R. Janáky

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 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).

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.

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 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.

Glutathione modulates the N-methyl-d-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells

The effects of reduced (GSH) and oxidized (GSSG) glutathione and dithiothreitol (DTT) and L-cysteine on the influx of 45Ca2+ were studied with cultured cerebellar granule cells. DTT slightly enhanced the basal influx but strongly activated the influx stimulated by glutamate or N-methyl-D-aspartate (NMDA). The effects on the kainate- or quisqualate-induced influx were less pronounced. Extracellular GSH had no effect on the basal influx of Ca2+. A concentration of 0.5 mM GSH slightly activated the glutamate- and NMDA-induced influx while GSSG was inhibitory. The enhancement by DTT and cysteine of the responses to excitatory amino acids was attenuated by GSH and GSSG. We propose that both the accessibility and redox state of the functional sulfhydryl groups in NMDA receptor-ionophores may be regulated by endogenous glutathione. These effects are attributed to the gamma-glutamyl moiety and sulfhydryl group in the tripeptide molecule.

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.

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, γ-D-glutamylglycine and γ-D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of γ-glutamyl peptides were more potent displacers than γ-D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. γ-L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas γ-L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. γ-L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent γ-L-glutamyl peptides (glutathione, γ-L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.

Endogenous γ-L-glutamyl and β-L-aspartyl peptides and excitatory aminoacidergic neurotransmission in the brain

Abstract The effects of γ-L-glutamyl- and β-L-aspartyl di- and tripeptides on glutamatergic neurotransmission were tested in vitro. Of the peptides, γ-L-glutamylglutamate was the most effective inhibitor, comparable to glutamate, of both Na+-independent and Cl − Ca 2+ -activated binding/transport of glutamate. γ-L-glutamylglutamate was most effective in the midbrain and hypothalamus and γ-L-glutamylaspartate in the hippocampus when tested on the Na+-independent binding. The Cl − Ca 2+ -dependent binding/transport of glutamate was affected by γ-glutamylaspartate most strongly in the hippocampus. γ-L-glutamylglycine and β-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 γ-L-glutamylglutamate and -aspartate and the release of D-aspartate also by γ-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.

Modulation of glutamate agonist-induced influx of calcium into neurons by γ-l-glutamyl and β-l-aspartyl dipeptides

Abstract γ- l -Glutamate and β- l -aspartate dipeptides, present in the mammalian brain with a yet unknown function, were shown to affect the influx of Ca 2+ into cultured cerebellar granule cells. The most active peptides, γ- l -glutamyl- l -aspartate, γ- l -glutamyl- l -glutamate and γ- l -glutamylglycine, enhanced the basal influx but inhibited the glutamate-activated influx of Ca 2+ in a dose-dependent manner. γ- l -Glutamyl- l -aspartate, the strongest inhibitor of the glutamate-activated influx of Ca 2+ , exhibited selective Mg 2+ -dependent antagonism in the N- methyl- d -aspartate (NMDA)-activated influx of Ca 2+ . This finding may explain its previously shown deleterious effects on the long-term memory. On the other hand, γ- l -glutamyl- l -aspartate enhanced alone the entry of Ca 2+ into neurons. This effect was antagonized by the non-NMDA antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), suggesting a non-NMDA receptor-mediated action, that may also be involved in excitotoxicity in some neurodegenerative disorders.

Regulation of glutamatergic neurotransmission in the striatum by presynaptic adenylyl cyclase-dependent processes

The aim here was to examine the possible roles of adenylyl cyclase- and protein kinase A (PKA)-dependent processes in ionotropic glutamate receptor (iGluR)-mediated neurotransmission using superfused mouse striatal slices and a non-metabolized L-glutamate analogue, D-[3H]aspartate. The direct and indirect presynaptic modulation of glutamate release and its susceptibility to changes in the intracellular levels of cyclic AMP (cAMP), Ca(2+) and calmodulin (CaM) and in protein phosphorylation was characterized by pharmacological manipulations. The agonists of iGluRs, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate, stimulated the basal release of D-[3H]aspartate, while N-methyl-D-aspartate (NMDA) was without effect. Both the AMPA- and kainate-mediated responses were accentuated by the beta-adrenoceptor agonist isoproterenol. These facilitatory effects were mimicked by the permeable cAMP analogue dibutyryl-cAMP. The beta-adrenoceptor antagonist propranolol, the adenylyl cyclase inhibitor MDL12,330A, the inhibitor of PKA and PKC, H-7, and the PKA inhibitor H-89 abolished the isoproterenol effect on the kainate-evoked release. The dibutyryl-cAMP-induced potentiation was also attenuated by H-7. Isoproterenol, propranolol and MDL12,330A failed to affect the basal release of D-[3H]aspartate, but dibutyryl-cAMP was inhibitory and MDL12,330A activatory. In Ca(2+)-free medium, the kainate-evoked release was enhanced, being further accentuated by the CaM antagonists calmidazolium and trifluoperazine, though these inhibited the basal release. The potentiating effect of calmidazolium on the kainate-stimulated release was counteracted by both MDL12,330A and H-7. We conclude that AMPA- and kainate-evoked glutamate release from striatal glutamatergic terminals is potentiated by beta-adrenergic receptor-mediated adenylyl cyclase activation and cAMP accumulation. Glutamate release is enhanced if the Ca(2+)- and CaM-dependent, kainate-evoked processes do not prevent the excessive accumulation of intracellular cAMP.