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The effects of metabotropic glutamate receptor agonists on the basal and potassium (50 mM K+)-stimulated release of [3H]GABA from mouse hippocampal slices were investigated using a superfusion system. The group I agonist (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal GABA release and reduced the K+-evoked release by a mechanism antagonized by (RS)-1-aminoindan-1,5-dicarboxylate in both cases. The group II agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine failed to have any effect on the basal release, but inhibited the stimulated release. This inhibition was not affected by the antagonist (2S)-2-ethylglutamate. The group III agonists L(+)-amino-4-phosphonobutyrate and O-phospho-L-serine inhibited the basal GABA release, which effects were blocked by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Moreover, the suppression of the K+-evoked release by L(+)2-amino-4-phosphonobutyrate was apparently receptor-mediated, being blocked by (RS)-2-cyclopropyl-4-phosphonophenylglycine. The results show that activation of metabotropic glutamate receptors of group I is able to potentiate the basal release of GABA, whereas activation of groups I and III receptors reduce K+-stimulated release in mouse hippocampal slices.

The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K+-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca2+-independent, a Ca2+-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na+-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl- channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultane ously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels.

Taurine is held to function as a modulator and osmoregulator in the central nervous system, being of particular importance in the immature brain. In view of the possible involvement of excitatory pathways in the regulation of taurine function in the brain, the interference of glutamate receptors with taurine release from different tissue preparations in vitro and from the brain in vivo is of special interest. The release of taurine from the brain is enhanced by glutamate receptor agonists. This enhancement is inhibited by the respective receptor antagonists both in vitro and in vivo. The ionotropic N -methyl- D -aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor agonists appear to be the most effective in enhancing taurine release, their effects being receptor-mediated. Kainate is less effective, particularly in adults. Of the glutamate receptors, the NMDA class seems to be the most susceptible to modulation by nitric oxide. Nitric oxide also modulates taurine release, enhancing the basal release in both immature and mature hippocampus, whereas the K + -stimulated release is generally inhibited. Metabotropic glutamate receptors also participate in the regulation of taurine release, group I metabotropic glutamate receptors potentiating the release in the developing hippocampus, while group III receptors may be involved in the adult. Under various cell-damaging conditions, including ischemia, hypoxia and hypoglycemia, taurine release is enhanced, together with an enhanced release of excitatory amino acids. The increase in extracellular taurine upon excessive stimulation of glutamate receptors and under cell-damaging conditions may serve as an important protective mechanism against excitotoxicity, being particularly effective in the immature brain.

In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K+-stimulated release of [3H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA(B) and GABA(C) receptors. The involvement of GABA(A) receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K+-evoked taurine release. Furthermore, several GABA(B) receptor effectors were able to inhibit K+-stimulated taurine release in the adults, while the GABA(C) receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds.

The interactions of S-nitrosoglutathione (GSNO) with the ionotropic glutamate receptors were studied on synaptic membranes isolated from the pig cerebral cortex. GSNO displaced the binding of [3H]glutamate, 3-[(R)-2-carboxypiperazin-4-yl][3H]propyl-1-phosphonate ([3H]CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist, and [3H]kainate, with IC50 values in the low micromolar range. It failed to displace (S)-5-fluoro-[3H]willardiine, a selective agonist of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Reduced and oxidized glutathione were almost as effective as GSNO in glutamate and CPP binding. Of the three, GSNO was the most potent in kainate binding. They all stimulated [3H]dizocilpine binding in a concentration-dependent manner. This effect was additive to that of glycine and not mimicked by NO donors such as S-nitroso-N-acetylpenicillamine, 5-amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1) and nitroglycerin. We assume that GSNO may act as an endogenous ligand at the NMDA and non-NMDA classes of glutamate receptors. In this manner it may facilitate NO transfer and target its delivery to specific sites in these receptors.

We review here the possible mechanisms of neuronal degeneration caused by L-cysteine, an odd excitotoxin. L-Cysteine lacks the omega carboxyl group required for excitotoxic actions via excitatory amino acid receptors, yet it evokes N-methyl-D-aspartate (NMDA) -like excitotoxic neuronal death and potentiates the Ca2+ influx evoked by NMDA. Both actions are prevented by NMDA antagonists. One target for cysteine effects is thus the NMDA receptor. The following mechanisms are discussed now: (1) possible increase in extracellular glutamate via release or inhibition of uptake/degradation, (2) generation of cysteine α-carbamate, a toxic analog of NMDA, (3) generation of toxic oxidized cysteine derivatives, (4) chelation of Zn2+ which blocks the NMDA receptor-ionophore, (5) direct interaction with the NMDA receptor redox site(s), (6) generation of free radicals, and (7) formation of S-nitrosocysteine. In addition to these, we describe another new alternative for cytotoxicity: (8) generation of the neurotoxic catecholamine derivative, 5-S-cysteinyl-3,4-dihydroxyphenylacetate (cysdopac).

Metabotropic glutamate receptors have recently been envisaged as involved in both potentiation and prevention of ischemic and excitotoxic neuronal damage. The release of the inhibitory amino acid taurine is markedly enhanced in ischemia in both the immature and mature mouse hippocampus. The modulation of [3H]taurine release by metabotropic receptor agonists and antagonists was studied in hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. Agonists of group I, II and III metabotropic glutamate receptors generally reduced the ischemia-induced release in adult animals. In the immature hippocampus the group I agonists (S)-3,5-dihydroxyphenylglycine and (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate, which mainly enhance neuronal excitation, potentiated initial taurine release in ischemia. Ionotropic glutamate receptor agonists also enhance the ischemia-induced taurine release in developing mice. This glutamate-activated taurine release may thus constitute an important protective mechanism against excitotoxicity in the immature hippocampus.

