GLUTAMATE UPTAKE INHIBITION BY OXYGEN-FREE RADICALS IN RAT CORTICAL ASTROCYTES

Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the developme nt of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be inte rdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cor tical astrocytic cultures to either xanthine + xanthine oxidase (X/XO) , a free radical-generating system, or hydrogen peroxide (H2O2) result s in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximate to 60% of its co ntrol value. In parallel no detectable release of lactate dehydrogenas e occurs. X/XO effect is abolished in the presence of a mixture of sca venger enzymes (superoxide dismutase + catalase) or by the disulfide-r educing agents glutathione and dithiothreitol (DTT), but not by lipoph ilic antioxidants or ascorbate. The time course of inhibition shows an almost linear decline of glutamate transport during cell exposure to free radicals, while upon their inactivation the decline stops but est ablished inhibition persists for at least 1 hr. In this situation, app lication of DTT significantly restores transport function. These data suggest that free radicals inhibit glutamate uptake primarily by long- lasting oxidation of protein sulfhydryl (SH) groups. Chemical modifier s of free SH groups, such as p-chloromercuribenzoate and N-ethylmaleim ide, also induce uptake inhibition. Na+/K+ ATPase is a known target of oxygen radicals and may be involved in glutamate uptake inhibition. I ndeed, ouabain, a blocker of the pump, reduces uptake in astrocytes. H owever, its effect is largely additive with that of radicals. Electrop hysiological recording of astrocytic resting conductance shows, in som e cells, a Ba2+-insensitive, inward current in response to H2O2. Howev er, in the majority of the cells, the oxidant has no effect on membran e current or voltage. In the same cells, application of glutamate in t he presence of inhibitors of ionotropic EAA receptors elicits a large inward current representing electrogenic uptake. In six of seven teste d cells, H2O2 significantly inhibited such current. These results indi cate that inactivation of Na+/K+ ATPase can be only part of the mechan ism by which oxygen radicals inhibit glutamate uptake and that a direc t action on glutamate transport is likely. In all, our data suggest th at free radicals may induce extracellular accumulation of glutamate by reduction of glial uptake. In pathologies such as ischemia/reoxygenat ion or amyotrophic lateral sclerosis, where evidence for both oxidativ e stress and EAA uptake disfunction exists, this mechanism may link ox ygen radical toxicity to excitotoxicity and represent an important ste p in the genesis of neurotoxic damage.