THE GLUTATHIONE LEVEL OF RETINAL MULLER GLIAL-CELLS IS DEPENDENT ON THE HIGH-AFFINITY SODIUM-DEPENDENT UPTAKE OF GLUTAMATE

The dependence of intracellular glutathione, an important radical scav enger, on the extra cellular glutamate and cystine concentration and t he velocity of the high affinity sodium/glutamate transporter was stud ied in freshly-isolated Muller glial cells of the guinea-pig, kept in vitro for up to 11 h. To this end the relative Muller cell glutathione levels were measured using the fluorescent dye monochlorobimane, usin g different concentrations of glutamate and cystine in Ringer solution . In some experiments L-buthionine-[S,R]-sulfoximine, a blocker of glu tathione synthesis, or L-trans-pyrrolidine-2,4-dicarboxylic acid and L -alpha-aminoadipic acid, inhibitors of glutamate uptake, were added. T he Muller cells maintained about 80% of the normal glutathione level w hen maintained in Ringer solution containing 100 mu M glutamate for 11 h. When under these conditions 100 mu M cystine was added, the glutat hione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without gl utamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solu tion. Likewise, application of L-buthionine[S,R]-sulfoximine (5 mM) le ad to a strong run down of the glutathione level even in glutamate/cys tine (100 mu M)-containing solution. A similar suppressing effect was observed using L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-a minoadipic acid in the presence of 100 mu M cystine and glutamate. We conclude that the intracellular glutamate concentration of the Muller cells is determined by the extracellular glutamate concentration and t he velocity of the sodium/glutamate uptake. Consequently, cystine upta ke into Muller cells, which is performed by the cystine/glutamate anti porter, is fuelled by the sodium/glutamate transporter with intracellu lar glutamate. Both glutamate and cystine are also substrates for glut athione synthesis. The glutathione level is logically limited by the c apacity of the sodium/glutamate transporter to provide glutamate intra cellularly for, first, cystine uptake and, second, direct insertion in to glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the gl utathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate c oncentrations are discussed. (C) 1997 IBRO. Published by Elsevier Scie nce Ltd.