolism in Vivo . . . . . . . . . . . Effects of Carbon Dioxide on Brain Intermediary Metabolism . . . . . . . . . . Glucose Metabolism by Developing Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . Brain Metabolism during Seizures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47 49
58 65 71 78
N,N-Dimethyltryptamine:An Endogenous Hallucinogen
STEVEN A. BARKER, JOHN A. MONTI, AND SAMUEL T. CHRISTIAN
I. 11. 111. IV. V. VI. VII. VIII.
Introduction . . .. . . . . . . Biosynthesisof ................................. Metabolism of Tolerance to D DMT and 5-Hydroxytryptamine DMT and Dopamine. . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . , . . . . , . . . . . DMT at the Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An Explanation for Hallucinatory Phenomena References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
83
85 91 101 101 103 104 106 107
vi
CONTENTS
Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiography
L . CHARLES MURRIN I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I1. Anatomical Studies of Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111. Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111 114 121 161 161
Neurotoxins as Tools in Neurobiology
E . G . MCGEERA N D P . L . MCGEER
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Toxins with Some Specificity for Certain CNS Neurons . . . . . . . . . . . . . . . 111. Toxins with Specificity for Certain Types of Receptors . . . . . . . . . . . . . . . . IV . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
173 175 179 201 201
Mechanisms of Synaptic Modulation
WILLIAM SHAIN AND DAVID0. CARPENTER
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 I1 . General Mechanisms Regulating Transmitter Action . . . . . . . . . . . . . . . . . 207
111. Presynaptic Factors Regulating Transmitter Release . . . . . . . . . . . . . . . . . . IV . Modulatory Effects of Transmitters on Postsynaptic Membranes . . . . . . . . V . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228 238 244 244
Anatomical. Physiological. and Behavioral Aspects of Olfactory Bulbectomy in the Rat
B. E . LEONARD AND M . TUITE
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Anatomical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Physiological and Behavioral Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Biochemical and Pharmacological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
251 252 260 277 283
vii
CONTENTS
The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System
LOUIS SOKOLOFF
I. Introduction . . . . . . . . . . . . . . . . . ........ 11. Theory ........................... 111. Experimental Procedure for Measurement of Local Cerebral ................ Glucose Utilization . . . . . . . . . . . IV. Rates of Local Cerebral Glucose Conscious State . . . . . . . . ......................... V . Effects of General Anesthe VI. Relation between Local Function ism . . . . VII. Mechanism of Coupling of Local Functional Activity and Energy Metabolism . . . . . . .................................. ................ VIII. Applications of the Deoxyglucose Method . IX. Recent Technological ............. X. Concluding Remarks ................. References . ................................. INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENT VOLUMES .........................................
296 301 304 314 315 328 330 330 335 341
This Page Intentionally Left Blank
CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.
STEVEN A. BARKER, The Neurosciences Program and the Department of Psychiatry, University of Alabama in Birmingham, Birmingham, Alabama 35294 (83) DAVID0. CARPENTER, Division of Laboratories and Research, N e w York State Department o f Health, Albany, N e w York 12201 (205) SAMUEL T. CHRISTIAN, The Neurosciences Program and the Department ofpsychiatry, University of Alabama in Birmingham, Birmingham, Alabama 35294 (83) B. E . LEONARD, Department o f Pharmacology, University College, Galway, Republic of Ireland ( 25 1 )
E. G . MCGEER, Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University o f British Columbia, Vancouver, British Columbia, Canada V6T lW5 (173) P. L. MCGEER, Kinsmen Laboratory of Neurological Research, Department o f Psychia t y , University o f British Columbia, Vancouver, British Columbia, Canada V6T lW5 (173) ALEXANDER L. MILLER, * Department o f Psychiatry, Harvard Medical School, and Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178 (47) JOHNA. MONTI,The Neurosciences Program and the Department of Psychiatry, University o f Alabama in Birmingham, Birmingham, Alabama 35294 (83) L. CHARLES MURRIN, Department o f Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68105 (1 1 1 ) ARNESCHOUSBOE, Department o f Biochemistry A , Panum Institute, University of Copenhagen, D K - 2 2 0 0 Copenhagen N., Denmark (1) WILLIAM SHAIN, Division of Laboratories and Research, N e w York State Department of Health, Albany, N e w York 12201 (2 0 5 ) LOUISSOKOLOFF, Laboratory o f Cerebral Metabolism, National Institute of Mentul Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, Maryland 20205 (287) M. TUITE, Department of Pharmacology, University College, Galway, Republic of Ireland ( 25 1 ) Present address: The University of Texas Health Science Center, San Antonio, Texas 78284
ix
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TRANSPORT AND METABOLISM OF GLUTAMATE AND GABA IN NEURONS AND GLIAL .CELLS By A r m Schourboo Dopofimont of I)lochomlstry A Panum Instltuto Unlvonlty of Coponhag.n Coponhagon, Donmark
I. Introduction . . . . . . . . . . . . ............ .... 11. Transport of Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Brain Slices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... B. Neuronal Transport .................... C . Glial Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. D. Substrate Specificity of the Transport Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Metabolism of Glutamate ...................... A. Whole Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.GlialCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Transport of GABA ....................... A. Brain Slices ........... B. Neuronal Tr ................................................. C . Glial Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Substrate Specificity of the Transport Systems . . . . . . . . . . V. Metabolism of GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Whole Brain B.Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Glial Cells VI. Concluding R ............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 3 3 3 5 8 10 10 10 13 16 16 17 19 23 26 26 31 32 33 34
1. introduction
Glutamate and y-aminobutyric acid (GABA) have a unique position among the amino acids in brain, being present in very high concentrations ranging from 5 to 15 and 1 to 10 pmollgm wet weight, respectively, with great topographical variations (Berl and Purpura, 1960; Roberts, 1962; Agrawal et al., 1966, 1968; Levi ctal., 1967; van den Berg, 1970; Baxter, 1970; Pate1 and Balizs, 1970; Krnjevif, 1970) and they both show an increase in the concentration during postnatal ontogenesis (Berl and Purpura, 1960; Agrawal et al., 1966, 1968). Furthermore, apart from a recent report of a high concentration of GABA in the pancreatic islets of Langerhanns (Okada et al., 1976) GABA 1 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 22
Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISBN 0-12-366822-0
2
A R N E SCHOUSBOE
is considered to be present in only trace amounts in tissues other than the central nervous system (Haber et al., 1970a). These two amino acids are intimately related in their participation in brain metabolism since they together with the L-glutamate decarboxylase (EC 4.1.1.15; GAD), the GABA-transaminase (EC 2.6.1.19; GABA-T), and succinic semialdehyde dehydrogenase (EC 1.2.1.16) constitute the so-called GABA shunt (McKhann ct al., 1960; Machiyama et al., 1970; Baxter, 1970) which allows the oxidative metabolism in brain to bypass the a-ketoglutarate dehydrogenase step of the tricarboxylic acid cycle (TCA). The quantitative importance of the GABA shunt has been assessed by McKhann et al. (1960), who concluded that 40% of the TCA flux goes through this bypass. Machiyama et al. (1970) and Balbs ct al. (1970), however, have showed, taking into consideration that the GABA metabolism is compartmentalized (cf. Berl and Clark, 1969), that the GABA shunt accounts for only about 10% of the flux through the TCA cycle. The compartmentation of glutamate and GABA metabolism implies that it occurs in different cellular and subcellular structures (cf. Balbs et al., 1973a,b; van den Berg et al., 1975) and GAD and GABA-T, which synthesize and degrade GABA, have been shown to reside in synaptosomes and free mitochondria respectively (Weinstein et al., 1963; Salganicoff and De Robertis, 1963, 1965; van Kempen et al., 1965; Baldzs et al., 1966; Waksman et al., 1968; Fonnum, 1968; Neal and Iversen, 1969; Reijnierse et al., 1975; Walsh and Clark, 1976a; Schousboe et al., 1977d), and the latter enzyme seems to be located in the mitochondria1 inner membrane (Schousboe ct al., 1977d) rather than in the matrix as originally suggested by Salganicoff and De Robertis (1965). The significance of the different cell types in glutamate and GABA metabolism will be discussed in Sections I11 and V. In addition to the important role of glutamate and GABA in brain metabolism, these amino acids have been shown by several authors to act, respectively, as excitatory and inhibitory transmitters in the CNS (Curtis and Watkins, 1960; Curtis et al., 1960; Krnjevif and Phillis, 1963; KrnjeviC and Schwartz, 1967; Obata et al., 1967; Curtis and Johnston, 1970; Hosli et al., 1973; Geller and Woodward, 1974; KrnjeviC, 1974; Curtis and Johnston, 1974; Snyder ct al., 1975; Curtis, 1975, 1979). The inactivation of these transmitters is generally thought to be brought about by sodium-dependent high-affinity uptake systems (Snyder et al., 1970; Curtis et al., 1970; Iversen, 1971; Logan and Snyder, 1971; Bennett et al., 1972, 1974; Curtis and Johnston, 1974; Schousboe, 1978a,b, 1979a) rather than enrymatic degradation (Elliott and van Gelder, 1958; Curtis and Johnston, 1974; Schousboe, 1978a). The importance of the different cell types in these processes and the characteristics of the different transport systems will be discussed in Sections I1 and IV.
