PEROXYNITRITE INHIBITS GLUTAMATE TRANSPORTER SUBTYPES

The reuptake of glutamate in neurons and astrocytes terminates excitat ory signals and prevents the persistence of excitotoxic levels of glut amate in the synaptic cleft. This process is inhibited by oxygen radic als and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide ( O-.(2)-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstit uted in liposomes. ONOO- reduces selectively the V-max of transport; i ts action is fast (reaching greater than or equal to 90% within 20 s), dose-dependent (50% inhibition at 50 mu M), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid ant ioxidant vitamin E in the liposomal membranes. Therefore, it likely de pends 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 a lso inhibits reconstituted transport, and its action matches that of O NOO- 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 fo rmation in synaptosomal cytosolic fraction. Overall, our results sugge st that the glutamate transporters contain conserved sites in their st ructures conferring vulnerability to ONOO- and other oxidants.