Cysteine sulphinate (CSA) as an excitatory amino acid transmitter candidate in the mammalian central nervous system

Progress in NeurobiologyVol. 35, pp. 313 to 323, 1990

0301-0082/90/$0.00+ 0.50 © 1990PergamonPress plc

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CYSTEINE SULPHINATE (CSA) AS A N EXCITATORY AMINO ACID TRANSMITTER CANDIDATE IN THE M A M M A L I A N CENTRAL NERVOUS SYSTEM ROGER GRIFFITHS Department of Biochemistry and Microbiology, University of St. Andrews, St. Andrews, Fife KY16 9AL, Scotland, U.K. (Recewed 29 March 1990) CONTENTS

1. 2. 3. 4. 5.

6. 7. 8.

9. 10. 11. 12.

Abbreviations Introduction Biosynthesis of neuroactive SAAs Essential characteristics of neurotransmitters Presence, synthesis and metabolism of CSA in nerve endings Release Inactivation Receptor interaction Identity of action CSA and second messenger systems CSA and cAMP formation CSA and inositol phosphate formation Conclusion Acknowledgements References

ABBREVIATIONS

ACPD L-AP4 CA CSA EAA HCA HSA NMDA SC

Tram- l-aminocyclopentane- 1,3-dicarboxylate L-Aminophosphonobutyrate Cysteate Cysteinesulphinate Excitatory amino acid Homocysteate Homocysteine sulphinate N-Methyl-D-aspartic acid S-Sulpho-cysteine

1. INTRODUCTION A large number of compounds that occur naturally in the brain have been reported to excite neurons following activation of excitatory amino acid receptors (Watkins, 1986). Although the majority of evidence points towards glutamate (and aspartate) being the major fast-acting neurotransmitters at excitatory synapses in the mammalian central nervous system (CNS), it is generally accepted that, in a number of cases, the endogenous molecules which activate excitatory amino acid (EAA) receptors under normal and pathological conditions have yet to be definitely identified. Substantial evidence exists to support the hypothesis that some of these compounds, such as the acidic sulphur amino acids (e.g. Dunlop et al., 1990), the tryptophan metabolite, quinolinate (Stone and Perkins, 1981), and certain acidic peptides (Zaczek et al., 1983) may function

313 313 314 315 315 315 315 316 317 319 319 319 32O 320 320

as neurotransmitters and/or neuromodulators in the mammalian CNS (see also reviews by Collingddge and Lester, 1989; Hansen and Krogsgaard-Larsen, 1990). Cysteine sulphinate (CSA) and a number of structurally-related sulphur-containing amino acids (SAAs) are well-established neuronal excitants (e.g. Curtis and Watkins, 1960, 1963; Mewett et al., 1983). In addition to CSA, the other SAAs of neurochemical interest (see Fig. l) include cysteate (CA), the longer chain homologues homocysteine sulphinate (HSA) and homocysteate (HCA), and the thio-ester, Ssulpho-cysteine (SC). These compounds which have been identified as endogenous constituents of mammalian brain (Do et al., 1986; Kilpatrick and Morley, 1986) bear a strong structural resemblance to the major excitatory amino acids, glutamate and aspartate, and also exhibit similar excitatory (Curtis and Watkins, 1960, 1963; Mewett et al., 1983), cytotoxic (Olney et al., 1971; Kim et al., 1987) and epileptogenic (Turski, 1989) actions. This ability of the SAAs to mimic the excitotoxic actions of glutamate has resulted in an upsurge of interest over recent years, and generated a number of independent studies aimed at characterization of the neurochemical actions and properties of these endogenous compounds. Of all the SAAs, evidence to support a transmitter candidacy appears strongest for CSA. Before reviewing this evidence it is appropriate to present an initial and brief overview of the established and proposed biosynthesis, metabolism and inter-relationships

313

R. GRIFFITHS

314

CO0"

/~CO0-

*NH 3

*NH3

L-HSA

L-HCA

( ( .C coo L-CSA

L-CA

L-SC

FIG. 1. Structures of the endogenous excitatory sulphurcontaining amino acids. Abbreviations: L-HSA, L-homocysteine sulphinate; L-HCA, L-homocysteate;L-CSA,L-cysteine sulphinate; L-CA, L-eysteate; L-SC, S-sulpho-L-cysteine. of the SAAs.