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
3
II. Tranrport of Glutamato
A. BRAINSLICES Thirty years ago it was shown by Krebs and co-workers that brain slices are able to concentrate glutamate from the incubation medium (Stern ct al., 1949; Terner ct al., 1950). Since then a large number of papers have accumulated concerning the characteristics of this uptake, which is energy dependent, requires sodium ions, and consists of both high- and low-affinity components (Stern ct al., 1949; Takagaki ct al., 1959; Tsukada ct al., 1963; Blasberg and Lajtha, 1966; Blasberg, 1968; Margolis and Lajtha, 1968; Levin ct al., 1970; Arnfred and Hertz, 1971; Banay-Schwartz ct al., 1971, 1974a,b, 1976; Cohen and .Lajtha, 1972; Balcar and Johnston, 1972a,b, 1973; Levi and Raiteri, 1973; Lajtha and Sershen, 1975a,b; Benjamin and Quastel, 1976; Schousboe ct al., 1976a; Teller et al., 1977). Since it is the sodium-dependent high-affinity uptake system that is believed to be involved in the termination of the transmitter activity of glutamate (Logan and Snyder, 1971; Bennett ct al., 1972, 1974; Curtis and Johnston, 1974; Snyder ct al., 1975; Iversen ct al., 1975) only this uptake system will be discussed in terms of the relative importance of neurons and glial cells in this process. Kinetic characteristics of high-affinity transport systems in brain slices and homogenates have been summarized in Table I together with similar data for the neuronal and glial transport systems.
B. NEURONAL TRANSPORT Neuroblastoma cells in culture ‘(Walum and Weiler, 1978) and granule cells isolated from cerebellum (Campbell and Shank, 1978) have high-affinity uptake systems for glutamate, and autoradiographic studies on spinal cord, brainstem, and brain cortex cultures have clearly shown that [3H]glutamate is accumulated in neurons (Balcar and Hauser, 1978; Hosli and Hosli, 1978a,b). Similar studies on brain homogenates have indicated that synaptosomes are the most important site of the uptake in this preparation (Beart, 1976; Iversen and Storm-Mathisen, 1976), and the glutamate uptake seems to be confined to unique synaptosomal fractions (Wofsey ct al., 1971; Logan and Snyder, 1972; Bennett ct al., 1972; Honegger et al., 1974). Also the observations that high-affinity uptake of glutamate in hippocampus is reduced after axotomy (Storm-Mathisen, 1977) or after injection of kainic acid (Fonnum and Walaas, 1978), which is a strong neurotoxic agent (Olney et al., 1974), indicate that glutamate is taken up in nerve endings, and Divac ct al. (1977)
4
ARNE SCHOUSBOE
TABLE I KINETIC CONSTANTS FOR HIGH-AFFINITY UPTAKE OF GLUTAMATE INTO BRAIN SLICES, SYNAPTOSOMES, A N D DIFFERENT NEURONAL AND GLIAL PREPARATIONS
Tissue preparation Brain slice Brain slice Brain homogenate Brain homogenate Synaptosomes Synaptosomes Synaptic vesicles Cerebellar glomeruli Dorsal spinal roots Ventral spinal roots C-1300 Neuroblastoma cells Bulk-prepared glia Bulk-prepared glia Dorsal root ganglia Dorsal root ganglia C-6 Astrocytoma cells C-6 Astrocytoma cells MGM-LM Astrocytoma cells NN-Glia cells 138-MG Glioma cells Cultured astrocytes (mouse brain) Cultured astrocytes (mouse brain) Cultured astrocytes (mouse brain) Cultured astrocytes (rat brain)
Km (pM)
20 30 36 20 30 1.9 34 5 19 25 33 12 12 20 21 66 15 20 14 65 220
'ma."
(nmol/min/mg protein)
Reference
2.5' 0.6 2.4 2.4 6.3
Balcar and Johnston, 1972b Benjamin and Quastel, 1976 Logan and Snyder, 1971 Logan and Snyder, 1972 Levi and Raiteri, 1973 Bennett cf al., 1974 Lahdesrnaki cf al., 1977 Wilson cf al., 1976 Roberts and Keen, 1973 Roberts and Keen, 1973 Walum and Weiler, 1978 Henn cf al., 1974 Henn, 1976 Roberts and Keen, 1974 Roberts, 1976b Henn cf a [ . , 1974 Faivre-Bauman cf al., 1974 Stewart cf al., 1976 Balcar ct al., 1977 Walum and Weiler, 1978 Schousboe cf al., 1977b
-
0.03 0.2 0.044 0.026
-
0.07 0.06
-
3.8 2.7 0.7
8
Schousboe cf al., 1977c
30-90
30-75
50
59
Hertz cf al., 1978c
10-20
4-6
Balcar and Hauser, 1978
Most of the values are quoted verbatim from the literature. The V,, values for brain slices (Benjamin and Quastel, 1976), brain homogenates (Logan and Snyder, 1971, 1972), synaptosomes (Levi and Raiteri, 1973), and dorsal mot ganglia (Roberts and Keen, 1974) have been calculated from Lineweaver-Burk or Hofstee plots in those papers. The V, values for brain slices and some other preparations have been recalculated from nmol/min/gm wet weight on the basis of a protein content of brain cortex slices of 95 mg/gm wet weight (Schousboe, 1972). In most cases the temperature was 37OC. The temperature was 25OC.
have shown a high-affinity uptake of glutamate in corticostriatal nerve terminals which is reduced after lesions. Only few quantitative studies of glutamate transport in synaptosomes have been performed (cf. Table I), and it is accordingly difficult to assess the importance of this uptake system. From the available data it appears, however, that this uptake can only account for a minor proportion of the glutamate uptake observed in brain slices. This is in
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
5
agreement with autoradiographic studies of intact brain cortex (McLennan, 1976), brain slices (Hokfelt and Ljungdahl, 1972), and cerebellar cultures (Lasher, 1974) which show that almost all labeled glutamate is localized over glial elements. The question of the significance of the synaptosomal highaffinity transport system is further complicated by the demonstration of Levi and Raiteri (1974) that most of this uptake can be accounted for by a 1:l homoexchange process which can be of no physiological importance for removal of glutamate from synaptic clefts (Levi and Raiteri, 1976). The transpprt process in nerve terminals in vivo may, however, function as a net uptake since the membrane potential is preserved (cf. Sellstrom ct af., 1976), and under certain conditions incubated synaptosomes are able to take up glutamate in a concentrative manner (Bradford ct af., 1975). Although the sodium dependence of the synaptosomal uptake (Bennett ct al., 1972, 1974; Levi and Raiteri, 1976; Wheeler and Hollingsworth, 1978) as well as the uptake in cultured neurons (Balcar and Hauser, 1978) is well established, only the latter studies report on the stoichiometry of the sodium requirement. It appears that two sodium ions are required for glutamate uptake in synaptosomes as well as in cultured neurons (Wheeler and Hollingsworth, 1978; Balcar and Hauser, 1978). The study of the sodium dependence of glutamate transport in brain slices by Balcar and Johnston (1972a) indicates, however, that the sodium dependence is noncooperative but since this uptake represents not only the neuronal or synaptosomal uptake it is not presently possible to draw any final conclusion of the number of sodium ions co-transported with glutamate in synaptosomes (cf. Schousboe, 1981).
C . GLIALTRANSPORT Using bulk-prepared glial cells, Hamberger (197 1) showed that glutamate is accumulated into such cells and Henn et al. (1974) demonstrated that this uptake exhibited high-affinity kinetics and was sodium dependent. Such an uptake system has also been demonstrated in glial cells isolated from cerebellum (Campbell and Shank, 1978), dorsal root ganglia satellite glial cells (Schon and Kelly, 1974a; P. J. Roberts, 1974, 1976b; Roberts and Keen, 1974), different glioma cell lines in culture (Henn cf af., 1974; FaivreBauman ct af., 1974; Haber and Hutchison, 1976; Stewart et af., 1976; Logan, 1976; Pfeiffer ct af., 1977; Balcar ct af., 1977; Walum and Weiler, 1978), and astrocytes in primary cultures of rat or mouse brain (Schousboe ct af., 1977b,c; Hertz ef af., 1978a,c; Balcar and Hauser, 1978). Kinetic data from these studies have been summarized in Table I. The data show that the
6
A R N E SCHOUSBOE
capacity for glutamate uptake exhibited by glial cells and especially the astrocytes in primary culture, which in other aspects seem to be a good model for astrocytes in uivo (Hertz, 1977; Schousboe, 1977, 1978b), is superior to the neuronal uptake and it can easily account for the major part of glutamate uptake in brain slices. This agrees well with several radioautographic studies of whole brain and spinal cord and cerebellar cultures which show that [JHIglutamate to a large extent is accumulated into glial cells (Hosli et al., 1975; Hosli and Hosli, 1976b, 1978a,b; McLennan, 1976; Hokfelt and Ljungdahl, 1972; Lasher, 1974). In dorsal root ganglia [3H]glutamate is localized exclusively over satellite glial cells as demonstrated by Schon and Kelly (1974a) using autoradiography at the light microscopic level. The findings also give support to the repeated suggestion that glial cells may be involved in the removal of amino acids from synaptic clefts (Henn and Hamberger, 1971; Schrier and Thompson, 1974; Hutchison et al., 1974; Henn, 1975, 1976; Haber and Hutchison, 1976; Hamberger et al., 1975; Schousboe et al., 1976a,c, 1977a,b; Schousboe, 1977, 1978a,b, 1979a). These results also more specifically lend support to the hypothesis propounded by Benjamin and Quastel (1972, 1974, 1975) that glial cells are the site of metabolism of glutamate released from neurons (cf. Section 111). The importance of the glial transport system for the removal of glutamate from synaptic clefts is further strengthened by the demonstration of Hertz et al. (1978~)that glutamate uptake in astrocytes represents a net inward transport and not a homoexchange process as in the case of synaptosomal uptake (Levi and Raiteri, 1974). Moreover, there appears to be a correlation between the extent of the glial glutamate uptake in a specific brain region and the presumed quantitative importance of that particular brain region in glutamatergic transmission (Schousboe and Divac, 1979). As mentioned earlier, glutamate uptake into different glial preparations has been found to be sodium dependent (Henn et al., 1974; Faivre-Bauman et al., 1974; Balcar el al., 1977; Schousboe el al., 1977b; Balcar and Hauser, 1978). It should, however, be mentioned that glutamate uptake into glial cells in spinal cord cultures studied by autoradiography seems to be sodium independent (Hosli and Hosli, 1976b). There is some uncertainty regarding the K, for sodium since Balcar et al. (1977) have reported a value of 139 mM for NN cells whereas Schousboe et al. (1977b) found a value of 18 mM for astrocytes in primary cultures. The latter value is close to the K , given by Balcar and Johnston (1972a) for the sodium dependence of glutamate transport in brain slices. If the data of Schousboe el al. (1977b) are replotted in a Hill plot (Fig. 1) it can be seen that a straight line with a slope of 1.12 is obtained, indicating that one sodium ion is co-transported with glutamate in astrocytes. In a more recent study by Balcar and Hauser (1978), in which, essentially,
TRANSPORT AND METABOLISM OF GLUTAMATE AND
GABA
7
Log I No' I
FIG. 1. Hill plot (Hill, 1913; Atkinson, 1966) of the sodium dependence of glutamate uptake into cultured astrocytes showing log[u/(Vmax- u ) ] as a function of the logarithm of the sodium concentration; u indicates initial uptake rates at the different sodium concentrations, Vm, the initial uptake rate at infinitely high-sodium concentration and the glutamate concentration of 100 pi4 used in the experiments. T h e uptake rates were corrected for the unsaturable component of the uptake (Schousboe el al., 1977b). The line was fitted to the experimental points by regression analysis and is described by the equation:y = 1 . 1 2 ~- 1.30 (r = 0.972). (Results were recalculated from Schousboe ct al., 1977b.)