Glutathione (γ-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na + -independent binding of [ 3 H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. l -Glutamate, pyroglutamate, quinolinate, ( S )-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione were weak inhibitors at concentrations of 0.5 or 1 mM. d -Glutamate, l - and d -aspartate, glutamine, quisqualate, kynurenate, other N -methyl- d -aspartate receptor ligands and non- N -methyl- d -aspartate receptor ligands failed to displace [ 3 H]glutathione. Except for weak inhibition by d -serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N -methyl- d -aspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S -nitrosoglutathione, γ- l -glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers ( ic 50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S -alkyl derivatives of glutathione ( S -methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). l -Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N -methyl- d -aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, γ- l -glutamylleucine and γ- l -glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain.

The release of taurine from cultured cerebellar granule neurons was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and in the presence of free radicals. The effects of both ionotropic and metabotropic glutamate receptor agonists on the release were likewise investigated. The release of [3H]taurine from the glutamatergic granule cells was increased by K+ (50 mM) and veratridine (0.1 mM), the effect of veratridine being the greater. Hypoxia and ischemia produced an initial increase in release compared to normoxia but resulted in a diminished response to K+. Hypoglycemia, oxidative stress and free radicals enhanced taurine release, and subsequent K+ treatment exhibited a correspondingly greater stimulation. A common feature of taurine release in all the above conditions was a slow response to the stimulus evoked by K+ and particularly to that evoked by veratridine. All ionotropic glutamate receptor agonists potentiated taurine release, but only the action of kainate seemed to be receptor-mediated. Metabotropic receptor agonists of group I slightly stimulated the release. The prolonged taurine release seen in both normoxia and cell-damaging conditions may be of importance in maintaining homeostasis in the cerebellum and reducing excitability for a longer period than other neuroprotective mechanisms.

Taurine release in the developing hippocampus is markedly potentiated in ischemia. The mechanisms of the ischemia-induced release were studied in hippocampal slices from seven-day-old mice using a superfusion system. The basal release of [ 3 H]taurine was significantly increased in media under normal conditions, but the ischemia-evoked release decreased in Na + -free media, indicating the participation of Na + -dependent transport processes. The involvement of taurine transporters in the release was confirmed with the structural analogs, hypotaurine and β-alanine. These amino acids potentiated the release by trans-stimulation, but not in Na + -free media. In the absence of Ca 2+ , the basal taurine release was markedly increased in normoxia but diminished in ischemia, indicating that a part of basal taurine release in ischemia is Ca 2+ dependent. On the other hand, the K + stimulation of taurine release was preserved in Ca 2+ -free medium. The phospholipase and protein kinase inhibitors had no effect on ischemia-induced taurine release, nor did the chloride channel blockers 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (2 mM) and diisothiocyanostilbene-2,2′-disulfonate (0.1 mM) affect the release in ischemia. The increase in extracellular levels of taurine in the immature hippocampus in ischemia may serve as an important protective mechanism against excitotoxicity, to which the developing brain is particularly vulnerable, and contribute to the resistance of the immature brain to hypoxia.

Acute hepatic encephalopathy (HE) is associated with disturbances in motor functions, but the underlying mechanisms remain obscure. Considerable experimental evidence suggests that motor activity is modulated by striatal dopamine neurons whose discharge is under glutamatergic control, mostly through activation of N -methyl- d -aspartate (NMDA) receptors. In this study we used intrastriatal microdialysis to compare the effects of infusion of 10 mM NMDA or 50 mM KCl as a general release stimulus, on the extracellular levels of endogenous dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in control rats and in rats with acute HE induced by repeated administration of thioacetamide. The basal levels of DA and DOPAC were not significantly altered by HE, while the HVA level was reduced. HE did not significantly affect the NMDA-or KCl-evoked increase in extracellular DA. Infusion of NMDA or KCl led to a decrease in extracellular DOPAC, and HE did not modulate these effects. However, HE attenuated the NMDA- but not the KCl-induced reduction in extracellular HVA. The results point to the impairment of modulation of striatal DA discharge and metabolism by glutamate acting at NMDA receptors, contributing to the motor disturbances in HE.

Rats were treated with a hepatotoxin thioacetamide (TAA) and examined 21 days later, when they showed moderate fatty metamorphosis of the liver and morphological changes in brain indicative of excitotoxic neuronal damage, but no evident biochemical or neurophysiological symptoms of hepatic encephalopathy (HE). High-performance liquid chromatography (HPLC) analysis of extracellular amino acids in striatal microdialysates of TAA-treated rats revealed a significant increase in the excitatory amino acids glutamate (Glu) and aspartate (Asp) and their amino acid metabolites glutamine (Gln) and alanine (Ala). Microdialysis in the presence of 50 mM K+ triggered in TAA-treated rats an accumulation of Asp and Glu, and diminished the accumulation of Gln. These effects were virtually absent in control rats. None of the treatments affected the accumulation of the nontransmitter amino acid leucine (Leu). The above changes mirror those previously described in symptomatic HE and are likely to contribute to excitotoxic damage. The basal microdialysate content of taurine (Tau), an amino acid with antioxidant and volume regulatory properties, was 60% lower in TAA-treated rats than in control rats despite its increased blood-to-brain transport. The decrease in extracellular Tau may thus reflect Tau redistribution to adjacent central nervous system (CNS) cells manifesting a cell-protective response. Stimulation with 50 mM K+ increased extracellular Tau in control rats by 182% and in TAA-treated rats by 322%. Stimulation with 100 microM N-methyl-D-aspartate (NMDA) increased extracellular Tau in control rats by 27 % and in TAA-treated rats by as much as 250%. The increase of K+- or NMDA-dependent Tau release may reflect improved cell volume regulation and neuroprotection and contribute to attenuation of neurologic symptoms in rats with liver failure.