various

neuroexcitatory

and

inhibitory

2. BIOSYNTHESIS OF NEUROACTIVE SAAs The biosynthetic route of ncuroactive SAAs in mammalian brain originates from catabolism of the essential amino acid, methioninc (Fig. 2). In a series M[THIONINE

of enzymatic reactions, methionine is demethylated to homocysteine, which lies at a metabolic branchpoint. In humans about half of the homocysteine formed is directed through trans-sulphuration to yield cysteine and other intermediates, the remainder being remethylated to methionine. Although mcthionine and cysteine are metabolised by a variety of reactions and pathways (not all present in brain) to at least two dozen intermediates and products, only pathways of immediate relevance to this review will be discussed. A number of excellent reviews on mammalian sulphur amino acid metabolism are recommended for further reference (e.g. Huxtable, 1986; Gditith, 1987). In brain, CSA is formed following oxidation of cysteine by the enzyme, cysteine dioxygenase. This reaction is believed to be the major pathway of cysteine catabolism in mammals. CSA undergoes further rapid metabolism either by decarboxylation to hypotaurine or transamination to fl-sulphinyl pyruvate. The metabolic partitioning between transamination and decarboxylation shows considerable species variation. In the rat, the specific activity of CSA transaminase is much greater than that of CSA decarboxylase in cerebral cortex and striatum, thus suggesting that the pathway through fl-sulphinyl pyruvate represents the major route of CSA metabolism in rat CNS. There is some doubt as to whether CA represents a true enzymaticallyformed metabolite of CSA. It has been suggested that CA is present in the diet as a result of non-enzymatic oxidation of cysteine and the actions of enteric bacteria. Furthermore, it has been reported that non-enzymatic oxidation of CSA to CA is the reason for the difficulty encountered in obtaining precise concentrations of CSA (Baba, 1987). In view of these findings it is unclear whether decarboxytation of CA is a metabolic route of taurine synthesis. The

]

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T 2SO4

dl°xygenasllI IIOMOCYSTEIN(l SULPHINIC ACID Il

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[ 3-AMIN0-PAOPANE SULPHINIC ACiD

~[

cystelne 1 i HOMOCY$TEICl ACIO

S-SULPHOCYSTEIN(

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~ 3-AMINO-PROPANE SULPHONIC ACIO

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FiG. 2. Biosynthetic pathways for the neuroactive sulphur amino acids. The excitatory SAAs are highlighted by the bold boxes. The (?) symbol denotes a lack of evidence to support enzymic interconversion of metabolitvs.

CSA ASANEXCITATORYAMINOAOD TRANSMITTER neuroactive SAA, SC, lies along the metabolic pathway between fl-sulphinyl pyruvate and its endproduct, inorganic sulphate. SC is a compound of neuropathological interest since it is implicated as the toxic agent in the neurodegenerative genetic disease, sulphite oxidase deficiency (Olney et al., 1975). Recent work on characterization of SC-evoked release of the amino acid transmitters, glutamate and aspartate, from primary cultures of granule cells (Dunlop et al., 1989a, 1990) coupled with a knowledge of its high affinity for EAA receptor sites in binding studies (Murphy and Williams, 1987) suggest the mechanism of its excitotoxic actions. The regional distribution of HSA and HCA in rat brain has been established (Do et al., 1986a; Kilpatrick and Mozley, 1986); however, their biosynthetic route from homocysteine is obscure and can only be assumed by analogy to the metabolism of cysteine. These oxidized products of homocysteine may be of only limited importance under normal circumstances and assume a more significant role in disorders of homocysteine metabolism (Perry, 1974; Mudd and Levy, 1983). Notwithstanding this suggestion, a great deal of recent evidence exists to suggest a role for HSA, but particularly HCA, as endogenous EAA agonists (Do et al., 1986b, 1988; Cu6nod et al., 1986; Lehmann et al., 1988). Much of this work has been undertaken in striatum where HCA appears to be a selective agonist at NMDA receptors. Perusal of the literature however indicates that using both electrophysiological and biochemical approaches, HSA and HCA agonist responses are mediated by NMDA and non-NMDA receptors in different brains regions (Davies et al., 1982; Dunlop et al., 1989a, 1990). It has been recognised that SAA metabolism in brain may be ultimately involved in the regulation of excitability by excitatory and inhibitory amino acids (Huxtable, 1986). These amino acids exist in a mutually close metabolic relationship in that decarboxylation of EAAs yields those that exhibit inhibitory properties. Since the concentration ratios of all these intermediates can be markedly altered by slight modification of enzyme activity, it is conceivable that they may represent major determinants of brain excitability.