the same type of culture system was used, it was, however, concluded that glutamate transport requires two sodium ions. This discrepancy cannot presently be explained. As shown in Table 11, uptake of glutamate into astrocytes in primary cultures is enhanced by 10-25 mM potassium whereas higher (50 mM) concentrations have no effect. The lack of effect of 50 mM potassium agrees with TABLE I1 EFFECT OF POTASSIUM AND CALCIUM ON HIGH-AFFINITY UPTAKE OF GLUTAMATE AND GABA INTO CULTURED ASTROCYTES'
Control
K'
Caz'
(mM)
(mM)
5 5 10 25 50
1.0 0 1.0 1.0 1.o
Uptake (% of control f SEM)'
GABA
Glutamate 100 100 159 163 93
5.1 9.0 f 16.1 f 16.7 f 5.5 f f
(15) (8) (10)'
(lOy (10)
100 86 117 66 53
6.3 (9) 7.1 (8) f 12.3 (6) f 3.7 (8)d f 5.6 (7)d f f
From Schousboe cf al., 1977a,b. Values are expressed as percentages f SEM of the value obtained after incubation for 5 min in a physiological medium containing either 100 phf glutamate or 50 phf GABA. Corrections were made for the unsaturable components of the uptakes. Numbers of experiments are given in parentheses. ' p < 0.005. d p
< 0.001.
8
ARNE SCHOUSBOE
findings on glutamate uptake in brain slices by Arnfred and Hertz (1971), Weiss and Hertz (1974), Banay-Schwartz et 01. (1975), and Schousboe cf 01. (1976a) but disagrees with the inhibitory effect observed by Balcar and Johnston (1972a). The stimulatory effect is in sharp contrast to the inhibitory effect of potassium on glutamate uptake into C-6 astrocytoma cells (FaivreBauman et al., 1974). The possible physiological implications of a differentiated effect of moderately high potassium concentrations on glutamate and GABA transport (Table 11) will be discussed in Section IV, C. It is also shown in Table I1 that calcium is not required for glutamate uptake into astrocytes in primary cultures (Schousboe et al. , 1977b). This agrees with results obtained on the NN glial cell line (Balcar ef al., 1977) but C-6 astrocytoma cells seem, however, to require calcium for glutamate uptake (Faivre-Bauman et al., 1974).
D. SUBSTRATE SPECIFICITY OF THE TRANSPORT SYSTEMS The substrate specificity of high-affinity glutamate uptake into synaptosomes, dorsal root ganglia glial cells, glioma cells, and astrocytes in primary culture has been investigated by different authors (Roberts and Watkins, 1975; Balcar et al., 1977; Schousboe ef al., 1977b), and some of the results have been summarized in Table 111. It can be seen that L-aspartate generally inhibits glutamate uptake, whereas D-glutamate only seems to inhibit glutamate uptake in NN cells and astrocytes. GABA, 0-alanine, and diaminobutyric acid, which are known to inhibit GABA transport (cf. Table VI), have no effect on glutamate transport. Also kainic acid, which is a strong excitant analog of glutamic acid that causes degeneration of cell bodies of cholinergic and GABA-ergic neurons in striatum (Olney ef al., 1974; Johnston et al., 1974; Coyle and Schwarcz, 1976; McGeer and McGeer, 1976; Schwarcz and Coyle, 1977), has no effect on glutamate transport in synaptosomes or in glial cells of peripheral or central origin (Roberts and Watkins, 1975; I. Divac and A. Schousboe, unpublished). The only compounds that have been found to act as selective inhibitors of glutamate uptake in glia compared to synaptosomes are the hydroxamic acid derivatives of aspartate and glutamate, of which the L-aspartic acid-0-hydroxamate appears to be the most potent inhibitor, preferentially inhibiting the synaptosomal uptake system for glutamate (Roberts and Watkins, 1975). Much more work is needed in order to characterize the glutamate transport systems with the intention of finding inhibitors which are potent selective inhibitors of one of the transport systems. This has been done rather successfully for the GABA transport systems (Schon and Kelly, 1974b; Iversen and Kelly, 1975; Bowery cf al., 1976; Schousboe el al., 1978, 1979b; Schousboe, 1979a,b). Such in-
TABLE 111 EFFECTOF GLUTAMATE ANALOGS ON HIGH-AFFINITY UPTAKEOF GLUTAMATE I N BRAIN SLICES AND DIFFERENT NEURONAL AND GLULPREPARATIONS Inhibition of uptake (76)'' Analog
Brain slices
Synaptosomes
D-Glutamate L-Aspartate ma-Aminoadipate L-Glutamine GABA Glutarate 3-Aminoglutarate r-Cysteate 4-Fluoroglutamate L-Aspartic acid 0-hydroxamate L-Glutamic acid y-hydroxamate Kainate
11 79 0 0 0 0 88 100 79
0 95
-
Dorsal mot ganglia
NN Glia
Astrocytes
0
70 69
34 60 30 0 0 0
74
-
-
0 0
98
0 0 83
91 100 0
54 84 0
-
-
0 0
49 99
-
0
The inhibitor concentrations were lo-' M for uptake in brain slices (Balcar and Johnston, 1972a) and M in studies of synaptosomes and dorsal mot ganglia (Roberts and Watkins, 1975). NN-glial cells (Balcar cf al., 1977). and cultured astrocytes (Schousboe cf al., 1977b; I. Divac and A. Schousboe, unpublished).
10
ARNE SCHOUSBOE
hibitors may be useful both for the study of the individual transport systems in more complex preparations, such as whole brain or brain slices, and for the design of compounds that can be used to manipulate the extraneuronal concentration of these transmitter amino acids, which may be involved in the development of certain neurological disorders such as Huntington’s chorea and Parkinson’s disease (cf. Coyle ct al., 1977; Schousboe et al., 1978; Schousboe, 1979; Krogsgaard-Larsen et al., 1979a; Section IV, D).
111. Motabollrm of Glutamato
A. WHOLEBRAIN Glutamate plays a key role in brain metabolism since it links amino acid metabolism with the TCA cycle and since it is the major precursor of the inhibitory transmitter, GABA (cf. van den Berg, 1973). Furthermore, its metabolism is quite rapid (Berl and Clarke, 1969) and takes place in at least two distinct metabolic pools (Berl, 1973) which have been correlated with the different cell types in the brain (Balhs ct al., 1973b; van den Berg et al., 1975). This is normally referred to as the compartmentation of glutamate metabolism, a subject which has been extensively reviewed in recent years (Berl and Clark, 1969; Berl, 1973; van den Berg, 1973; Balizs et al., 1973a,b; Quastel, 1975; Pate1 and Balizs, 1975; van den Berg ct al., 1975; van Gelder, 1978; Berl ct al., 1978) and which accordingly will not be dealt with in this paper. The glutamate metabolism involves several enzymes, the most important of which are GAD, glutamic-oxaloacetic transaminase (EC 2.6.1.1; GOT), glutamate dehydrogenase (EC 1.4.1.3; GLDH), glutamine synthetase (EC 6.3.1.2), and glutaminase (EC 3.5.1.2). These all have relatively high activities in the brain, G O T being by far the most active (Wu, 1963; Bayer and McMurray, 1967; Wu ct al., 1973; van Gelder, 1974; Kvamme and Svenneby, 1975; Ozand ct al. , 1975; Wu ct al. , 1976; Sadasivudu et al., 1977), but since GAD is the enzyme that is directly involved in the conversion of glutamate to GABA more emphasis will be laid upon a description of this enzyme.
