Neurochemical Characterization of Cysteine Sulfinic Acid, an Excitatory Amino Acid, in Hippocampust Akemichi BABA Departmentof Pharmacology,Faculty of PharmaceuticalSciences, Osaka University,1-6 Yamada-oka,Suita, Osaka 565, Japan Accepted September16, 1986

Abstract-In this communication, I have summarized our studies on the possible roles of cysteine sulfinic acid (CSA) in the central nervous system (CNS), from these observations, CSA was suggested to be a neurotransmitter. We reported the presence of CSA in the CNS and subsequently characterized Na+-dependent high affinity uptake and depolarization-induced release of CSA. Depolarization induced release of [' 4C] CSA from the preloaded hippocampal slices was specifically attenuated by benzodiazepines and GABA agonists. Synaptic membranes have a Na+-independent specific binding site for cysteic acid, an analogue of CSA, which may be a possible binding site for CSA. This binding site seemed to be distinct from that for glutamate. To assess CSA as a physiologically active candidate which is distinct from glutamate, two neurochemical experiments were performed: one experiment determined the enhancement by excitatory amino acids of depolarization-induced release of [3H]GABA from the preloaded slices, and the other one monitored the cyclic AMP formation by excitatory amino acids in hippocampal slices. In both studies, differences in the responses to the various antagonists indicate that CSA receptors are distinct from glutamate receptors. Furthermore, we proposed that excitatory amino acid receptors which are sub sequently linked to adenylate cyclase are functionally related to the CI channel.