3. ESSENTIAL CHARACTERISTICS OF NEUROTRANSMITTERS A number of criteria must be satisfied before a given substance can be accepted unequivocally as functioning as a "true" neurotransmitter (McGeer et al., 1978). Briefly, these requirements are the presence, synthesis and metabolism, release, inactivation, identity of action and receptor activation by the proposed transmitter. CSA has been shown to satisfy many of these criteria; however, to the author's knowledge, the mapping of a neuronal pathway for CSA remains a major obstacle in unequivocal allocation of a neurotransmitter status. Evidence to support the transmitter candidacy of CSA is discussed in the following sections, with greater emphasis being given to reviewing the more recent and topical findings regarding its neurochemical actions.

315

4. PRESENCE, SYNTHESIS AND METABOLISM OF CSA IN NERVE ENDINGS High affinity uptake systems for cysteine (the precursor of CSA) have been shown to be located in the synaptosomal plasma membrane (Hwang and Segal, 1979; Misra, 1980), while the presence of cysteine dioxygenase, the CSA synthesising enzyme, has also been reported in synaptosomes (Agrawal et a/., 1971; Misra and Olney, 1975; Pasantes-Morales et al., 1977; Recasens et al., 1978). Enzymes involved in the degradative pathway of CSA have also been localised in nerve endings by immunochemical methods (Recasens and Delaunoy, 1981; Chan-Palay et al., 1982). The heterogeneous distribution of CSA in gross dissections of rat brain has been demonstrated (Baba et al., 1980; Ida and Kuriyama, 1983) with estimates of the mean brain concentration of CSA ranging between 0.1-0.2 mM.

5. RELEASE Demonstration of a depolarisation-induced release provides further support to the hypothesis that CSA may play a role as a neurotransmitter. The release of both endogenous CSA and of exogenously-supplied radiolabelled CSA has been studied. Using a sensitive hplc method, Do et al. (1986a) were able to demonstrate that endogenous CSA could be released from slices of rat brain in a Ca2+-dependent manner following K+-induced depolarisation. This release of CSA was observed in slices derived from cortex, hippocampus and mesodiencephalon. Making use of a superfusion method in an attempt to avoid reuptake of any released CSA, Iwata et al. (1982a) and Baba et al. (1983) demonstrated the K +- and veratridineevoked release of [14C]CSA from pre-loaded rat brain cortical slices and crude (P2) synaptosomal fractions which were partially Ca2+-dependent. The release of L-[3H]CSA from pre-loaded slices of various rat brain regions in response to either K +- or veratrine-induced depolarisation has also been investigated (Recasens et al., 1984b). These workers showed a regional variability in the calcium-dependence of CSA release and, in general, the depolarisation-induced release pattern was heterogenous.

6. INACTIVATION Sodium-dependent, high-affinity transport of CSA has been demonstrated and kinetically characterised in primary cultures and cell lines of neurons and glia (Abele et aL, 1983), in membrane vesicles (Recasens et a[., 1982) and in synaptosomes (Iwata et al., 1982a) using radiolabelled CSA as the transport substrate, in synaptosomes (Grieve et al., 1990) using hplc to measure net CSA uptake and also by quantitative autoradiography (Parsons and Rainbow, 1984) by mapping the distribution of [35S]CA, a structural analogue of CSA, and making the assumption that the Na+-dependent binding sites identified were identical to those of CSA. In all the kinetic studies of CSA transport, Km values of essentially the same order

316

R. GRIFFITHS

(6-60/aM) were obtained. The range of values determined can be explained on the basis of differing methodology and the nature of the preparation employed. However, some major discrepancies occur with regard to the nature of transporters. Thus, some workers (Iwata et al., 1982a; Recasens et al., 1982) showed a biphasic dependence of radiolabelled CSA uptake with regard to varying CSA concentrations, indicative of both high and low-affinity transporters of CSA. However, using a wide range of CSA concentrations and compensating for non-specific attachment and/or diffusion, Grieve et al. (1990), using a hplc method, could demonstrate only a single relatively high affinity transport site (Kin = 57/aM). The physiological requirements for a low-affinity (Kin = 200-400 #M) CSA transporter are unclear. It is possible that this dubious low-affinity transport is of no functional significance and may represent only the affinity that CSA exhibits for another (related?) transporter. In agreement with Grieve et al. (1990), other workers (Abele et al., 1983) observed only a single, high-affinity transporter (Kin = 5-80 pM) in primary cultures and cell lines, calculated from the uptake data employing radiolabelled CSA. In order to resolve these discrepancies, one aspect of current work in the author's laboratory (unpublished) involves a study of the transport kinetics of CSA (and other neuroactive SAAs) using hplc analysis following SAA transport assays undertaken using primary cultures of different neurons (cortex and cerebellar granule cells), astrocytes and synaptosomal fractions. Mutual inhibition transport kinetic studies show that glutamate, aspartate, CA and CSA are competitive inhibitors of each other's transport, providing data consistent with CSA sharing a common transporter With the dicarboxylic acidic amino acids (Iwata et al., 1982a; Erecinska and Troeger, 1986; Wilson and Pastuszko, 1986; Griffiths et al., 1989; Grieve et al., 1990).