The release of preloaded D-[3H]aspartate, an unmetabolizable analogue of L-glutamate, was studied in superfused hippocampal slices from 7-day-old and 3-month-old (adult) mice under various cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals and metabolic poisons. The release was generally markedly enhanced in most of the above conditions, the responses being greater in adults than in developing mice. The presence of dinitrophenol had the most pronounced effect at both ages, followed by NaCN- and free-radical-containing media and ischemia. Hypoxia did not affect release in the immature hippocampus. Under most conditions K+ stimulation (50 mM) was still able markedly to enhance D-aspartate release. This potentiation under cell-damaging conditions in both adult and developing hippocampus signifies that increased L-glutamate release contributes to excitotoxicity and subsequent cell death. The mechanisms of ischemia-induced release of D-aspartate were analyzed in the adult hippocampus using ion channel inhibitors and modified superfusion media. The induced release proved to be partly Ca(2+)-dependent and partly Ca(2+)-independent. The results obtained with Na+ omission and homo- and heteroexchange with D-aspartate and L-glutamate demonstrated that a part of the release in normoxia and ischemia is mediated by the reversal of Na(+)-dependent glutamate transporters. The Na+ channel blockers amiloride and riluzole reduced the ischemia-induced release, also indicating the involvement of Na+ channels. In addition to this, the enhanced release of D-aspartate may comprise a swelling-induced component through chloride channels.

The effects of lamotrigine and carbamazepine on the release of preloaded D-[3H]aspartate and the involvement of nitric oxide were studied with mouse cerebral cortical slices in a superfusion system. Lamotrigine inhibited the veratridine-evoked release, whereas the K+-stimulated release was attenuated more strongly by carbamazepine than by lamotrigine. These effects were accentuated by the N-methyl-D-aspartate receptor antagonist L-2-amino-5-phosphonovalerate and the nitric oxide synthase inhibitor L-nitroarginine, but diminished by the nitric oxide donor sodium nitroprusside. The results show that in addition to the blockade of voltage-sensitive Na+ (and Ca2+) channels, NO-mediated mechanisms are probably involved in the anticonvulsant actions of carbamazepine and, in particular, those of lamotrigine.

The release of the inhibitory amino acid β-alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by β-alanine itself and the structural analogs taurine and γ-aminobutyrate. It was dependent on Na+, but independent of Ca2+ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced β-alanine release at both ages, but more markedly in the adults. The great amounts of β-alanine, together with the inhibitory amino acids taurine and γ-aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.

The release of preloaded [3H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K+ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2′-disulphonate) and SITS-(4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate) sensitive CI− channels, since these blockers were able to reduce both basal and K+ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K+ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.

Taurine release in the hippocampus is markedly potentiated in various cell-damaging conditions, including ischemia and excitotoxic damage produced by glutamate. The increase in the levels of taurine may provide an important protective mechanism against excitotoxicity. The mechanisms of the enhanced release were now studied in mouse hippocampal slices using a superfusion system. The basal release of [3H]taurine was significantly increased in Na+-deficient media in normal conditions, whereas the ischemia-evoked release was decreased, indicating the participation of Na+-dependent transport processes. The involvement of taurine transport carriers in the release was confirmed with the structural analogs, hypotaurine and beta-alanine. These amino acids potentiated the release by trans-stimulation in normoxia. In Na+-free conditions, this heteroexchange was not discernible, the carriers not being functional without Na+. In ischemia, the marked potentiation of taurine release by hypotaurine and beta-alanine further indicates that the Na+-requiring transporters also operate in ischemia. The effects of membrane disruption on taurine release due to activation of phospholipases were estimated using phospholipase and protein kinase inhibitors, which had no marked effects on hippocampal taurine release. The chloride channel blockers, 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonate (SITS) and diisothiocyanostilbene-2,2'-disulphonate (DIDS), reduced the ischemia-induced release, suggesting that taurine diffusion through an anion channel is partially responsible for the enhanced release in ischemia.

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K+ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K+ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K+ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H2O2, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca2+-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on 45Ca2+ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na+-independent binding of L-[3H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[3H]methyl-4-isoxazolepropionate [3H]AMPA (IC50 values: 0.8-15.9 microM). S-Alkylation of glutathione rendered the derivatives unable to inhibit [3H]kainate binding. The NMDA-sensitive binding of L-[3H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-(3)H]propyl-1-phosphonate ([3H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [3H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [3H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of 45Ca2+ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their gamma-glutamyl moieties.