There is substantial evidence that glutamate product, taurine, has inhibitory effects in the and aspartate may be excitatory neuro CNS. Since CSA has a strong excitatory transmitters within the central nervous system effect in the central neurons when applied (CMS) (for review, see refs. 1 and 2). The iontophoretically, like glutamate and aspar recent development of specific antagonists tate do, it was classified as an excitatory of excitatory amino acids has revealed at amino acid (5). Among the metabolites least three pharmacologically distinct classes shown in Fig. 1, only CSA which was injected of receptors for excitatory amino acids in the intraventricularly caused severe behavioral vertebrate CNS (3, 4). In view of the electro and EEG seizures of which occurrences were physiological studies, some other amino acids markedly reduced by a simultaneous injection are also neurotransmitter candidates in the of taurine (6, 7). Therefore, if CSA has an CNS. excitatory function and taurine an inhibitory Cysteine sulfinic acid (CSA) is an inter function in the CNS, the metabolism of mediate of the metabolic pathway of taurine cysteine to taurine may be significant for biosynthesis (Fig. 1), in which the final neuronal functions. By contrast to the case of taurine (8-12), little is known about the t This was presented at the 59th General Meeting neurochemical and pharmacological char of the Japanese Pharmacological Society, April acteristics of CSA. To determine the role of 1986, on the occassion of the author receiving the CSA in the CNS, it is necessary to charac Award for Encouragement of Young Investigator terize the neurochemical aspects of CSA in from the Japanese Pharmacological Society. the CNS. In this communication, I shall Fig. 1. Metabolic pathway of cysteine sulfinic acid in the central nervous system. briefly describe some neurochemical charac is much higher than that of CSA de terizations of CSA in the CNS, especially carboxylase in rat cerebral cortex and striatum, in the hippocampus, which were mainly showing that the pathway leading to pyruvate performed in our laboratory. is likely to be a major route for CSA 1. Metabolism, uptake and release metabolism in the CNS. Detailed character CSA is synthesized from cysteine by ization of the enzymes in CSA metabolism cysteine oxidase and is converted to taurine have been reviewed elsewhere (19). It by CSA decarboxylase. These enzymes are should be noted that the characterization of localized in synaptosomes (13). Synap cysteine oxidase of the brain remains to be tosomes can accumulate extracellular cysteine clarified. We examined the uptake and and synthesized CSA and taurine (14, 15). release of CSA by synaptosomes and slices Although the electrophysiological study had of rat cerebral cortex (20). Like many other revealed the excitatory effects of CSA in the substances, CSA is accumulated in synap central neurones (4), there had been no tosomes by a Na-dependent high affinity information on this amino acid. In 1980, we uptake system of which the Km value is 12 used an enzymatic cycling method and icM. None of the centrally acting agents had reported the presence and the distribution of any effect on the uptake of CSA at the con CSA in rat CNS (16). Later, H PLC methods centration of less than 10 W. Glutamate, were developed for the determination of CSA aspartate and cysteic acid are competitive in brain preparations (17, 18). CSA is distri inhibitors for the uptake of CSA, indicating buted unevenly in rat brain, but its content that CSA may be transported into synap is much lower than that of glutamate. Since tosomes by the high affinity carrier system oxidation of CSA to CA occurs non for acidic amino acids (20). enzymatically, it is difficult to estimate the One of the important criteria for neuro precise concentration of CSA. CSA is a transmitters is that the candidates are released substrate for both CSA decarboxylase and by depolarization in a Cat'-dependent CSA transaminase. The former enzyme manner. We, therefore, next examined the initiates a pathway leading to taurine; the release of [14C] CSA from the preloaded latter enzyme forms / -sulfinylpyruvate, which slices and synaptosomal fractions by a spontaneously decomposes to pyruvate (Fig. superfusion method (20). Like other putative 1). The specific activity of CSA transaminase transmitter substances, CSA is released from the preloaded preparations in a partly Ca2+ binding site for CSA that differs from dependent manner by depolarization. glutamate binding sites. We used [35S] Recently, Do et al. (18) reported that cysteic acid (CA), an analogue of CSA, as a depolariztion caused in vitro release of ligand for the binding assay (23). Scatchard endogenous excitatory sulfur-containing analysis of Na+-independent specific binding amino acids including CSA from slices of of CA indicated a single population of binding various rat brain regions. sites, with a Kd of 474 n M and a Bmax of Inhibition of CSA release by benzodiaze 3.29 pmol/mg protein. Displacement studies pines (21): When the pharmacological dose with various structural analogues showed of CSA is injected intraventricularly, it causes that among the excitatory amino acids strong EEG seizures which initiate in the tested, L-CSA was the most effective hippocampus and propagate to the cortex displacer, following by L-glutamate, L-CA (7). Therefore, in the following experiments, and L-aspartate. The K, value of L-CSA was the preparation used was the hippocampus. about 1/14 of that of L-glutamate. Two Benzodiazepines and meprobamate, but not conclusions can be derived from the obser chlorpromazine, diphenylhydantoin and vations described above. First, synaptic hexobarbital, at a concentration of 100 i M membranes have a Na+-independent specific significantly inhibited the depolarizatioin binding site for CA, which may be a possible induced release of [14C] CSA from the binding site for CSA. Second, this binding preloaded rat hippocampal slices without site seems to differ from that for glutamate. affecting the spontaneous release. The Recasens et al. (25, 26) used [3H] CSA as inhibition by diazepam is relatively specific, a ligand and also indicated the presence of because it also inhibited the release of distinct binding sites for CSA and glutamate glutamate without affecting the release of in synaptic membranes. In addition, our GABA, ACh, noradrenaline and dopamine. recent study showed that the binding site IC50 values of diazepam for high K+-induced for CSA has a complete dependency on Cl-, release of CSA and glutamate were about showing that the characteristics of CSA 20 ,uM and 7 pM, respectively. GABA is an binding are not identical to glutamate inhibitory neurotransmitter of basket cells binding (Baba et al., in preparation). Thus, which innervate granule cells, excitatory the radioligand binding assay indicates the amino acid neurones, in the hippocampus presence of a specific binding site for CSA in (22). As did the benzodiazepines, GABA and synaptic membranes. However, to assess miscimol at the concentration of 100 aM CSA as a physiologically active candidate also reduced the release of CSA. Bicuculline, which is distinct from glutamate, it is a GABA antagonist, which by itself had no necessary to find some specificities in the significant effect, antagonized the inhibitory actions of CSA. This was investigated in the effects of diazepam and GABA. Similar following two neurochemical experiments. results were obtained with the glutamate 3. Neurochemical effects release. These results indicate the modulation Potentiation of GABA release: Glutamate by GABA innervation of the release of and similar substances are likely to be the excitatory amino acids in rat hippocampal neurotransmitters of several afferent and formation, and they also suggest that some efferent pathways, and GABA seems to be of the pharmacological effects of diazepam a neurotransmitter for basket cell inhibition may be a consequence of inhibition of of pyramidal and granule cells in the hip excitatory amino acid transmission. pocampal formation (22). To differentiate 2. Binding site for CSA (23, 24) CSA neurones, if any, from glutamate Among the excitatory amino acids, the neurones, we characterized the stimulation of Na+-independent specific binding of gluta GABA release by excitatory amino acids mate to synaptic membranes has been especially CSA in rat hippocampal slices studied extensively (for review, see refs. 2 (27). Excitatory amino acid agonists, such as and 4). It is, therefore, of interest to examine CSA, glutamate, kainate (KA) and quis whether synaptic membranes have a specific qualate (CA), significantly potentiated the high K* (25 mM)-induced release of [3H] However, our studies on the mechanisms of GABA from the preloaded hippocampal slices CSA-induced formation of cAMP in guinea at concentrations above 0.1 mM. Among pig hippocampal slices (31-34) conflict these agonists, CSA was the most potent. with this hypothesis. CSA (0.1-10 mM) N-methyl-DL-aspartate (N MA), another greatly increased the cAMP level in hip agonist, did not enhance the release. In pocampal slices. CSA was more potent contrast, CSA had no significant effect on than glutamate and aspartate, and its effect the high K+-induced release of ACh. In was selectively antagonized by 0.1 to 30 mM addition, the stimulatory effect of CSA on taurine. This inhibition by taurine was the release of GABA was negated by observed in hippocampal slices but not in tetrodotoxin and was not observed in the cortical slices (31). To identify the receptors crude synaptosomal fractions of the hip that are involved in the stimulatory effect of pocampus. These results indicate the CSA and in the inhibitory effect of taurine on neuronal circuit is absolutely required for the cAMP formation, various agonists and stimulatory effect of CSA. antagonists of excitatory amino acids were In view of the electrophysiologial studies, examined for their possible effect on cAMP N MA, KA and QA are selective agonists to formation (32). Excitatory amino acid three different types of receptors for exci agonists such as N MA, KA and QA stimulated tatory amino acids (3), which exhibit the formation of cAMP in the slices. Effects differential sensitivities to blockage by of these agonists were more pronounced various antagonists. Table 1 summarizes the in the hippocampus than in the cerebral effects of various excitatory amino acid cortex. N MA was less effective than KA and antagonists on CSA and glutamate-induced QA. Taurine markedly reduced the stimulatory release of GABA from the preloaded slices effects of KA and QA without affecting that (ref. 27 and A. Baba et al., in preparation). It is of N MA. With respect to the effects of the clear that the stimulatory effects of CSA and antagonists, whereas D-a-aminoadipate itself glutamate have different sensitivity to the showed an agonist-like effect, CSA-induced antagonists. Thus, CSA receptors involved in formation of cAM P was significantly the enhancement of GABA release seems to antagonized by D-a-aminoadipate and also be distinct from glutamate receptors. by glutamate diethylester. These antagonists However, since these antagonists are had no effect on the stimulation by glutamate relatively nonspecific, examinations must be and aspartate (32). These studies clearly carried out using more specific antagonists demonstrate the heterogeneity of excitatory for a further evaluation of the receptors for amino acid receptors that are involved in the CSA. formation of cAM P. Enhancement of cyclic AMP (cAM P) Unlike other transmitters, it is not likely formation: Excitatory amino acids markedy that excitatory amino acid receptors are increased the formation of cAMP in directly coupled with adenylate cyclase. In incubated brain slices (28, 29). The mecha fact, neither of the excitatory amino acids nisms by which excitatory amino acids activate adenylate cyclase activity in a cell elevate the cAMP level in brain slices were free system from the hipppocampus under believed to be common, i.e., common any conditions. As has been suggested (35, receptors or release of adenosine (28-30). 36), the stimulated formation of cAMP by