7. RECEPTOR INTERACTION The actions of the EAAs are mediated by at least five receptor subtypes (Watkins and Evans, 1981; Lodge and Collingridge, 1990) which transduce their signals both by the classical actions as mediators of fast excitatory responses and also via stimulation of inositol phospholipid metabolism. The EAA receptor subtypes are generally designated with respect to the response of selective pharmacological agonists. Receptor nomenclature has recently been modified in line with current availability of agonists with greater selectivity for the variety of EAA receptor subtypes (see Lodge and Collingridge, 1990). The three most studied subtypes are the ionotropic, N-methyl-o-aspartate [NMDA]-, domoate (formerly kainate)- and ~-amino-3-hydroxy5-methyl-4-isoxazoleproprionic acid [AMPA] (previously quisqualate)-receptors. More recently, a quisqualate-activated (but AMPA-insensitive) EAA receptor has been recognised which, when activated, leads to generation of the second messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) as a result of plasma membrane phosphoinositide

metabolism. The physiological role of this so-called "metabotropic" receptor, which appears to be selectively activated by the agonist, trans-l-aminocyclopentane-l,3-dicarboxylate (trans-ACPD), has yet to be elucidated. A fifth (L-aminophosphonobutyrate, L-AP4) receptor, of possible pre-synaptic origin, is proposed, although in the absence of appropriate agonists and antagonists, little new information is available regarding its role in CNS function. Initial demonstration of specific Na+-independent binding (receptor) sites for CSA on synaptic membranes was reported (Iwata et al., 1982b; Iwata and Baba, 1983; Recasens et al., 1982, 1983, 1984a,b) prior to the development of many of the currently available EAA receptor antagonists. Recasens et al. (1982) demonstrated a specific and saturable binding of [3H]CSA to rat brain membranes which occurred by a single high-affinity process (Kd = 100 nM; Bmax= 2.4 pmol/min/mg protein), and furthermore, showed a heterogenous regional distribution of [3H]CSA binding. In subsequent studies (Recasens et al., 1983, 1984a,b), these workers compared the binding of [3H]CSA and [3H]glutamate in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to evaluate whether these two amino acids bound at distinct sites. It was concluded that [aH]CSA and [3H]glutamate binding processes were complex but the evidence pointed to the existence of several binding sites some of which were shared by both substances. More importantly, the results indicated that there were binding sites which were unique to CSA, thus enhancing its transmitter candidacy in the CNS by implying that CSA may have a distinct receptor. Following the initial classification of the ionotropic EAA receptors into NMDA, kainate and quisqualate subtypes (reviewed by Watkins and Evans, 1981), it was a number of years before the subclass specificity of the SAAs were reported (Pullan et al., 1987; Murphy and Williams, 1987), although selectivity for EAA receptors had been previously suggested for some of the SAAs (Davies et al., 1982; Mewett et al., 1983). This "delay" coincided with renewed interest in the SAAs as endogenous neuroexcitatory agonists of physiological and pathological interest whilst also reflecting the use of the SAAs as probes to characterise further the EAA receptors. In radioligand binding studies, Murphy and Williams (1987) studied the inhibition by SAAs of [3H]AMPA and [3H]kainate binding to rat brain synaptic membranes. Their studies showed that, of the two receptor subtypes investigated, the SAAs interact preferentially with the AMPA receptor. Of the L-SAAs utilised in their study, CSA was less potent than either CA, HSA, HCA and especially SC in displacing either [3H]AMPA or [3H]kainate, exhibiting K~ values of 8.5#M and >I00#M, for the respective sites. However, it is known that CSA is transported with high-affinity via the L-glutamate carrier in the pre-synaptic membrane, and therefore, would be rapidly cleared from the synaptic cleft and also the EAA receptor sites. This might explain the low displacer potency of CSA in binding studies and furthermore, indicates a potential underestimation in quantifying a Ki value. Moreover, the rank order of SAA potency in displacing the radiolabelled agonists