B. NEURONS The so-called large compartment of glutamate metabolism is supposed to be comprised of neuronal perikarya and nerve terminals (Balizs ct al.,
TRANSPORT AND METABOLISM OF GLUTAMATE AND
GABA
11
1973b), and neuroblastoma cell lines contain a high concentration of glutamate (Drummond and Phillips, 1977; Passonneau et al., 1977). This is in keeping with the view of Benjamin and Quastel (1972) that the major pool of glutamate is associated with neurons. This compartment contains all of the above-mentioned enzymes (Table IV) but the glutamine synthetase seems to be localized primarily in glial cells (Utley, 1964; Martinez-Hernandez et al., 1977) or more specifically in astrocytes (Schousboe et al., 1977b; Norenberg and Martinez-Hernandez, 1979; Schousboe, 1981). The result of Rose (1968) on bulk-prepared neurons and glial cells (Table IV) showed, however, the opposite localization of this enzyme but the recoveries in the fractions were very low, particularly for glutamine synthetase (Rose, 1968). The glutamate decarboxylase is, on the other hand, located in neurons as judged from the immunohistochemical localization recently performed by Roberts and coworkers (Saito et al., 1974c; McLaughlin et al., 1974; Ribak et al., 1976) and by the very low activity of this enzyme reported in cultured astrocytes by Schousboe et al. (1977b) and Wu et al. (1979). The possible existence of a glial GAD will be discussed in Section 111, C. The neuronal or synaptosomal GAD has been studied extensively in crude or semipurified preparations (Roberts and Frankel, 1951a,b; Roberts and Simonsen, 1963; Roberts et al., 1964; Susz et al., 1966; Wood, 1975; Tapia and Meza-Ruiz, 1975,1976; Wu et al., 1976; Bay6n et al., 1977a,b; Miller et al., 1978), and efforts have been made to purify the enzyme by affinity chromatography (Possani et al., 1977; Yamaguchi and Matsumura, 1977). The enzyme has been purified to homogeneity from mouse brain (Wu et af., 1973) and characterized both immunologically (Saito et al., 1974a; Wu et al., 1976) and kinetically (Wu and Roberts, 1974). It has a molecular weight of 85,000 and a K, for glutamate of 0.7 mM and for pyridoxal phosphate of 0.05 pM. The pH optimum is 7.0. The enzyme was found to consist of two physically indistinguishable subunits (Wu et al., 1973; Wu, 1976) but the possibility of the existence of even smaller subunits (MW 15,000) has been suggested (Matsuda et al., 1973). More recently, it has been reported (Blindermann et al., 1978) that the enzyme has been purified also from human brain. This enzyme has a molecular weight of 140,000 and consists of two identical subunits. The K, for glutamate of 1.3 m M is similar to that of the mouse brain enzyme. The differences between the mouse and rat brain enzymes are in disagreement with the finding of Saito et al. (1974a) that the enzymes from human and mouse brain were almost indistinguishable in the microcomplement fixation test, which is able to pick up very small differences in protein structure (Wilson et al., 1964). The human brain enzyme was, however, reported to contain a substantial cysteine sulfinate decarboxylase activity (Blindermann et al., 1978) and it is somewhat unclear whether this represents an impurity or a double function of the enzyme. In this context it should be mentioned that Wu (1977b) and Wu et al.
ACTlMnES O F
Enzyme activity (nmoWmidmg protein)
TABLE IV ENZYMES INVOLVED I N GLUTAMATE METABOLISM I N DIFFERENT PREPAUTIONS OF NEURONS AND GLULCELLS
C-1300 or M 1 Neuroblastoma cells
GAD
Bulk-prepared neurons
0.07'
0.20' 1Id
GOT GLDH Glu-S'
a
8Md 326d
2.5" 17'
-
Passonneau et al., 1977.
' Roth-Schechter et al., 1977.
' Sellstr6m el al., 1975. Rose, 1968.
' Hamberger et al., 1978.
23d 12.2'
Astrocytes in primary culture
C-6 Astrocytoma cells or NN glial cells
< 0.05f < 0.09'
0.02'; 0.0086 0.04h; 0.2'
206J 16.6' 12.3f 25.9f
Schousboe et al., 1977b. Wilson et al., 1972. Schrier and Thompson, 1974. ' Nicklas and Browning 1978. Glu-S: Glutamine synthetase.
f
8
J
11"; 21'
Bulk-prepared glia
0.12r lld
356d 124d
55.8'
1'
13d 14.8'
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
13
(1978a,b) have claimed that two forms of cysteic acid decarboxylase exist. One is distinct from GAD and the other is attributable to GAD, being active both on glutamate and on cysteic acid, the precursor of taurine Uacobsen and Smith, 1968). This means that the glutamate decarboxylase might be active on three different substrates, namely glutamic acid, cysteine sulphinic acid, and cysteic acid, in keeping with the suggestion of Davison (1956) that these substrates are acted upon by one and the same enzyme. The latter two substrates have, however, no effect on GAD purified from synaptosomes or heart (Wu, 1977a), indicating that more than one enzyme exists. Due to the proposed role of taurine as an inhibitory neurotransmitter (Curtis and Watkins, 1960, 1965; KrnjeviC and Puil, 1976), it is highly desirable to solve this problem since a purified cysteic acid decarboxylase void of GAD activity (Wu et al., 1978a,b) could be used for the production of antibody by which “taurinergic” neurons might be marked, employing immunohistochemical techniques analogous to those employed for the visualization of GABA-ergic neurons (Barber and Saito, 1976; Wood et al., 1976). Of the remaining glutamate-metabolizing enzymes, GLDH and glutaminase appear to be the most important ones for metabolism of glutamate in the neurons (Weil-Malherbe and Gordon, 1971; Benjamin and Quastel, 1974; Bradford and Ward, 1976; Quastel, 1978; Hamberger et al., 1978). The dehydrogenase is, however, also present in astrocytes cultured from dissociated rodent brain (Schousboe et al., 1977b; Roth-Schechter et al. , 1977) although the activity may be somewhat lower than in whole brain (cf. van Gelder, 1974; Sadasivudo et al., 1977). Surprisingly, the glutaminase, which has been thought to be primarily a neuronal or synaptosomal enzyme (Bradford and Ward, 1976; Dienel et al., 1977; Dennis et al, 1977; Hamberger et al., 1978), has recently been found in cultured astrocytes at a substantial activity (Schousboe et al., 1979a). The functional implications of this will be discussed in Section 111, C. C. GLIALCELLS
Although the major pool of glutamate by indirect evidence has been proposed to reside in the neurons (Benjamin and Quastel, 1972; Quastel, 1978), astrocytes cultured from rat or mouse brain have been shown to contain glutamate at concentrations similar to those found in the brain in vivo (Schousboe et al., 1975, 1977c; Drummond and Phillips, 1977; Hertz et al., 1978a). Also bulk-prepared glial cells have relatively high glutamate contents compared to neurons isolated in bulk (Rose, 1968; Nagata et al., 1974; Sellstrom et al., 1975), and glioma cell lines generally have higher glutamate contents than neuroblastoma cell lines (Schubert et al., 1975). In contrast to
14
ARNE SCHOUSBOE
this, the NN glial cell line has been reported to have a very low content of glutamate (Mokrasch, 1971). The C-6 astrocytoma cells seem, on the other hand, to have contents of glutamate similar to those reported for astrocytes in primary culture (Drummond and Phillips, 1977; Passonneau ct af., 1977; Nicklas and Browning, 1978). The apparent high content of glutamate in glial cells suggests that this cell type is important for glutamate metabolism, which agrees with the high capacity for glutamate uptake (Section 11, C) and the reported high activities of GOT, GLDH, glutamine synthetase (Table IV), and glutaminase (Fig. 2) in astrocytes in primary cultures (Schousboe cf af., 1977b, 1979a; RothSchechter cf af., 1977; Hertz ct af., 1978a). The question of the presence of GAD activity in glial cells seems to be somewhat more complex. It was reported by Haber ct af. (1970a-c) that nonneuronal tissues (e.g., kidney and heart) and glioma cells contained a glutamate decarboxylase which, in contrast to the brain enzyme, had a mitochondrial localization and was stimulated by chloride, pyruvate, and aminooxyacetic acid. This enzyme was named nonncusonuf GAD or GAD 11. Later, however, it was also shown that some of the properties of this GAD I1 could be ascribed to the presence of an impurity in the [l-14C]glutamate used in the assay (Miller and Martin, 1973), and it was shown by Miller and Martin (1976) that the decarboxylase in mitochondria did not differ from that of synaptosomes in terms of kinetic properties. Furthermore, Walsh and Clark (1976b) pointed out that the simultaneous operation of glutamate dehydrogenase and a-ketoglutarate dehydrogenase may lead to a COZ production from [14C]glutamate synonymous with the action of a glutamate decarboxylase. Using an assay in which [WIGABA was measured instead of W O Z , Drummond and Phillips (1974) showed that GAD activity in kidney is indistinguishable from the neuronal enzyme in terms of sensitivity to chloride or aminooxyacetate. Also
FIG. 2. Glutaminase activity (nmol/min/mg protein) in newborn (A) and adult (B) mouse brain and cultured mouse astrocytes (C) measured at 5 mM phosphate (open area) and 20 mM phosphate (open plus hatched area). (From Schousboe el ol., 1979a.)
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
15
Wu and Roberts (1973) concluded that kidney GAD was not stimulated by aminooxyacetate. The presence of glutamate decarboxylase activity in nonneuronal tissues such as kidney and heart seems, however, to be indisputably correct since it has been confirmed by MacDonnell and Greengard (1975), Wu (1978), and Wu et al. (1978d) that these tissues contain GAD at an activity of about 5-10% of the activity in brain. Moreover, Wu (1977a) has purified a glutamate decarboxylase from heart which, judged from immunological evidence (e.g., microcomplement fixation), is different from the neuronal enzyme. Its kinetic characteristics are, however, also different from the enzyme described by Haber el al. (1970a-c) since it was found to be inhibited by pyruvate. O n the other hand, the enzyme purified from heart was found to be stimulated by chloride as first reported by Haber et al. (1970a.b) for GAD 11. The question whether this enzyme might reside primarily in glial cells still remains to be answered, but a production of antibody against the heart enzyme might eventually lead to a visualization of this enzyme in glial cells. The very low activity of glutamate decarboxylase reported for primary cultures of astroblasts or astrocytes by Wilson et al. (1972), Schousboe ct al. (1977b), and Wu et al. (1979) indicates, however, that it might be difficult to visualize the enzyme using immunohistochemical techniques. The most important glutamate-metabolizing enzyme in glial cells seems to be the glutamine synthetase. Degeneration studies of Utley (1964) indicated that this enzyme was mainly glial, and recently Martinez-Hernandez et al. (1977) and Riepe and Norenberg (1977) have shown, employing immunohistochemical techniques, that the enzyme is localized exclusively over glial cells in brain and retina. Electron microscopic studies of rat brain have revealed an astrocytic localization (Norenberg and Martinez-Hernandez, 1979) that agrees with the high activity of this enzyme in astrocytes in primary culture reported by Schousboe et al. (1977b) and Hertz et al. (1978a). It should, however, be mentioned that the C-6 astrocytoma cell line has a very low glutamine synthetase activity (Nicklas and Browning, 1978) but such cells are generally not quantitatively comparable with astrocytes in primary culture (cf. Hertz, 1977; Schousboe, 1977, 1978b). The possible use of glutamine synthetase as an astrocytic marker enzyme has been discussed by Schousboe (1981). The high glutamine synthetase activity in astrocytes is in excellent agreement with the view that glial cells take up glutamate released from neurons and metabolize it to glutamine, which is then transferred back to the neurons (Benjamin and Quastel, 1972, 1974, 1975). This implies, however, that glutaminase, which converts glutamine to glutamate, should reside primarily in the neurons (Salganicoff and De Robertis, 1965; Bradford and Ward, 1976; Bradford et al., 1978; Hamberger et al., 1978). It was, therefore, somewhat surprising that a high glutaminase activity (Fig. 2) and a rather efficient low-affinity glutamine transport system were recently observed in cultured astrocytes by Schousboe et al. (1979a). As illustrated in Fig.
