The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide (·O2/-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO- reduces selectively the V(max) of transport; its action is fast (reaching ≥90% within 20 s), dose-dependent (50% inhibition at 50 μm), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO- with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO-. H2O2 also inhibits reconstituted transport, and its action matches that of ONOO- on all respects; however, this is observed only with 5-10 mM H2O2 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM) does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO- and other oxidants.
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