CSA As ANEXCITATORYAMINOACIDTRANS~UTTER correlates inversely with the Ki values of the various SAAs as competitive inhibitors of the high-affinity, Na+-dependent dicarboxylic amino acid transporter in synaptosomes and in primary cultures of neurons and astrocytes (Griffiths et al., 1989). An extensive study was undertaken by Pullan et al. (1987) in which receptor selectivity (in synaptic plasma membranes) and excitotoxic potential (in chick retinal slices) of the SAAs were evaluated. Although showing a rank order of potency in displacing radiolabeiled receptor site agonists, which was in general agreement with that of Murphy and Williams (1987), significant quantitative differences in the magnitude of the K~ values are apparent. Nevertheless, the study of Pullan et al. (1987) showed that CSA acted as a broad-spectrnm agonist at all EAA receptors with a potency in functional assays (Na + flux and excitotoxic index) greater than that of glutamate. Further evidence to support EAA receptor activation by CSA is presented below as part of identifying its transmitter-like actions.

8. IDENTITY O F ACTION Although glutamate is accepted as being the predominant EAA in brain, additional EAAs may play similar roles. Indeed, the neurotransmitters which elicit the release of glutamate, D-[3H]aspartate (Davies and Johnston, 1976; Malthe-Sorenssen et al., 1979, Storm-Mathisen, 1984) and GABA have not been unequivocally identified, at least in hippocampus and cortex, while there is also uncertainty regarding some excitatory pathway innervation (via mossy fibres) to granule cells of the cerebellum (Freeman et aL, 1983). Although electrophysiological studies have shown the excitatory nature of CSA in spinal cord (Curtis and Watkins, 1960, 1963), in cortex (Thomson, 1986) and in caudate nucleus (Cu6nod et aL, 1986; Turski et aL, 1987) little attention has been directed to a biochemical/pharmacological evaluation of CSA as an inducer of neurotransmitter release. However, recent investigations from the laboratories of Griffiths and Schousboe (Dunlop et aL, 1989a,b, 1990) and from Schramm (Minc-Golomb et aL, 1989), have addressed this question. These workers, in addition to Weiss (1988), have demonstrated that CSA, as well as other SAAs, evokes the release of excitatory and inhibitory amino acid transmitters in the absence of additional depolarising agents. Griffiths and co-workers have utilised primary cultures of different neurons (viz. cortical [gabaergic] neurons and cerebellar granule cells [giutamatergic]) and also synaptosome fractions, as complementary models to study SAA-evoked release. In primary cultures, CSA evoked a dose-dependent, saturable and Ca2+-dependent release of D-[3H]aspartate and [3H]GABA from respectively, granule cells and cortical neurons (Dunlop et al., 1989a, 1990). Furthermore, CSA was found to depolarise the plasma membrane when the equilibrium distribution of the lipophilic [3H]TPP+ cation was used as an indicator of membrane potential change. This observation, coupled with the sensitivity of CSA-evoked Ca 2+dependent release to the presence of selective, JPN 35/4--F