16
ARNE SCHOUSBOE
2, the glutaminase in astrocytes has the same activity both at low- and highphosphate concentrations as has the enzyme in brain homogenates from adult mice. Therefore, to avoid a futile cycle using up ATP in the cell there must be a mechanism to regulate the activities of the two enzymes. Since glutamine synthetase has a microsomal localization (Sellinger and De Balbian Verster, 1962) and the glutaminase-a mitochondrial localization (Errera and Greenstein, 1949; Salganicoff and De Robertis, 1965; Neidle el al., 1969; Dienel et al., 1977; Dennis ct al., 1977; Schousboe el al., 1979a), a possible regulation may be the transport of glutamine into the mitochondria and this transport seems to be energy dependent (Minn and Gayet, 1978). Another possibility may be that glutamate, which is abundant in astrocytes and astrocytoma cells (Schousboe et al., 1975, 1977c; Drummond and Phillips, 1977; Passonneau et al., 1977; Nicklas and Browning, 1978; Hertz et al., 1978a), may regulate the glutaminase activity since it is an inhibitor of the purified enzyme (Svenneby, 1971) as well as of the synaptosomal glutaminase (Bradford et a!., 1978) and the glutaminase in nonsynaptosomal mitochondria (Minn and Gayet, 1977). Recently it has also been observed that glutaminase in astrocytes is almost completely inhibited at a glutamate concentration of 5 mM (E. Kvamme and A. Schousboe, unpublished), which is close to the actual concentration normally present in the cells (Schousboe et al., 1975, 1977c; Hertz et al., 1978a). It is, however, an open question as to what extent glutamate transport into mitochondria may affect this regulation of the enzyme activity in the astrocytes, but glutamate readily enters mitochondria of nonsynaptosomal origin isolated from whole brain (Dennis et al., 1976; Minn and Gayet, 1977) either by aid of the glutamate-aspartate translocase (Azzi et al., 1967) or the glutamate-hydroxy transport system (Meijer et al., 1972). The latter transport system has a K, for glutamate of 1.6 mM (Minn and Gayet, 1977). In addition to the ability to convert glutamate into glutamine and vice versa, glial cells have the capacity to transaminate and to oxidatively deaminate glutamate (Table IV) since the activities of G O T and GLDH in astrocytes (Schousboe, 1977; Schousboe et al., 1977b; Roth-Schechter et al., 1977; Hertz et al., 1978a) and astrocytoma cells (Passonneau et al., 1977; Nicklas and Browning, 1978) are comparable to the corresponding enzyme activities in brain (Schousboe, 1977, 1978b). IV. Tronrport of GABA
A . BRAINSLICES
That brain slices are able to accumulate GABA from the incubation medium was first demonstrated by Elliott and van Gelder (1958) and this phenomenon, which these authors related to a storage mechanism for GABA,
TRANSPORT AND METABOLISM OF GLUTAMATE AND
GABA
17
has since been observed by a large number of investigators (Tsukada ct al., 1960, 1963; Blasberg and Lajtha, 1966; Machiyama et al., 1967, 1970; Iversen and Neal, 1968; Hokfelt ct al., 1970; Shiu and Elliott, 1973; Schousboe et al., 1976a; Banay-Schwartz et al., 1976, 1977). This accumulation of GABA in slices of spinal cord or brain cortex has been kinetically characterized and it consists of both high- and low-affinity uptake systems (Blasberg and Lajtha, 1966; Iversen and Neal, 1968; Iversen and Johnston, 1971; Beart ct al., 1972; Cohen and Lajtha, 1972; Beart and Johnston, 1973; Balcar and Johnston, 1973; Levi and Raiteri, 1973; Bond, 1973; Johnston and Davies, 1974; Cohen, 1975) of which the high-affinity uptake system is probably involved in termination of the transmitter activity of GABA (Snyder ct al., 1970; Curtis et al., 1970, 1976; Iversen, 1971; Curtis and Johnston, 1974; Schousboe, 1978a,b, 1979a). Like the corresponding transport system for glutamate (Section I,A), this uptake system is energy and sodium dependent (Balcar and Johnston, 1972a; Teller et d . , 1977) and also the stimuluscoupled release of GABA from synaptosomes seems to be energy dependent (Nelson-Krause and Howard, 1978). The kinetic constants of the highaffinity transport system in the central and peripheral nervous system of different species have recently been summarized by Martin (1976), and only a few representative values for brain cortex have, therefore, been given in Table V together with values for the neuronal and glial components of this uptake. The relative importance of these two uptake systems for the removal of GABA from synaptic clefts will be discussed in Sections IV, B and C).
B. NEURONAL TRANSPORT Measurements of GABA in isolated single neurons from spinal cord, cerebrum, and cerebellum (Otsuka et al., 1971; Okada and Shimada, 1976; Wu, 1978) have revealed high neuronal concentrations of GABA and studies on neuroblastoma cells (Hutchison et al., 1974; Schubert, 1975), bulkprepared neurons (Henn and Hamberger, 1971; Sellstrom and Hamberger, 1975), and cultured neurons from cerebellum and spinal cord (Hosli ct al., 1972, 1975; Lasher, 1974, 1975; Burry and Lasher, 1975, 1978a,b; Hosli and Hosli, 1976a, 1978a,b) have clearly shown that GABA is accumulated into neurons via high-affinity transport (Table V). It should, however, be mentioned that some of the available neuroblastoma cell lines seem to lack the ability to accumulate GABA (Schubert, 1975; Balcar et al., 1978). Furthermore, electron microscopic studies of autoradiographs of brain sections incubated with [sH]GABA (Hokfelt and Ljungdahl, 1970, 1971; Bloom and Iversen, 1971; Iversen and Bloom, 1972; Schon and Iversen, 1972; Makara et al., 1975) or uptake experiments performed after specific lesions of GABAergic pathways (Storm-Mathisen, 1975) have presented evidence that GABA
18
ARNE SCHOUSBOE
TABLE V FOR HIGH-AFFINITY UPTAKE OF GABA INTO BRAIN SLICES, KINETIC CONSTANTS SYNAPTOSOMES, AND DIFFERENT NEURONAL AND GLIAL PREPARATIONS"
Tissue preparation Adult brain slices Neonatal brain slices Synaptosomes Synaptosomes Synaptosomes Synaptosomes Cerebellar glomeruli Cerebellar glomeruli Cerebellar glomeruli Dorsal spinal roots Ventral spinal roots Bulk-prepared neurons Cultured cerebellar neurons NB 41 Neuroblastoma cells Bulk-prepared glial cells Bulk-prepared glial cells Superior cervical ganglia Sensory ganglia Sensory ganglia Cultured cerebellar glial cells C-6 Glioma cells C-6 Glioma cells C-6 Glioma cells Cultured astrocytes mouse brain Cultured astrocytes mouse brain
Km (PM)
11-31 5-43 13 4 0.42 4 9.6 10 15 24 33 0.72 0.33 0.15 0.27 0.6
7 10 9.7 0.29 32 0.22 50 40 45
'ma
(nmol/min/mg protein)
Reference
0.36- 1.76' 0.05' 2.2 1.lb
Martin, 1976 Martin, 1976 Levi and Raiteri, 1973 Martin, 1976 Henn and Hamberger, 1971 Hitzemann and Loh, 1978b Wilkin el a[., 1974 Wilson cf al., 1976 Hamberger ef al., 1976 Davies and Johnston, 1974 Davies and Johnston, 1974 Henn and Hamberger, 1971 Lasher, 1975 Hutchison et a l . , 1974 Henn and Hamberger, 1971 Henn, 1976 Bowery and Brown, 1972 Schon and Kelly, 1974b Roberts, 1976b Lasher, 1975 Schrier and Thompson, 1974 Hutchison el al., 1974 Henn, 1976 Schousboe el al., 1977a Hertz cf al., 1978b
1.3 1.6 1.5
-
0.07 0.05 0.2 1-0.84 0.0021
0.002b 0.02' 0.03 0.0005-0.002 0.023 0.0014 0.35
0.90
' Most of the values are quoted verbatim from the literature. The V,, for synaptosomes (Levi and Raiteri, 1973) is calculated from Fig. 4 in that article. The V,, values for brain slices have been recalculated from nmol/min/gm wet weigh: on the basis of a protein content of brain cortex slices of 95 values from Lasher (1975) have been converted from mg/gm wet weight (Schousboe, 1972). The V,, nmol/mg DNA on the basis of the DNA content in rat brain reported by Zamenhof cl 01. (1972). In most cases the temperature was 37'C. The temperature was 25-27'C. The results obtained on superior cervical ganglia and sensory ganglia represents peripheral glial cells since all GABA in the ganglia is located in glial cells (Beart el al., 1974; Roberts, 1976b). is taken up into presynaptic nerve terminals. This uptake site has been confirmed by studies of GABA uptake into synaptosomes (Table V) that have been shown to have a high-affinity uptake system (Henn and Hamberger, 1971; Levi and Raiteri, 1973; Martin, 1976). It is, however, a question as to what extent this uptake may be responsible for the inactivation of GABA since it has been demonstrated that the major part of the high-affinity uptake into synaptosomes is due to a 1: 1 homoexchange process (Levi and Raiteri, 1974; Simon ct al., 1974; Raiteri et al., 1975; Levi et al., 1976a,c) that can be
TRANSPORT AND METABOLISM OF GLUTAMATE AND
GABA
19
of no physiological importance for removal of GABA from synaptic clefts. Under in vivo conditions where the membrane potential is preserved there may, however, be a concentrative net uptake as shown by Sellstrom et al. (1976). In addition, Ryan and Roskoski (1977) have reported that at least 30% of the uptake of radioactive GABA in synaptosomes is due to a nkt uptake and not to a homoexchange process. The high-affinity GABA uptake into neurons is sodium dependent (Henn and Hamberger, 1971; Hutchison et al., 1974; Sellstrom and Hamberger, 1975) and the kinetics of the sodium dependence have been most extensively studied in synaptosomes (Martin and Smith, 1972;Martin, 1973; Simon et al., 1974; Sellstrom et al., 1976; Blaustein and King, 1976) and it has been shown that two or three sodium ions are required per GABA molecule transported. Furthermore, it appears that one potassium ion needs to be transported in the other direction (Martin, 1976;Sellstrom et af., 1976). The transport is, on the other hand, inhibited by high concentrations (25 mM) of potassium (Sellstrom and Hamberger, 1975; Martin, 1976; Blaustein and King, 1976). Using the general expression [Eq. (l)] for the equilibrium ratio of internal (i) and external ( 0 ) GABA derived from the expression for the equilibrium constant: GABA, GABAo