The inhibitory amino acid taurine has been held to function as a modulator and osmoregulator in the brain, being of particular importance in the immature brain. The release of preloaded [3H]taurine was now studied in hippocampal slices from developing (7-day-old), adult (3-month-old) and ageing (6–24-month-old) mice focussing on the effects of agonists of ionotropic glutamate receptors. N-methyl- d -aspartate (NMDA), kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) potentiated taurine release concentration-dependently at each age, more so in the immature than in the adult and ageing hippocampus. The effect of kainate was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the developing and aged hippocampus and those of AMPA and NMDA by 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and dizocilpine a(MK-801) at every age studied. This indicates the involvement of NMDA and AMPA receptors in taurine release throughout the life-span of mice, while the kainate-receptor-mediated release does not appear to function in adults. The increased hippocampal taurine release evoked by ionotropic glutamate receptors could act neuroprotectively, counteracting by several mechanisms the harmful effects of the simultaneous release of excitatory amino acids. The substantial release of taurine in the immature hippocampus might be particularly significant in view of the vulnerability of brain tissue to excitotoxicity at early age.

The involvement of arginyl residues in the binding of ligands to different ionotropic glutamate receptors, to the modulatory glycine site in the N-methyl-D-aspartate (NMDA) receptor and to the NMDA-governed ionophore was assessed with porcine cortical synaptic membranes. The arginyl residues were covalently modified with phenylglyoxal. The binding and protection experiments showed that arginine residue(s) are directly involved in the binding of ligands to the agonist sites of all ionotropic glutamate receptors and to the modulatory glycine site but not to the ion channel in the NMDA receptor complex.

Taurine is a neuromodulator and osmoregulator in the central nervous system, also protecting neural cells against excitotoxicity. The effects of the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA), kainate and 2-amino-3-hydroxy-5-methyl-4-imidazolepropionate (AMPA) on [3H]taurine release from hippocampal slices from 3-month-old and 7-day-old mice were studied in cell-damaging conditions. Neural cell injury was induced by superfusing the slices in hypoxic, hypoglycemic and ischemic conditions and by exposing them to metabolic poisons, free radicals and oxidative stress. The release of taurine was greatly enhanced in these conditions at both ages, except in oxidative stress. In normal conditions the three glutamate agonists potentiated taurine release in the immature hippocampus in a receptor-mediated manner, but kainate receptors did not participate in the regulation in the adults. The ability of the agonists to evoke taurine release varied in the cell-damaging conditions, but the glutamate-receptor-activated release was generally operating in the immature hippocampus. This glutamate-receptor-evoked massive release of taurine could have significant neuroprotective effects, particularly in the developing hippocampus, countering the harmful actions of the simultaneously liberated excitatory amino acids.

Hepatic encephalopathy (HE) is characterized by symptoms pointing at disturbances in glutamatergic neurotransmission in the brain, particularly in the striatum. The binding parameters of ligands specific for different recognition sites in theN-methyl-d-aspartate (NMDA) receptor complex and the distribution of the receptor subunit mRNAs (NR1, NR2A-D) were assessed in rats with acute HE induced with a hepatotoxin, thioacetamide (TAA). The binding of: In HE rats,B max of NMDA-displaceable glutamate binding was increased in the cerebral cortex and hippocampus, but slightly decreased in the striatum. In this region, the binding affinity was also slightly increased. In HE,B max of [3H]dizocilpine binding was unchanged in the striatum and cerebral cortex, but substantially decreased in the hippocampus. Pretreatment with phorbol ester enhanced the binding of dizocilpine more in HE than in control rats.B max of [3H]TCP binding was decreased in the cerebral cortex and striatum, but increased in the hippocampus. The different responses of these two phencyclidine site antagonists to HE may be indicative of a conformational change within the ion channel and/or the presence of microdomains reacting differently to extrinsic factors. HE did not affect glycine binding, but potentiated the maximal stimulation of [3H]dizocilpine binding by glycine in the cerebral cortex. The results emphasize the brain region and domain specificity of the responses of the NMDA receptor complex to HE.

Possible involvement of histidyl residues in the binding of ligands to ionotropic glutamate receptors and to modulatory sites on the N-methyl-D-aspartate (NMDA) receptor was assessed in porcine cortical synaptic plasma membranes after covalent modification with diethyl pyrocarbonate (DEPC). Binding of [3H]glutamate to the NMDA sites was enhanced but to the 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors unaffected by 1 and 5 mM DEPC. Binding of 3-[(R)-carboxypiperazin-4-yl]-[1,2-(3)H]propyl-1-phosphonate ([3H]CPP) was reduced in a dose-dependent manner by DEPC and the activation of binding by 1-hydroxy-3-amino-2-pyrrolidone (HA-966) blocked by 10 mM DEPC. DEPC reduced the strychnine-insensitive binding of [3H]glycine and the glycine- and glutamate-activated binding of [3H]dizocilpine. Protection experiments indicated that histidyl residues are directly involved in the binding of glycine (but not HA-966) and allosterically modulate the binding of glutamate, CPP and dizocilpine. The results corroborate the existence of agonist- and antagonist-preferring sites or conformational states of the NMDA receptors.