317

excitatory amino acid antagonists for both the NMDA and non-NMDA receptors, is consistent with a CSA-stimulated, receptor-mediated release of neurotransmitter arising as a consequence of membrane depolarisation. In both cortical neurons and granule cells, CSA also evoked a component of release which was insensitive to the presence of external Ca 2+. This Ca 2+independent component of release, which may assume much significance under pathological conditions of extracellular amino acid accumulation, can be inhibited (manuscript in preparation, but see Dunlop and Griffiths, 1990) by non-transportable blockers of both the glutamate and GABA Na +dependent, high-affinity transporters, viz. dihydrokainate (Johnston et al., 1979) and SKF 89976-A (Larsson et al., 1988), respectively. Furthermore, this component of release can be abolished when Na + are replaced by ions such as Li + or choline. Taken together, these observations are consistent with the Ca2+-independent component of CSA-evoked release being attributable to thermodynamic reversal of the transporters which normally serve, in the direction of uptake, to physiologically inactivate or "clear" released transmitter from the synaptic space by a Na+/neurotransmitter symport (Kanner, 1983). A summary of our recent studies aimed at characterising the nature of CSA-evoked release from primary cultures is presented in Fig. 3. The CSA-evoked release of neurotransmitter amino acids has also been studied in some detail in synaptosome fractions prepared from rat cerebrocortex and cerebellum (Dunlop et al., 1989b). CSA was the most efficacious of a number of SAAs in evoking a release of D-[3H]aspartate of similar magnitude to that produced by L-glutamate or Laspartate. However, such release was wholly Ca 2+independent. In order to probe further the absence of Ca2+-dependency, analysis of endogenous release, measured by hplc, was determined. This revealed the presence of both Ca2+-dependent and -independent components of CSA-evoked release of endogenous L-glutamate but only a Ca2+-independent component of endogenous L-aspartate release. The CSAevoked release of exogenously-supplied [3H]GABA from synaptosomes exhibited Ca2+-dependent and -independent components of release, the Ca 2+dependent component only being apparent after pre-loading for 60 min with [3H]GABA. This timedependency of loading for demonstration of depolarisation-induced [3H]GABA release has been observed by Nicholls' group in a series of elegant papers aimed at elucidation of the molecular mechanisms of amino acid neurotransmitter release (see Nicholls (1989) for review). In synaptosomes, the magnitude of CSA-evoked release of exogenously-supplied D-[3H]aspartate and [3H]GABA, and of endogenous L-glutamate, L-aspartate and GABA, is unaffected by up to 1 mM concentrations of EAA receptor antagonists, thus demonstrating the non-involvement of currently recognised EAA receptor subtypes in mediating the release (Dunlop et al., 1989b). Since depolarisation of the plasma membrane could be demonstrated by use of [3H]TPP+, these observations indicate that CSA-evoked release could be triggered in part by

MECHANISMSOF ~A-RVOKEDStELEASEOF AMINO ACID NEUROTRANSMrrTERS

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(d)

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CSA

CSA

~

2K +

~

CSA

t RELEASE

RELEASE

FIG. 3. Characterisation of CSA-evoked release of amino acid transmitters from primary cultures of cortical neurons and cerebellar granule cells. Mechanisms of CSA-evoked release of amino acid transmitters [1], where T = GABA or glutamate, are shown in (a). Activation of EAA receptors [1] by CSA leads to opening of ligand-gated cation channels [2] which allows entry of Na +. The ensuing depolarisation of the plasma membrane causes opening of voltage-gated Ca 2+ channels [3] which is then thought to result in a Ca2+-dependent exocytotic release of T [4]. Depolarisation may also occur following electrogenic co-transport of CSA with Na + via the high-affinity transporter [5] as a result of thermodynamic reversal of the transporter and release of T in a Ca2+-independent manner [6]. An ion pump, the Na+/K+-ATPase [7], is crucial in maintaining the (inside) negative potential. A Ca2+-independent component of SAA-evoked release can be demonstrated in the presence of voltage-gated Ca 2÷ channelblocker, for example, Co 2+ (b). This component of release can be demonstrated to arise from reversal of the plasma membrane transporter as shown by the attenuation of T release (c, dashed arrow) in the presence of either the GABA transport blocker, SKF 89976-A, or the glutamate transport blocker, dihydrokainate, or by replacement of Na + with Li + or choline ions. The involvement of EAA receptors in both the Ca2+-dependent and -independent release of T is demonstrated by the attenuation of CSA-evoked release in the presence of EAA receptor antagonists selective for the NMDA- and the non-NMDA receptor subtypes (d). Blockade of the EAA receptor binding site would prevent opening of the ligand-gated Na + channels and consequently inhibit release. Any observed release would be a result of reversal of the transporter and independent of the presence of Ca 2+. The illustrations are solely schematic representations of the proposed mechanisms and are not meant to convey any suggestion of the localisation of components in respect of the nerve cell body or nerve ending. 318