=
(e)n (%)rn
exp(rn-n)F
AE RT
(1)
in which F, R, and T have their usual meanings, A E is the membrane potential, and rn and n refer to the number of potassium and sodium ions transported, respectively. Martin (1976) and Sellstrom et al. (1976) have estimated the presynaptic ratio GABA,/GABAo to be approximately 100,000 under in vivo conditions for the membrane potential and the intra- and extracellular concentrations of Na+ and K +. This ratio is in reasonably good agreement with the GABA concentrations of 40-100 mM in GABA-ergic terminals calculated by Fonnum (1973), Fonnum and Walberg (1973a,b),and Simon el af. (1974). O n the other hand, most of this GABA is stored in vesicles (Kuriyama et al., 1968, 1969;Kuriyama, 1976) and can accordingly not contribute as free GABA to the concentration gradient across the synaptic membrane. For osmotical reasons it would also seem highly unlikely to have such a high concentration of free GABA.
C. GLIALTRANSPORT High-affinity transport of GABA also occurs in glial cells, as first demonstrated by Henn and Hamberger (1971)in bulk-prepared glial cells, and later in a variety of glial preparations including glioma cell lines (Hut-
20
A R N E SCHOUSBOE
chison ct al., 1974; Schrier and Thompson, 1974; Schubert, 1975), glial cells in spinal cord explant cultures (Hosli et al., 1972), in primary cultures of cerebellum (Lasher, 1974, 1975; Burry and Lasher, 1975, 1978a,b; Hosli and Hosli, 1976a; 1978a,b) and cerebrum (Schousboe et al., 1977a; Hertz et al., 1978b), and in glial cells in rat retina (Neal and Iversen, 1972; Marshall and Voaden, 1974a; Lake and Voaden, 1976) and peripheral ganglia (Bowery and Brown, 1972; Young ct al., 1973; Schon and Kelly, 1974a,b; Roberts, 1976a,b; Kelly and Dick, 1978). The glial uptake in rat retina is somewhat peculiar since in other species, such as rabbit, the uptake seems to be located primarily in neurons (Ehinger and Falck, 1971; Lam and Steinman, 1971; Marshall and Voaden, 1974b). Kinetic characteristics of the glial transport systems have been summarized in Table V, and it can be seen that only the transport system in cultured astrocytes exhibited a Vmaxcomparable to that found in brain slices. The C-6 astrocytoma cells had much lower capacity for GABA transport, which was also the case for neuroblastoma cells (Hutchison et al., 1974), compared to neurons cultured from cerebellum (Lasher, 1975). Surprisingly, glial cells cultured from cerebellum seem to have a low capacity for GABA transport. That also satellite glial cells have low capacities for GABA transport (Table V) may be in keeping with the fact that there is no GABA-mediated transmission occurring in peripherial ganglia (Iversen and Kelly, 1975). From the presence of a high-affinity uptake of GABA in a variety of glial preparations and the quantitative agreement between Vmaxfor GABA uptake in brain slices and astrocytes in primary culture (cf. Table V) it seems safe to conclude that glial cells and particularly astrocytes, which control a very large surface area around synapses (Wolff, 1970), are of major importance for the inactivation of GABA (Henn and Hamberger, 1971; Hamberger and Henn, 1973; Hutchison el al., 1974; Schrier and Thompson, 1974; Iversen and Kelly, 1975; Henn, 1976; Schrier, 1977; Schousboe et al., 1977a; Schousboe, 1977, 1978a,b, 1979a; Hertz ct al., 1978b). A crucial point in this conclusion is the demonstration (Hertz et al. , 1978b) of a net inward transport of GABA in astrocytes. As shown in Fig. 3 efflux of [SH]GABAfrom astrocytes cannot be stimulated by the addition of excess nonradioactive GABA to the washout medium. This strongly points against a homoexchange mechanism analogous to the one demonstrated in synaptosomes (Levi and Raiteri, 1974; Simon et al., 1974; Raiteri et al., 1975; Levi et al., 1976a,c), in peripheral ganglia glial cells (Roberts, 1976a), and in bulk-prepared glial cells (Sellstrom and Hamberger, 1976). In this context it should be noted that GABA uptake into C-6 astrocytoma cells (Schrier and Thompson, 1974; Schrier, 1977) and rat retina glial cells (Lake and Voaden, 1976) represents net uptake. From Eq. (1) and knowledge of the number of sodium and potassium ions required for GABA transport in astrocytes the equilibrium ratio between the
TRANSPORT AND METABOLISM OF CLUTAMATE A N D
GABA
21
Tine from slort of worhovt (min)
FIG. 3. Washout curves showing, as a function of time, release of radioactivity from astrocyte cultures loaded with ["CIGABA for 30 min. (a) The washout medium contained 25 f l ( 0 )GABA except during the period 42-66 min ( 0 ) when the GABA concentration was increased to 200 f l .(b) The washout medium contained 25 f l ( 0 )GABA except during the period 0-24 or 42-66 min ( 0 ) when the GABA concentration was increased to 2000 f l .(c) The washout medium contained 2.5 f l (0)GABA except during the period 42-66 min ( 0 ) when the GABA concentration was increased to 200 f l .The lack of effect of nonradioactive GABA under any of these conditions on the efflux of radioactive GABA strongly indicates that the GABA transport is not due to homoexchange. (From Hertz ct al., 1978b.)
intra- and extracellular concentration of GABA in these cells can be calculated. As shown in Fig. 4 the sodium dependence of GABA uptake is complex and at high concentrations of GABA (50 M )curves showing velocity of GABA uptake versus the sodium concentration are clearly sigmoid. It was recently concluded by Larsson ct al. (1980) that at least two sodium ions are required for GABA uptake into astrocytes, which makes the glial GABA
22
ARNE SCHOUSBOE
03
-
i
P
- a 2
.k
-f
; 01
50
100
INa4
150
(mM)
FIG. 4. Velocity of GABA uptake (nmol X min-' X mg:') as a function of the Na' concentration at the following GABA concentrations: 5 p44 ( O ) , 15 p44 (.), 50 @ 200 jd4 ,).I( 1000 f l (A). The velocities have been corrected for the unsaturable component of the uptake. Results are averages of five individual experiments, and the curves were drawn on the basis of the velocities obtained by computer analysis. Unpublished results of 0. M . Larsson, L. Hertz, and A. Schousboe.
(a),
transport comparable to that observed in synaptosomes (cf. Section IV, B). Furthermore, the uptake into astrocytes is independent of potassium (Hertz et al. 197813) although high concentrations of this ion inhibit the uptake (Table 11). If the intracellular sodium concentration is close to the estimated value for brain slices reported by Schousboe and Hertz (1971) of 50 pmol/gm wet weight and if the average membrane potential is -60 mV as reported by Moonen and Nelson (1978) and Kanje el al. (1978) for cultured astrocytes, a value of approximately 1000 for the equilibrium tissue/medium GABA ratio is obtained from Eq. (1). This value is higher than the actual value measured by Hertz et al. (1978b) and Wu et al. (1979) in astrocytes incubated in GABAcontaining media long enough to ensure equilibrium, indicating that a net uptake of GABA does occur in such cells. The intracellular/extracellular ratio of GABA that astrocytes are able to generate is possibly lower than that which can be generated by nerve endings. The uptake in glial cells may, however, still be quantitatively important since there is no or very little GABA production by the cell itself (Schousboe el al. 1977b; Hertz et al. 1978b; Tardy el al., 1978; Wu et al., 1979) in contrast to the nerve endings that contain GAD
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
23
(Weinstein et al., 1963; Salganicoff and De Robertis, 1963, 1965; van Kempen et al., 1965; Balizs et al., 1966; Fonnum, 1968) and since the activity of GABA-T, which is responsible for the degradation of GABA, is quite high in cultured astrocytes (Schousboe et al., 1977a; Schousboe, 1977, 1978a, 1979b; Tardy et al., 1978). The inhibitory effect exerted by 25 mM potassium on GABA uptake in astrocytes (Table 11) is in agreement with analogous findings on brain slices (Machiyama et al., 1967, 1970; Schousboe et al., 1976a), synaptosomes (Blaustein and King, 1976), and bulk-prepared neurons and glial cells (Sellstrom and Hamberger, 1975). It also agrees with the inhibitory effect of excess potassium on uptake of taurine, another inhibitory transmitter, into cultured astrocytes (Schousboe et al., 1976~).The observation that excess potassium increases glutamate uptake in astrocytes (Table 11) is intriguing since during excitation, in which the extraneuronal concentration of potassium is elevated (Somjen, 1975; Hertz and Schousboe, 1975), the uptake of the excitatory transmitter would be enhanced whereas the uptake of the inhibitory transmitter would be decreased, facilitating an overall inhibitory milieu. This might be a mechanism protecting neurons from excessive ,firing, which might otherwise be caused by the elevated extracellular potassium concentration (cf. Schousboe, 1978b). As can also be seen from Table 11, the GABA uptake in astrocytes was found to be independent of calcium (Schousboe et al., 1977a). This agrees with previous observations on bulk-prepared glial cells (Sellstrom and Hamberger, 1975) but is in contrast to results on different glioma cell lines which require calcium for GABA transport (Schubert, 1975).