The anticancer drug tamoxifen is widely used in breast cancer therapy. Tamoxifen has been reported to cause ocular toxicity and impairment of vision in epidemiological studies. To study the possible role of an excitotoxic mechanism in the ocular toxicity of tamoxifen, we investigated the effect of tamoxifen on retinal pigment epithelium (RPE) glutamate uptake in vitro. RPE, a layer of cells between photoreceptors and choroidal capillaries, contributes to the regulation of the concentration of the major excitatory amino acid, glutamate, in the sub-retinal space. Dysfunction in RPE glutamate uptake can lead to accumulation of extracellular glutamate and can cause various excitotoxic effects in the retina. The study was conducted by using cultured pig RPE cells. Six different tamoxifen citrate concentrations, ranging from lμM to 100μM, and [3H]-L-glutamate were added to the culture medium. To specify the glutamate uptake, 1mM dinitrophenol was added and a Na+-free culture was used. Due to the anti-oestrogenic character of tamoxifen, the possible effect of β-oestradiol on the glutamate uptake of RPE was also examined. The results show that glutamate uptake by RPE cells was reduced in the presence of tamoxifen, and that the reduction was dose-dependent. These results suggest that tamoxifen exposure could lead to the extracellular accumulation of glutamate. Disturbances in glutamate uptake can cause eye toxicity via an excitotoxic mechanism. The glutamate uptake of RPE cells was reduced under Na+-free conditions and was also reduced in the presence of dinitrophenol.

The release of [3H]GABA from hippocampul slices from adult (3-month-old) and developing (7-day-old) mice was studied in cell-damaging conditions in vitro using a superfusion system. Cell damage was induced by modified superfusion media, including hypoxia, hypoglycemia, ischemia, the presence of free radicals and oxidative stress. The basal release of GABA from the immature and mature hippocampus was generally markedly increased in all cell-damaging conditions. In 7-day-old mice the release was enhanced most in the presence of free radicals, 1.0 mM NaCN and ischemia, whereas in the adults 1.0 mM NaCN provoked the largest release of GABA, followed by ischemia and free radical-containing media. Potassium stimulation (50mM K+) was still able to potentiate the release in all cell-damaging conditions in both age groups. It was shown by superfusing the slices in Ca- and Na-free media that ischemia-induced GABA release was Ca-independent, occurring by a reversed operation of Na-dependent cell membrane carriers in both adult and developing hippocampus. Glutamate and its receptor agonists, N-methyl- d -aspartate (NMDA), kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), potentiated GABA release only in the immature hippocampus by a receptor-mediated mechanism. The enhancement by kainate and AMPA receptors also operated under ischemic conditions. The massive amount of GABA released simultaneously with excitatory amino acids in the mature and immature hippocampus may be an important protective mechanism against excitotoxicity, counteracting harmful effects that lead to neuronal death. The GABA release induced by activation of presynaptic glutamate receptors may contribute particularly to the maintenance of homeostasis in the hippocampus upon impending hyper-excitation.

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [3H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.

A study was made of the effects of reduced (GSH) and oxidized (GSSG) glutathione on the Na+-independent and N-methyl-D-aspartate (NMDA) displaceable bindings of glutamate, on the binding of kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ligands of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (±)-3-(2-car-boxypiperazin-4-yl)propyl-1-phosphonate (CPP). GSH and GSSG strongly inhibited the binding of glutamate, CPP and AMPA, kainate and glycine binding being less affected. Both peptides enhanced the binding of dizocilpine in a time- and concentration-dependent manner. This activatory effect was not additive to that of saturating concentrations of glutamate or glutamate plus glycine. The activation of dizocilpine binding by GSH and GSSG was prevented by the competitive NMDA and glycine antagonists DL-2-amino-5-phosphonovalerate and 7-chlorokynurenate. GSH and GSSG may be endogenous ligands of AMPA and NMDA receptors, binding preferably to the glutamate recognition site via their γ-glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.

Kynurenic acid (KYNA) modulates the glutamatergic tone by controlling neuronal glutamate (GLU) release. The present study tested the potential of the KYNA precursor, kynurenine (KYN) to counter increased extracellular GLU associated with the pathogenesis of hepatic encephalopathy accompanying acute liver failure (ALF).ALF was induced in adult rats by administration of a hepatotoxin, thioacetamide. KYNA and GLU were measured in the cerebral cortical microdialysates of control (saline-treated) and ALF rats using HPLC. The expression of mRNA coding for kynurenine aminotransferase II (KAT-II), the astrocytic enzyme converting KYN to KYNA, was assayed by real-time PCR.Cerebral cortical extracellular KYNA was increased in ALF rats not treated with KYN, consistent with a previously observed increase of cerebral cortical KATII activity in this ALF model. Single intraperitoneal administration of KYN (50 mg/kg, 120 min before microdialysate collection), produced a further substantial increase of extracellular KYNA, paralleled by a decrease of extracellular GLU. In cultured cerebral cortical astrocytes, the cells which in situ are the primary target of blood-derived ammonia and other toxins liberated due to ALF, elevation of KAT-II mRNA expression was noted upon their incubation with KYN and the KYN precursor, tryptophan (Trp), which is normally elevated by ALF.Administration of exogenous KYN to stimulate KYNA synthesis may help correcting excessive extracellular accumulation of GLU in cerebral cortex caused by ALF. The therapeutic potential of KYN in ALF appears to be fostered by increased expression of KAT-II in astrocytes upon exposure to KYN or Trp.

