Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives.

Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, gamma-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [(3)H]taurine evoked by K+-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K(+), 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K+. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by gamma-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and gamma-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life.

Effects of metabotropic glutamate receptor agonists and antagonists on D-aspartate release from mouse cerebral cortical and striatal slices.

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[3H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[3H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)alpha-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S,1'S,2'R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.

Mechanisms of L-cysteine neurotoxicity.

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 alpha-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).

Interference of S-nitrosoglutathione with the binding of ligands to ionotropic glutamate receptors in pig cerebral cortical synaptic membranes.

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.

Modulation of glutamate receptor functions by glutathione.

In addition to its well-known antioxidant effects, glutathione apparently has an additional double role in the central nervous system as a neurotransmitter and neuromodulator. A number of recent neurochemical, neuropharmacological and electrophysiological studies have yielded evidence on both functions. As an excitatory neurotransmitter, glutathione depolarizes neurons by acting as ionotropic receptors of its own which are different from any other excitatory amino acid receptors. As a neuromodulator, it displaces ionotropic glutamate receptor ligands from their binding sites and regulates calcium influx through N-methyl-D-aspartate receptor-governed ionophores. In brain slices glutathione has been shown to regulate the release of other transmitters, e.g., gamma-aminobutyrate and dopamine, mediated by N-methyl-D-aspartate receptors. In the present article, we review recent findings on the neuromodulatory actions of glutathione and discuss possible physiological and pathophysiological consequences.

Specific glutathione binding sites in pig cerebral cortical synaptic membranes.

Glutathione (gamma-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na+-independent binding of [3H]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 [3H]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, gamma-L-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers (IC50 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, gamma-L-glutamylleucine and gamma-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.

Glutathione and signal transduction in the mammalian CNS.

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Interference of S-alkyl derivatives of glutathione with brain ionotropic glutamate receptors.

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.

Ligand binding to porcine ionotropic glutamate receptors with chemically modified arginyl residues.

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.

Glutathione is an endogenous ligand of rat brain N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors.

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 ligand of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (+/-)-3-(2-carboxypiperazin-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 gamma-glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.

Role of histidyl residues in the binding of ligands to the porcine N-methyl-D-aspartate receptor.

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.

Endogenous gamma-L-glutamylglutamate is a partial agonist at the N-methyl-D-aspartate receptors in cultured cerebellar granule cells.

gamma-L-Glutamylglutamate (LGG), an endogenous constituent of the brain, reduced the glutamate-evoked increase in intracellular Ca2+ in cultured cerebellar granule cells. The extent and properties of this inhibition were different at different Mg2+ concentrations. The intracellular Ca2+ response to NMDA was slightly enhanced by 0.1 mM LGG in normal (1.3 mM) Mg2+ medium, but in Mg(2+)-free medium LGG was stimulatory at low (0.1-1 microM) NMDA and inhibitory at high (0.1-1 mM) NMDA concentrations. In the absence of Mg2+, LGG alone increased cytosolic free Ca2+ and depolarized the cells. These effects were potentiated by glycine and blocked by extracellular Mg2+, 2-amino-5-phosphonopentanoate (APV), 7-chlorokynurenate, 3-amino-1-hydroxypyrrolidin-2-one (HA-966) and 5,7-dinitroquinoxaline-2,3-dione (MNQX). The results indicate that LGG is a partial NMDA agonist. On the other hand, the non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) also inhibited the effects of LGG. This indicates an involvement of non-NMDA receptors in the actions of LGG. The consequent depolarization may also contribute to the activation of NMDA receptor-governed ionophores.

Endogenous gamma-L-glutamyl and beta-L-aspartyl peptides and excitatory aminoacidergic neurotransmission in the brain.

The effects of gamma-L-glutamyl- and beta-L-aspartyl di- and tripeptides on glutamatergic neurotransmission were tested in vitro. Of the peptides, gamma-L-glutamylglutamate was the most effective inhibitor, comparable to glutamate, of both Na(+)-independent and Cl-/Ca(2+)-activated binding/transport of glutamate. gamma-L-glutamylglutamate was most effective in the midbrain and hypothalamus and gamma-L-glutamylaspartate in the hippocampus when tested on the Na(+)-independent binding. The Cl-/Ca(2+)-dependent binding/transport of glutamate was affected by gamma-glutamylaspartate most strongly in the hippocampus. gamma-L-glutamylglycine and beta-L-aspartylglycine moderately inhibited the Na(+)-dependent uptake of L-glutamate and D-aspartate while the other peptides were only weak inhibitors. Reduced and oxidized glutathione enhanced the uptake of L-glutamate. The K(+)-stimulated release of L-glutamate was enhanced by gamma-L-glutamylglutamate and -aspartate and the release of D-aspartate also by gamma-L-glutamylglycine. The results indicate that both pre- and postsynaptic events in glutamatergic neurotransmission are modulated by these endogenous acidic oligopeptides.

Release of [3H]GABA evoked by glutamate agonists from hippocampal slices: effects of dithiothreitol and glutathione.

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.

Glutamate agonists and [3H]GABA release from rat hippocampal slices: involvement of metabotropic glutamate receptors in the quisqualate-evoked release.

The effects of glutamate agonists and their selective antagonists on the Ca(2+)-dependent and independent releases of [3H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca(2+)-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca(2+)-free medium. The release evoked by QA in Ca(2+)-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca(2+)-free medium. Verapamil inhibited the QA-activated release in both Ca(2+)-containing and Ca(2+)-free media. The effect of QA but not that of AMPA was blocked in Ca(2+)-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca2+ form intracellular stores.

Interactions of gamma-L-glutamyltaurine with excitatory aminoacidergic neurotransmission.

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.