SPECIFICITY OF THE TRANSPORT SYSTEMS D. SUBSTRATE Studies of the effects of 0-alanine and diaminobutyric acid on GABA uptake and release in brain slices, synaptosomes, and glial cells of peripheral origin (Iversen and Johnston, 1971; Simon and Martin, 1973; Simon et al., 1974; Sutton and Simmonds, 1974; Minchin, 1975; Schon and Kelly, 1974b, 1975; Iversen and Kelly, 1975; Raiteri et al., 1975; Levi etal., 1976b; Leach et al., 1976; Hammerstad and Lytle, 1976; Bowery et al., 1976; Brennan and Cantrill, 1978; Weitsch-Dick et al., 1978) have led to the conclusion that the former compound preferentially inhibits glial GABA uptake whereas the latter compound interferes primarily with the neuronal or presynaptic transport system. It should, however, be mentioned that results of Snodgrass el al. (1973) on the effect of P-alanine on GABA uptake into synaptosomes and results of Sellstrom and Hamberger (1975) on the effect of diaminobutyric acid on GABA uptake in synaptosomes and bulk-prepared neurons and glial
24
ARNE SCHOUSBOE
cells are not in agreement with this conclusion. Furthermore, Hitzemann and Loh (1 978b) have demonstrated a high-affinity uptake of 0-alanine in synaptosomes but this uptake showed a substrate specificity that was different from that of the GABA uptake. O n the other hand, studies by Schousboe and coworkers on GABA uptake into astrocytes in primary cultures have confirmed that /3-alanine but not diaminobutyric acid inhibits this uptake (Schousboe et al., 1978, 1979b; Schousboe, 1978a,b, 1979a,b). Schousboe and co-workers (Schousboe et af., 1978, 1979b, 1980) have recently performed a systematic study of the effect of a large number of GABA analogs on the high-affinity uptake of GABA into cultured astrocytes. For comparison “prisms” of brain cortex (0.5 x 0.1 X 0.1 mm) have been used as a model for the neuronal transport system since Riddall et a f . (1976) have reported that the glial uptake system is progressively destroyed as the dimensions of a brain slice decrease. Some of the results of this work have been summarized in Table VI together with similar results from studies on brain slices and peripheral ganglia glial cells by other investigators. It is seen that nipecotic acid and its derivatives (Fig. 5) are the most potent inhibitors of the glial uptake system, in agreement with the inhibitory effect of these compounds on GABA uptake in brain slices (Johnston et al., 1975a, 1976, 1977, 1979; Krogsgaard-Larsen and Johnston, 1975; Krogsgaard-Larsen, 1978; Brehm ct a f . , 1979). Except for (3RS, 4SR)-4-hydroxynipecotic acid, none of this class of compounds showed any appreciable selectivity for one of the transport systems. This compound seems to be a selective inhibitor of the glial uptake system (Schousboe et al., 1979b). A new selective glial uptake inhibitor was, however, discovered among the heterocyclic five-membered rings, namely P-proline (Schousboe et a f . , 1978). As seen in Fig. 6, its structure combines the P-alanine and the GABA molecules, and it was found to be a more potent inhibitor of GABA uptake in astrocytes than 0-alanine. A kinetic analysis of the inhibition indicates that it is a competitive inhibitor (Schousboe, 1979a). It can be seen from Table VI that it is the R form of 0proline that is inhibitory. This stereoselectivity of the carrier is apparent also for nipecotic acid and the 4-methyl derivatives of GABA and trans-4aminocrotonic acid. The latter finding indicates that the transport carrier distinguishes between the symmetrical hydrogen atoms on carbon atom 4 in the GABA molecule. This is analogous to the stereoselectivity of several enzymes that act on symmetrical molecules (Dixon and Webb, 1964). Another selective, although weak inhibitor was found among the seven-membered heterocyclic rings (Schousboe et af., 1980). RS-Perhydroazepine-3-carboxylic acid (Fig. 5) seems to be a weak inhibitor of the glial uptake system, whereas it has essentially no effect on the uptake into brain cortex “prisms” (Table VI). Among the compounds tested, RS-3-hydroxy-5-aminovaleric acid was found to be a selective inhibitor of the neuronal transport system like the
TRANSPORT AND METABOLISM OF GLUTAMATE AND
GABA
25
TABLE VI O N HIGH-AFFINITY UPTAKEO F GABA I N BRAINCORTEX MINISLICES, EFFECTSOF GABA ANALOGS PERIPHERAL GANGLIA GLIALCELLS,AND CULTURED ASTROCYTES
Analog
Minislices
Peripheral ganglia
750 S-4-Methyl-GABA R-4-Methyl-GABA 200 S-truns-4-Amino-4-methylcrotonic acid > 5000 R-tronr-4-Amino-4-rnethylcrotonic acid 160 RS-3-Hydroxy-5-aminovaleric 430 acid 1200; 504 S-2,4-Diarninobutyric acid @- Alanine > 5000; 21,OOW RS-@-Proline 1000 S-@-Proline 1700 2500 3RS, 4RS-4-Hydroxy-P-proline Muscimol 2500 1500 RS-4,5-Dihydromuscimol cis- 1,3-Arninocyclohexanecarboxylic acid 6Zd S-Nipecotic acid 500 R-Nipecotic acid 70 3RS, 4SR-4-Hydroxynipecotic acid 200 RS-N-Methylnipecotic acid 300 Guvacine 100 Isoguvacine > 5000 Isonipecotic acid > 5000 RS-Perhydroazepine-3-carboxylic acid 3000
Astrocytes 1000
120
>
5000
500 1400
> 5000 1000 320 1900 1800 2000 4000
1000 30 10 70
25
> 5000 > 5000 1000
Iversen and Johnston, 1971. Bowery and Brown, 1972. Schon and Kelly, 1974b. Bowery cf a l . , 1976. All other results from Schousboe cf al., 1978, 197913, 1980. The external GABA concentration was 1 pl4 in studies of Schousboe and co-workers, whereas studies in footnotes a-d used GABA concentrations in the range of 0.01-0.1 1M.
Guvacine
Niprcolrc acid
Isoguvacine
Piperidme - 4 c e r b o r y l i c acid
-
Prrhydroazepinr 3 - c a r b o r y l i c acid
FIG. 5. Structural formulas of some key six- and seven-membered heterocyclic compounds mentioned in Table VI. Piperidine-4-carboxylic acid is also called isonipecotic acid.
26
A R N E SCHOUSBOE
doon d""" H
H
A
0
p -Proline FIG. 6. Formulas of 0-proline illustrating its structural relation of GABA (A) and 8-alanine (B). (From Schousboe, 1979a.)
previously known compounds diaminobutyric acid (cf. earlier) and cis-3aminocyclohexanecarboxylic acid (Johnston and Stephanson, 1976; Bowery el al., 1976; Neal and Bowery, 1977; Hitzemann and Loh, 1978a). From the presently available evidence it seems safe to conclude that the neuronal and glial transport systems exhibit distinctly different specificity for the substrate. This difference may be useful not only as an analytical tool by which the individual transport systems may be studied in intact preparations such as whole brain or brain slices but it may also be of pharmacological interest since interference with one of the inactivation mechanisms for GABA might be beneficial in future treatment of some of the neurological disorders, such as schizophrenia, Huntington's chorea, Parkinson's disease, and epilepsy, in which the GABA system is thought to be involved (E. Roberts, 1974; Bird and Iversen, 1974; Meldrum, 1975; Tower, 1976; Hornykiewicz el al., 1976; Chase and Walters, 1976; McGeer and McGeer, 197613, 1979; Chase and Tamrninga, 1979).