Table 1. Summary of the effects of various compounds on the enhancement of [3H] GABA release from the preloaded hippocampal slices by cysteine sulfinate (CSA) and glutamate

Antagonism (+), No antagonism (-), Not determined (ND). DAA: D-a-aminoadipate, GDEE: Glutamate diethylester, K.A.: Kynurenic acid, S.B.: Secobarbital, APB: 2-Amino-4-phosphonobutyric acid. Table 2. Summary of the effects of various compounds on the CSA-induced formation of cyclic AMP in hippocampal slices

Potentiation (+), Suppression (-), No change (±), Not determined (ND). DAA: D-a-aminoadipate, GDEE: Glutamate diethylester, FK: Forskolin, LA: Linoleic acid.

CSA was also inhibited by adenosine lation of cAM P formation by CSA and deaminase and 2"-deoxyadenosine. Thus, glutamate were completely negated. The activation of the receptors by the agonist anion selectivity for the stimulation by CSA might be accompanied by a subsequent was in parallel to that for the CI channel. release of adenosine. Other approaches were Thus, the enhancement of cAMP formation made to evaluate excitatory amino acid by excitatory amino acids absolutely receptors which were involved in the for required the presence of CI-. Results of mation of cAMP (33, 34); the treatment of these studies are summarized in Table 2. slices with polyunsaturated fatty acid 4. Conclusion resulted an enhancement of the functional In the present communication, several coupling between adenyalte cyclase and neurochemical characteristics of CSA were norepinephrine and and adenosine-receptors described. These characteristics were In contrast, the treatment had no significant common to those observed for many other effect on the response to CSA and neurotransmitter candidates. Furthermore, the glutamate. This indicates that the coupling present communication indicates that the between excitatory amino acid receptors and neurochemical characteristics of CSA seem adenylate cyclase is qualitatively different to be distinct from those of glutamate. from those of other neurotransmitters. Our Recently, similar lines of evidence were recent study further provides new aspects of reported by others (18, 25, 26, 39). At excitatory amino acid-induced formation of present, the biochemical and pharmacological cAMP in hippocampal slices (Baba et al., in qualifications of ,"CSA neurones" are not preparation). Forskolin, an activator of clear. However, on the basis of the obser adenylate cyclase, has two distinct effects on vations described above, it is likely that CSA the adenylate cyclase system. 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