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 development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximate to 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase + catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic 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 established inhibition persists for at least 1 hr. In this situation, application 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 modifiers of free SH groups, such as p-chloromercuribenzoate and N-ethylmaleimide, also induce uptake inhibition. Na+/K+ ATPase is a known target of oxygen radicals and may be involved in glutamate uptake inhibition. Indeed, ouabain, a blocker of the pump, reduces uptake in astrocytes. However, its effect is largely additive with that of radicals. Electrophysiological recording of astrocytic resting conductance shows, in some cells, a Ba2+-insensitive, inward current in response to H2O2. However, in the majority of the cells, the oxidant has no effect on membrane current or voltage. In the same cells, application of glutamate in the presence of inhibitors of ionotropic EAA receptors elicits a large inward current representing electrogenic uptake. In six of seven tested cells, H2O2 significantly inhibited such current. These results indicate that inactivation of Na+/K+ ATPase can be only part of the mechanism by which oxygen radicals inhibit glutamate uptake and that a direct action on glutamate transport is likely. In all, our data suggest that free radicals may induce extracellular accumulation of glutamate by reduction of glial uptake. In pathologies such as ischemia/reoxygenation or amyotrophic lateral sclerosis, where evidence for both oxidative stress and EAA uptake disfunction exists, this mechanism may link oxygen radical toxicity to excitotoxicity and represent an important step in the genesis of neurotoxic damage.