C S A AS AN EXCITATORY AMINO ACID TRANSMrlTER

depolarisation-induced reversal of the synaptosomal plasma membrane transporters. Schramm and co-workers (Minc-Golomb et al., 1989) employed a tissue slice preparation to study CSA-evoked release from rat hippocampus. These workers were able to demonstrate a CSA-evoked release of exogenously-supplied D-[3H]aspartate and [14C]GABA, in the absence of additional depolarising agents, which was dose-dependent, saturable and displayed tetrodotoxin sensitivity. Moreover, the evoked-release of D-[3H]aspartate was shown to be Ca~+-independent unlike that of [~4C]GABA which was potentiated by removal of Ca 2+. Their demonstration of a CSA-evoked Ca2+-independent release of exogenously-supplied D-[3H]aspartate from slices is in agreement with that of Dunlop et al. (1989b), using synaptosome fractions, and with others investigating the evoked-release of D-[3H]aspartate from synaptosomes by alternate depolarising agents (Levi and Gallo, 1981, 1986; Wheeler, 1984) but contradicts a number of studies which demonstrate the depolarisation-induced Ca2+-dependent release of D[~H]aspartate from primary neuronal cultures (Drejer et al., 1983, 1986; Dunlop et al., 1989a, 1990). The reason for this preparation-related discrepancy in reports of the Ca2+-dependency of D-[3H]aspartate release is unresolved.

9. CSA AND SECOND MESSENGER SYSTEMS A number of reports have appeared which show that the EAAs, and in some cases CSA, stimulate the formation of second messengers such as the cyclic nucleotides, cyclic AMP (cAMP) and cyclic GMP (cGMP), and those derived from metabolism of plasma membrane phosphoinositide metabolism, inositol triphosphate and diacylglycerol.

10. CSA AND cAMP FORMATION It is well established that biogenic amines such as norepinephrine, histamine and adenosine stimulate marked accumulation of cAMP in vitro by direct interaction and activation of well-defined extraceUular receptors. The EAAs have also been shown to markedly elevate the formation of cAMP in incubated brain slices (Ferrendelli et al., 1974; Shimizu et al., 1974). However, it appears that EAAs, such as glutamate, may stimulate the accumulation of cAMP in brain slices indirectly following release of endogenous factors (Bruns et al., 1980). Since this stimulation by glutamate is blocked by adenosine antagonists or adenosine deaminase (Shimizu et al., 1974; Mah and Daly, 1976), it appears that adenosine which is known to be released from glutamate-treated slices (Pull and McIlwain, 1975) is primarily responsible for activation of the systems generating cAMP. An alternative proposal by Ferrendelli et al. (1974) and Shimizu et al. (1975) suggests that common receptors may be involved. It is unlikely that EAA receptors are directly coupled to adenylate cyclase (the cAMP-synthesising enzyme) since the EAAs do not activate adenylate cyclase in a cell-free system,

319

at least of hippocampal origin (Baba, 1987). In a series of publications (Baba et al., 1982, 1984a,b), the heterogeneity of EAA receptors involved in the CSA-stimulated formation of cAMP has been demonstrated. These workers also showed that CSA was more potent than either glutamate or aspartate in stimulating cAMP formation in guineapig hippocampal slices. Furthermore, on the basis of differences in stimulatory responses to CSA and glutamate/aspartate in the presence of various non-selective EAA antagonists, it was suggested that CSA receptors were distinct from glutamate receptors. This latter suggestion endorses the conclusions made from radioligand binding studies (Iwata et al., 1982b; Iwata and Baba, 1983; Recasens et al., 1982, 1984) in which distinct binding sites for CSA and glutamate were demonstrated. In attempting to further evaluate the EAA receptors involved in the CSA-stimulated formation of cAMP, other approaches were adopted. Thus, when hippocampal slices were treated with polyunsaturated fatty acids (e.g. iinoleic acid) in the presence of either norepinephrine or adenosine, an enhanced coupling betweeen adenylate cyclase and norepinephrineand adenosine-receptors was observed, whereas the treatment had no significant effect on the response to either CSA or glutamate, thus indicating a qualitative difference between the coupling of EAA receptors and adenylate cyclase from those of other transmitters (Baba et al., 1984a,b). More recently, further differences between these systems have been demonstrated. Thus, Baba et al. (1988) showed that forskolin (an activator of adenylate cyclase) at 0.1-10#}a concentrations markedly attenuated cAMP stimulation induced by CSA although potentiating the stimulatory effects of histamine and adenosine. The studies of CSA-stimulated formation of cAMP have been extended by Minc-Golomb et al. (1989), who showed that forskolin plus isobutyl methyl xanthine (IBMX; a phosphodiesterase inhibitor) strongly enhanced the CSA-stimulated release of exogenously-accumulated D-[3H]aspartate and [14C]GABA from rat brain hippocampal slices, whereas forskolin plus IBMX alone, caused little release of the radiolabelled amino acids. These results show clearly that formation of cAMP has a highly significant potentiating effect on the CSA-evoked release of these two transmitters, presumably by initiating phosphorylation of some specific single component. The inability of forskolin plus IBMX alone to elicit significant release of the transmitter amino acids is consistent with the presence or requirement of an additional messenger to cAMP.