The influx of taurine into cerebral cortical slices was studied with 3-day-old and 3-month-old mice in different ionic environments in incubation medium. In standard Krebs-Ringer medium the influx comprised two saturable uptake components, high- and low-affinity, and non-saturable penetration. In isoosmotic medium potassium stimulation abolished the high-affinity uptake in both age groups. In hyperosmotic medium the high-affinity uptake disappeared totally in 3-day-old mice and partially in 3-month-old mice. The high-affinity uptake was also obliterated in hypoosmotic medium and in the absence of chloride ions in both age groups. The low-affinity uptake was abolished by potassium stimulation in 3-month-olds and strongly inhibited in 3-day-olds. Hypoosmotic and chloride-free media also inhibited the low-affinity uptake at both ages. Non-saturable influx was greatly diminished in chloride-free media. The taurine uptake systems are thus strongly inhibited in incubation conditions which simultaneously evoke apparent release of taurine from cerebral cortical slices. This inhibition contributes to the magnitude of the estimated release, which in vitro represents overflow of released taurine molecules which escape recapture by the membrane carriers. In vivo the same mechanism may underlie the delayed and spreading neuromodulatory actions of taurine.

Intrastriatal administration of ammonium ions (“ammonia”) via a microdialysis probe overactivates N -methyl- d -aspartate (NMDA) receptors, which results in cGMP accumulation in the microdialysates. Co-administration of a potent glycine site-specific NMDA receptor antagonist CGP 78608 ([(1 S )-1-[[(7-bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]ethyl]phosphonate) significantly reduced (at 20 nM) or abolished (at 100 nM) ammonia-dependent cGMP synthesis. Since NMDA receptor activation is an important causative factor in ammonia neurotoxicity, the present results suggest the glycine site of the receptor to be a potential valuable target for protective intervention.

Taurine release in mouse hippocampal slices is regulated by several neurotransmitter receptor systems. The ionotropic glutamate receptors and the adenosine receptor A(1) are the most effective. The effect of N-methyl-D-aspartate receptors is mediated via activation of the pathway involving nitric oxide and 3',5'-cyclic guanosine monophosphate. The activation of excitatory amino acid receptors causes at the same time an increase in taurine release. The activation of adenosine A(1) receptors also potentiates taurine release. The taurine released may counteract any excitotoxic effects of glutamate, particularly in the developing hippocampus.

Alcohol consumption by mothers during pregnancy causes a fetal alcohol syndrome associated with massive neuronal apoptosis. We have recently shown that taurine at a dose of 2 g/kg saves about 50% of dying cerebellar neurons from ethanol-induced apoptosis in 7-day-old mice. However, a further increase in the taurine dose to ethanol-treated mice had a toxic and in some cases lethal effect. In the present work we studied the toxic effects of taurine and ethanol coadministration in three age groups: 7-day-old, adult (5 to 6 months old), and old (12 to 13 months old) mice. Taurine and ethanol were injected in two half-doses: taurine at 0 and 4 h and ethanol at 1 and 3 h. The minimal 100% lethal doses in coadministration of taurine and ethanol were the following: 7-day-old mice-6 g/kg taurine + 5 g/kg ethanol, adult mice-10 g/kg of taurine + 8 g/kg of ethanol, and old mice-above 6 g/kg of taurine + 6 g/kg of ethanol. All mice treated with taurine or ethanol alone survived. The adult and old mice dying from the combined toxicity of taurine and ethanol showed a marked fall in blood glucose, which may be one reason for lethality. A comparison of the lethal doses of taurine and ethanol coadministration in different age groups allows us to conclude that the adverse effect of the combined toxicity of taurine and ethanol is age dependent.

Taurine release in mouse hippocampal slices is regulated by several neurotransmitter receptor systems. The ionotropic glutamate receptors and the adenosine receptor A1 are the most effective. The effect of N-methyl-d-aspartate receptors is mediated via activation of the pathway involving nitric oxide and 3′,5′-cyclic guanosine monophosphate. The activation of excitatory amino acid receptors causes at the same time an increase in taurine release. The activation of adenosine A1 receptors also potentiates taurine release. The taurine released may counteract any excitotoxic effects of glutamate, particularly in the developing hippocampus.

Alcohol consumption by mothers during pregnancy causes a fetal alcohol syndrome associated with massive neuronal apoptosis. We have recently shown that taurine at a dose of 2 g/kg saves about 50% of dying cerebellar neurons from ethanol-induced apoptosis in 7-day-old mice. However, a further increase in the taurine dose to ethanol-treated mice had a toxic and in some cases lethal effect. In the present work we studied the toxic effects of taurine and ethanol coadministration in three age groups: 7-day-old, adult (5 to 6 months old), and old (12 to 13 months old) mice. Taurine and ethanol were injected in two half-doses: taurine at 0 and 4 h and ethanol at 1 and 3 h. The minimal 100% lethal doses in coadministration of taurine and ethanol were the following: 7-day-old mice—6 g/kg taurine + 5 g/kg ethanol, adult mice—10 g/kg of taurine + 8 g/kg of ethanol, and old mice—above 6 g/kg of taurine + 6 g/kg of ethanol. All mice treated with taurine or ethanol alone survived. The adult and old mice dying from the combined toxicity of taurine and ethanol showed a marked fall in blood glucose, which may be one reason for lethality. A comparison of the lethal doses of taurine and ethanol coadministration in different age groups allows us to conclude that the adverse effect of the combined toxicity of taurine and ethanol is age dependent.

