Specific glutathione binding sites in pig cerebral cortical synaptic membranes

Glutathione (gamma-glutamylcysteinylglycine) is a neuromodulator at glutama te receptors, but may also act as a neurotransmitter at sites of its own. T he Na+-independent binding of [H-3]glutathione to pig cortical synaptic mem branes was characterized here using glycine, cysteine analogs, dipeptides a nd glutathione derivatives, and ligands selective for known glutamate recep tors. L-Glutamate, pyroglutamate, quinolinate, (S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3 dione were weak inhibitors at co ncentrations of 0.5 or 1 mM. D-Glutamate, L- and D-aspartate, glutamine, qu isqualate, kynurenate, other N-methyl-D-aspartate receptor ligands and non- N-methyl-D-aspartate receptor Ligands failed to displace [3H]glutathione. E xcept for weak inhibition by D-serine (0.5 mM), glycine and other ligands o f 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 up take sites were generally inactive. Glutathione, oxidized glutathione, S-ni trosoglutathione, gamma-L-glutamylcysteine, cysteinylglycine, cysteine, cys teamine and cystamine were the most potent displacers (IC50 values in the m icromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione (S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). L-Homocysteinate and aminomethanesulfonate exhibit ed a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl-D-aspartate receptor glycine co-activatory site, was a pot ent activator of glutathione binding. At 1 mM, some dipeptides also slightl y activated the binding, gamma-L-glutamylleucine and delta-L-glutamyl-GABA being the most effective. The specific binding sites for glutathione in bra in synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. Th e oxidation or alkylation of the cysteine thiol group reduces the binding a ffinity. The strong activation by thiokynurenate may indicate that the glut athione receptor protein contains a modulatory site to which co-agonists ma y 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 tr ansduction in the brain. (C) 1999 IBRO. Published by Elsevier Science Ltd.