11. CSA AND INOSITOL PHOSPHATE FORMATION An enhanced metabolism of membrane inositol phospholipids is believed to have a major role in the transduction of various chemical synaptic signals. As a consequence of the receptor-mediated activation of phospholipase C, plasma membrane phosphoinositides are hydrolysed, into diacylglycerol and inositol phosphates. Two independent cascades of events are triggered by these two metabolites: inositol

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phosphates trigger the intracellular mobilization of Ca 2+, while diacylglycerol stimulates protein phosphorylation by activating protein kinase C. In addition to the well-characterised agonist actions of acetylcholine, norepinephrine, 5-hydroxytryptamine and histamine in stimulating phoshoinositide metabolism (see Michell, 1975, for review), more recent studies have shown that EAAs (L-glutamate, L-aspartate and a number of their structural analogues) directly stimulate inositol phosphate synthesis in a number of preparations, such as synaptoneurosomes (Recasens et al., 1988), primary cultures of cerebellar granule cells (Nicoletti et al., 1986a) and striatal neurones (Sladeczek et al., 1985; Schmidt et aL, 1987), hippocampal (Nicoletti et al., 1986b) and cortical (Godfrey et aL, 1988) slices, and in Xenopus oocytes injected with rat brain m R N A (Sugiyama et al., 1987). However, the exact nature of the EAA receptor associated with the EAA-stimulated production of inositol phosphates has yet to be elucidated. Sladeczek et al. (1985) demonstrated that various EAAs, but not inhibitory or non-neuroactive amino acids, directly stimulate formation of inositol phosphates. The neuroactive SAAs, CSA, CA and HCA, were shown to stimulate formation of inositol phosphates, although being less potent than either L-glutamate or L-aspartate. In view of the excitotoxic actions of the SAAs and the proposed neurotoxic role of Ca 2+ in neuronal damage, further knowledge of the effects of SAAs on phosphoinositide metabolism is warranted, particularly since it is possible that SAA activation of the so-called "metabotropic" EAA receptor could result in, amongst other things, elevation of intracellular Ca 2÷ due to inositoi phosphate production. Excitatory amino acids have also been reported to produce stimulatory or inhibitory effects on phosphoinositide metabolism which is induced by other agonists. Thus, for example, inhibition by EAAs of norepinephrine-induced phosphoinositide hydrolysis in brain tissue has been demonstrated by some (Nicoletti et al., 1986c; Jope and Li, 1989) but not by others (Baudry et al., 1986; Schmidt et al., 1987). In a recent study (Li and Jope, 1989), it was shown that various SAAs, including CSA (which was of intermediate potency to cysteine and HCA), inhibited the norepinephrine-stimulated phosphoinositide hydrolysis in rat cortical slices. These observations imply that certain SAAs effectively modulate the response to norepinephrine of the phosphoinositide second messenger system in rat brain. The actual mechanism(s) involved are yet to be resolved, although it has been suggested (Li and Jope, 1989) that (i) excitotoxic damage by the SAAs, (ii) SAA activation of specific receptors which have direct or indirect modulatory functions, or (iii) uptake of SAAs with subsequent modulation at an intracellular site, may be involved.

12. CONCLUSION It is clear that a number of the neurochemical properties of CSA are common to those exhibited by more established neurotransmitters. Of all the excitatory SAAs, the evidence is most convincing for CSA

which appears to satisfy most, if not all, the criteria necessary for acceptance of transmitter status. It is proposed that, on the basis of the available information reviewed in this article, CSA is able to function as an EAA transmitter and may also participate in a number of modulatory interactions. Acknowledgements--The author acknowledges the efforts

of all co-workers in his laboratory, and particularly wishes to thank Professor Arne Schousboe (Panum Institute, Copenhagen) for frequent use of cell culture facilities. The personal research of the author was supported by project grants from The Wellcome Trust, The Scottish Home and Health Department and The Maitland Ramsay Trust (scholarship for J. Dunlop), and by travel and equipment grants from The Royal Society (European Exchange Program), The Carnegie Trust for the Universities of Scotland, Federation of European Biochemical Societies, The SmithKline (1982) Foundation and The William Ramsay Henderson Trust.

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