The studies were carried out on 3- and 7-day, 3-, 6-, 12-, 18- and 24-month-old mice. The levels of glutamate and aspartate increased in most brain areas in developing mice and then decreased gradually during ageing, the changes depending, however, on the brain region. The maximal velocity (V) of high-affinity uptake of [3H]glutamate was markedly reduced in cerebral cortical synaptosomes at the age of 24 months, indicating an age-related loss in the number of transport sites. The transport constant (Km) was also diminished in aged mice, indicating a compensatory increase in the affinity of the remaining transport sites. The basal and K(+)-stimulated (50 mM) release of endogenous glutamate and aspartate varied depending on the brain region. The responses to K+ stimulation generally increased during maturation, whereas the other age-related changes were more variable. The basal unstimulated release of glutamate remained fairly constant in the cerebral cortex during ageing, but K+ depolarization liberated more glutamate in 24-month-olds than in 3-month-olds. The decreased uptake capacity together with an increased release of glutamate may contribute to the degenerative changes associated with normal brain ageing and also to the pathogenesis of age-related neurodegenerative disorders.

The sodium-independent binding of beta-alanine to cerebral cortical membranes from adult (3- and 12-month-old) and developing (7-day-old) mice was characterized for the first time. The binding was saturable in each age group, consisting of only one component. The affinity for beta-alanine was highest and the number of available binding sites greatest in young animals. The binding was not affected by strychnine, but inhibited by beta-alanine itself, glycine, L-alanine and L-serine, the IC50 values being lower in immature mice. Glycine was shown to be a competitive inhibitor. The binding was also inhibited, albeit only in adults, by N-methyl-D-aspartate receptor antagonists acting at the glycine modulatory site and by some GABAergic substances. It is concluded that even though beta-alanine may possess binding sites of its own, particularly in the immature cerebral cortex, beta-alanine could at least partly bind to strychnine-insensitive glycine sites in the N-methyl-D-aspartate receptor complex.

The effects of dithiothreitol (DTT) and, reduced (GSH) and oxidized (GSSG), glutathione on the release of [3H]GABA evoked by glutamate and its agonists were studied in rat hippocampal slices. DTT had no effect on the basal release of [3H]GABA but it enhanced and prolonged the glutamate agonist-evoked release. This effect was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801), a noncompetitive NMDA antagonist, and blocked by Mg2+ ions. It was only slightly attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, and not affected by L-(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. The effect of DTT on the NMDA-evoked release of GABA was only slightly affected by extracellular Ca2+ but completely blocked by verapamil even in the absence of Ca2+. GSH and GSSG attenuated or abolished the effects of DTT on the agonist-induced release of [3H]GABA. The results imply that the enhanced and prolonged release of GABA evoked by the coexistence of DTT and excitatory amino acids and attenuated by endogenous GSH and GSSG is a consequence of sustained activation of the NMDA receptor-governed ionophores, which contain functional thiol groups. DTT, GSH and GSSG may regulate the redox state and accessibility of these groups. In addition to the influx of extracellular Ca2+, DTT mobilizes Ca2+ from intracellular pools distinct from those regulated by metabotropic glutamate receptors.

The in vitro effects of gamma-L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with gamma-D-glutamyltaurine as reference. gamma-L-Glutamyltaurine enhanced the K(+)-stimulated release of [3H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). gamma-L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than gamma-D-glutamyltaurine. The basal influx of Ca2+ into cultured cerebellar granular cells was not affected by 1 mM gamma-L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg2+ (0.1 mM) and Mg(2+)-free media. The glutamate-evoked increase in free intracellular Ca2+ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM gamma-L-glutamyltaurine. We propose that gamma-L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K(+)-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K(+) stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K(+) stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.

The strychnine-insensitive binding of [3H]glycine was characterized in purified cerebral cortical membranes from mice aged from 7 days to 22 months. The binding was saturable, exhibiting only one component during the whole life-span studied. The binding constant KD did not change during development and ageing, whereas the maximal binding capacity Bmax, calculated per protein content, increased up to the age of two weeks and then again in ageing animals (18- and 22-month-olds). The binding was similarly inhibited by the antagonists 7-chlorokynurenate, 3-amino-l-hydroxypyrrolidin-2-one (HA-966) and 6-cyano-7-nitroquin- oxalline-2,3-dione (CNQX) in 7-day-, 3-month- and 12-month-old mice. The inhibition caused by glycine, l -serine and β-alanine also remained unaltered during the whole life-span. β-Alanine was a noncompetitive inhibitor. The alterations in the maximal binding capacities during development and ageing could be of importance in the regulation of NMDA receptors, which have been implicated in synaptic potentiation, developmental processes and various pathological conditions.

The binding of N-[1-(2- thienyl)cyclohexyl]-[ 3 H]piperidine ([3H]TCP) to the phencyclidine-binding sites in the N-methyl-D-aspartate (NMDA) receptor complex-associated ion channel was characterized in cerebral cortical membranes from 3-day-old to 24-month-old mice. The binding was saturable, exhibiting only one binding component during the whole life-span studied. The maximal binding capacity Bmax, calculated per protein content, decreased during postnatal development until 3 months of age, remaining thereafter constant in ageing mice, thus indicating the greatest availability of phencyclidine-binding sites in the immature cerebral cortex. The binding contant KD increased during the first postnatal week, remained thereafter unchanged and increased again during the second year of life, indicating a decreased affinity of these receptor sites for the ligand. The general properties of the binding; potentiation by glutamate and NMDA, as well as by glycine in a strychnine-insensitive manner, prevailed during development and ageing, certain of the effects being however less pronounced in the immature brain. Taurine and β-alanine stimulated TCP binding, acting probably at the glycine modulatory site. The actions of these inhibitory amino acids were weak and inconsistent when compared to that of glycine. Since NMDA receptors have been suggested to be involved in neuronal plasticity and learning and memory processes, these modifications in the properties of cortical phencyclidine-binding sites might be of importance in the regulation of excitatory amino acid functions during development and ageing.