V. Motabolirm of GABA
A. WHOLE BRAIN As mentioned in Section I, GABA metabolism via the GABA shunt may account for approximately 10% of the flux through the TCA cycle (Balizs et al. 1970; Machiyama et al. 1970). The enzymes involved in the degradation of GABA are the GABA-transaminase and the succinic semialdehyde dehydrogenase. The activity of the latter enzyme is normally higher than the activity of the transaminase (De Boer and Bruinvels, 1977), making the transamination reaction rate limiting. Moreover, the K, of succinic semialdehyde dehydrogenase for succinic semialdehyde is very low (Albers and Koval, 1961; Pitts ct al., 1965; Kammeraat and Veldstra, 1968; Walsh and Clark, 1976a; De Boer and Bruinvels, 1977), meaning that the reversible transamination reaction (Bessrnan ct al., 1953; Pitts et al., 1965; van der Laan el
TRANSPORT A N D METABOLISM OF CLUTAMATE A N D
GABA
27
1978) becomes essentially irreversible under i n uivo conditions (Baxter, 1970; Roberts and Hammerschlag, 1972). This means that the major part of GABA is derived from glutamate via the a-decarboxylation catalyzed by GAD (Roberts and Hammerschlag, 1972) although other metabolic pathways for GABA production in nervous tissue have been described (Seiler et al., 1971; Seiler and Al-Therib, 1974; Kremzner et al., 1975; Seiler and Wagner, 1976). It is furthermore generally agreed that the GABA concentration is governed by the glutamate decarboxylase rather than the GABAtransaminase (Roberts and Kuriyama, 1968; Baxter, 1970; Fisher and Davies, 1976). In spite of this, there has been a considerable interest in the study of the transamination of GABA. The GABA-T has been investigated in brain homogenates (Roberts and Bregoff, 1953; Bessman et al., 1953; Baxter and Roberts, 1958; Pitts et al., 1965; Waksman and Bloch, 1968; De Boer and Bruinvels, 1977; White and Sato, 1978; van der Laan et al., 1978), partially purified preparations (Waksman and Roberts, 1965; Sytinsky and Vasiliev, 1970), and preparations of the enzyme purified to homogeneity (Schousboe el al., 1973, 1974; Cash et al., 1974; Maitre et al., 1974; BlochTardy et al., 1974; John and Fowler, 1976). Kinetic and physicochemical constants of GABA-T from the latter group of studies have been summarized in Table VII. It is seen that large species variations are found in K , values for GABA and a-ketoglutarate, which may reflect the finding by Saito ct al. (1974b) that the enzyme is strictly species specific in terms of protein structure (Fig. 7) and even more specific than the glutamate decarboxylase (Saito et al., 1974a). O n the other hand, all preparations have given relatively identical molecular weights of around 110,000. All investigators agree that the enzyme consists of two subunits but only Schousboe et al. (1974) have found subunits of unequal size. This, again, may reflect the species specificity. The high K , values for the enzyme from pig, rat, and rabbit brain (Block-Tardy et al., 1974; Maitre et al., 1974; John and Fowler, 1976) are in sharp contrast to the low K, (10-4 - 10-3 M) for both substrates reported for GABA-T in rat brain homogenates (Pitts et al., 1965) or mitochondria (Walsh and Clark, 1976a) and partly purified GABA-T from rat and human brain (White and Sato, 1978). However, Waksman and Roberts (1965) also reported very high K , values for a relatively pure preparation of the enzyme from mouse brain acetone powder. From a functional point of view, K , values of the order of lo-+ - 10m3M seem, however, most reasonable, since they reflect the concentrations of the substrates in the brain (Baxter, 1970; Cheng, 1971). From the K, values determined by Schousboe et al. (1973) it could be concluded that a-ketoglutarate might be a regulator of the enzyme activity i n uiuo, which could make a-ketoglutarate a key compound in the regulation of GABA levels since GAD is inhibited by this substance (Wu, 1972; Wu and Roberts, 1974). Another aspect of the molecular structure of GABA-transaminase that has received considerable interest is the possible existence of isoenzymes. It has a/.,
TABLE VII KINETIC AND PHYSICAL CONSTANTS FOR BRAINGABA-T PURIFIED TO HOMOGENEITY FROM DIFFERENT ANIMALS’
K, (d) Purification Mouse brain crude mitochondria Human brain crude mitochondria Pig brain acetone powder Rat brain crude mitochondria Rabbit brain homogenate a
From Schousboe, 1978a.
MW
GABA
a-KG
Native
Subunits
Optimum PH
Reference
1.1
0.25
109,000
53,000; 58,000
8.0
Schousboe cf QI., 1973, 1974
0.4
1.o
-
-
8.6
4.8
1.3
105,000
2
51,000
8.6
Cash ct al., 1974 Bloch-Tardy ef Qf.,
4.0 18.0
5.5 5.0
105,000 120,000
2 x 57,000 2 X 58,000
8.5
X
1974
Maitre cf d., 1975 John and Fowler, 1976
TRANSPORT AND METABOLISM OF CLUTAMATE AND
GABA
29
Pure transaminose ( ng)
Crude transominose (pg)
FIG. 7 . Fixation of complement (C’) in percent by different amounts of GABA-transaminase from different species. Purified transaminase (0) was from mouse brain and crude transaminase was from mouse brain ( O ) , rat brain (w), or guinea pig brain (A). (From Saito cf al., 1974b.)
been suggested by different authors (Waksman and Bloch, 1968; Buu and van Gelder, 1974; Bloch-Tardy ct al., 1974; Cash et al., 1974; Ho et al., 1975; Tardy ct al., 1976a,b) that GABA-T from several species including mice consists of isoenzymes. In the course of purification of the enzyme from mouse brain, Schousboe ct al. (1973) had no indication of the existence of isoenzymes but it could be argued that the enzyme was extracted from a crude mitochondrial fraction. In a later study (Wu et al., 1976) in which the enzyme was quantitatively extracted from whole brain of mice, GABA-T was shown to be homogeneous in terms of molecular size in contrast to GAD, which in the same study was shown to exist in a high-molecular-weight form. Moreover, studies of the molecular structure of mouse GABA-T during ontogenetic development (Fig. 8) and in different organs (Wu et al., 1978c) employing microcomplement fixation, which is extremely sensitive for small differences in protein structure (Wilson et al., 1964), have revealed no differences in pro-
30
ARNE SCHOUSBOE
U
L 20
1
.
1
I
I
30
60
90
120
I
punits GABA-1
FIG. 8. Fixation of complement (C') in percent by GABA-transaminase from whole brains of adult ( 0 )and 7-day-old (0) mice. One unit is defined as the activity catalyzing the formation of 1 pmol glutamate/min at 37OC. (From Schousboe, 1978a.)
tein structure. It seems, therefore, unlikely that the mouse enzyme consists of isoenzymes since the existence of isoenzymes normally is reflected by differences in the isoenzyme pattern both during ontogenesis and between different organs, e.g., lactate dehydrogenase (Wroblewski and Gregory, 1961; Fine et al., 1963; Bonavita et al., 1964; Maker et al., 1972). This apparent discrepancy between the latter studies and that of Buu and van Gelder (1974) is presently impossible to explain and the question of isoenzymes of GABA-T needs further experimentation to be finally resolved. In recent years there has been an extensive effort to make active site directed irreversible catalytic inhibitors of GABA-T such as ethanolamine-osulfate (Fowler and John, 1972; Fowler, 1973), y-acetylenic GABA (Metcalf and Casara, 1975; Jung and Metcalf, 1975; Jung et al., 1977b; Metcalf et al., 1979; Schechter et al., 1979), y-vinyl-GABA (Jung and Metcalf, 1975; Jung et al., 1977a; Lippert et al., 1977; Metcalf et al., 1979; Schechter et al., 1979), gabaculine (Kobayashi et al., 1976; 1977; Rando and Bangerter, 1976; 1977; Allan et al., 1977; Rando, 1977, 1979), and isogabaculine (Schechter et al., 1979) since elevated brain GABA levels protect against different types of seizures (Meldrum, 1975; Wood, 1975; Tapia, 1975). The GABA level per se is, however, not the only determining factor since the GAD activity is also of considerable importance (Wood and Peesker, 1974; Wood, 1975). The irreversible catalytic inhibitors of GABA-T are, therefore, superior to other classical inhibitors such as aminooxyacetic acid and other carbonyl trapping agents (cf. Tapia, 1975) since they lead to a much greater increase in brain GABA levels lasting for a longer period of time (Tapia, 1975; Jung et al.,
TRANSPORT A N D METABOLISM OF CLUTAMATE A N D
GABA
31
1977a,b). Another important property of y-vinyl GABA and y-acetylenic GABA is that they, in contrast to GABA itself (Elliott and van Gelder, 1958, van Gelder, 1965b), cross the blood-brain barrier u u n g et al., 1977a; Metcalf el af., 1979). This obviously is of major importance for the therapeutical use of such compounds.
B. NEURONS Employing a histochemical technique in which NADH, formed in the GABA-T and succinic semialdehyde dehydrogenase reactions, reacts with nitroblue-tetrazolium to form a blue precipitate (van Gelder, 1965a), it has been shown by several investigators that GABA-T is localized in neuronal cell bodies in cerebrum, cerebellum, spinal cord, and retina (van Gelder, 1965a,b; Kuriyama et al., 1966; Robinson and Wells, 1973; Hyde and Robinson, 1974a-d, 1976a,b). The localization in cerebellum has been confirmed by Barber and Saito (1976) using an immunohistochemical technique based on the availability of monospecific antibody prepared against the purified mouse brain enzyme (Saito et al., 1974b). From the studies of Robinson and Wells (1973) and Hyde and Robinson (1974b), in which several anatomical parts of cerebrum and cerebellum were stained for GABA-T activity, it is apparent that the activity generally is higher in neurons than in glial cells, and van Gelder (1965a) found only staining in neurons, but it should be emphasized that the histochemical technique only gives a semiquantitative estimate of GABA-T activity. Table VIII summarizes GABA-T activities in different neural preparations. Bulk-prepared neurons have lower activity than brain homogenates and bulk-prepared glial cells (Sellstrom et al., 1975) but C-1300 neuroblastoma cells appear to have higher GABA-T activity than C-6 astrocytoma cells (Passonneau ct af., 1977). The activities in both of these cell lines are, on the other hand, much lower than the activity in the brain (Passonneau et al., 1977), and it is often difficult to make quanTABLE VIII ACTIVITY OF GABA-T I N DIFFERENT PREPARATIONS OF NEURONS AND GLIAL CELLS ~~
~
Cell type C-1300 neuroblastorna cells Bulk-prepared neurons Astrocytes in primary culture C-6 astrocytorna cells C-6 astrocytorna cells Bulk-prepared glial cells
GABA-T activity (nmol/rnin/rng protein)