Effects of glutamate agonists and their selective antagonists on the Ca2+-dependent release of [3H]GABA from rat hippocampal slices were studied in a superfusion system. The release was enhanced by glutamate, kainate, quisqualate, N -methyl- d -aspartate and glycine in a concentration-dependent manner. In general, (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) strongly antagonized the effects of all agonists tested in low-Mg2+ (0.1mM) media. 6-Cyano-7-nitroquinoxaline-2, 3-dione did not inhibit the effects of N -methyl- d -aspartate, quisqualate, glycine or N -methyl- d -aspartate together with glycine in 0.1 mM Mg2+ media. Extracellular Mg2+ failed to block the release elicited by the agonists alone. However, the action of N -methyl- d -aspartate was potentiated by glycine only in low-Mg2+ media. We suggest that under the present experimental conditions the activation of N -methyl- d -aspartate/lowaffinity quisqualate receptors located presynaptically on excitatory fibre terminals and/or postsynaptically on GABAergic interneurons is essential in hippocampal GABA release. A functional interaction is proposed between glycine and extracellular Mg2+ in the modulation of the quisqualate N -methyl- d -aspartate-gated ionophores involved in this process.

This article deals with the release of GABA, glycine and taurine from the brain stem under normal conditions and in ischemia. The release mechanisms, the effects of glutamate and adenosine receptors, and the roles of nitric oxide and second messengers are reviewed.

Background Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period. Methods The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC). Results Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls. Conclusions We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

The effects of Mg2+ on the glutamate-, kainate-, N-methyl-D-aspartate- and quisqualate-induced influx of 45Ca2+ were studied in cultured cerebellar granule cells. The N-methyl-D-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg2+. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg2+, whereas N-methyl-D-aspartate was most effective in totally Mg(2+)-free media. D-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca2+ influx by 1 mM quisqualate in 0.1-mM Mg2+ medium. The effect of 10 microM quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-D-aspartate, (RS) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and metabotropic receptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg2+ medium. The efficacy of quisqualate in inducing influx of Ca2+ and the selectivity of antagonists for different receptors are also modified by extracellular Mg2+.

The release of newly loaded [3H]GABA was studied in slices of different brain regions derived from rats in which acute hepatic encephalopathy (HE) was induced with a hepatotoxin thioacetamide. HE increased both spontaneous and high (50 mM) ammonium chloride-evoked GABA release in cerebral cortical slices by 38% and 50%, respectively. No effects of HE were noted in cerebellar or striatal slices. An increased release of GABA in the cerebral cortex may contribute to the endogenous benzodiazepine-mediated enhancement of GABAergic tone, which is thought to be partly responsible for the pathophysiological mechanism of HE.

Adult mice were exposed to long-term phencyclidine (PCP) treatments as animal models for psychosis. The drug was administered via osmotic minipumps implanted subcutaneously in the backs of the mice. The treatments for 7 days with 2.5 and 1 mg/d/mouse and the treatment for 3 days with 1 mg/d/mouse differentially affected the release of dopamine and D-aspartate from striatal and frontal cortical slices. In frontal cortical slices the potassium-stimulated release of dopamine increased, whereas the release of D-aspartate varied with the PCP dose. In striatal slices the release of D-aspartate was either decreased or unchanged, whereas the release of dopamine was mostly unchanged. The 3-day treatment with PCP followed by the 3-day period of withdrawal increased the potassium-stimulated release of dopamine from frontal cortical slices and decreased that from striatal slices. In brain slices from untreated mice PCP increased the release of dopamine in vitro, whereas the release of D-aspartate was not affected. It seems that PCP has region-specific effects on both dopaminergic and glutamatergic transmission in the central nervous system, and it may thus serve as an interesting experimental model for further research on psychosis.

Adenosine is a neuromodulator which inhibits the synaptic release of neurotransmitters. However, only little is known of the effects of inhibitory neurotransmitters and modulators on adenosine release. We studied these effects with hippocampal slices from developing (7-day-old) and young adult (3-month-old) mice under normoxic and ishemic conditions. In normoxia, adenosine release was about 3-fold greater in adults than in developing mice. beta-Alanine and L-serine (both 0.1 mM) significantly reduced the release of [3H]adenosine in normoxia in developing mice. Glycine, beta-Alanine and L-serine (all 0.1 mM) had similar effects in ischemia. Taurine depressed concentration- dependently the release in developing mice but had no effect in adults. The simultaneous release of taurine and adenosine may constitute an important protective mechanism under ischemic conditions.

Acute ethanol administration causes extensive apoptosis throughout the nervous system. We studied the protective effect of taurine on alcohol-induced apoptosis in the cerebellum of developing mice. Taurine rescued a part of immature neurons by markedly reducing caspase-3 immunoreactivity and the number of TUNEL-positive cells in most cerebellar lobules.