Trotti D, Rossi D, Gjesdal O, Levy L M, Racagni G, Danbolt N C, Volterra A
Center of Neuropharmacology, Institute of Pharmacological Sciences, University of Milan, Italy.
J Biol Chem. 1996 Mar 15;271(11):5976-9. doi: 10.1074/jbc.271.11.5976.
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 Vmax of transport; its action is fast (reaching > or = 90% within 20 s), dose-dependent (50% inhibition at 50 microM), 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 H202 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.
神经元和星形胶质细胞对谷氨酸的再摄取可终止兴奋性信号，并防止突触间隙中谷氨酸水平达到兴奋性毒性阈值。该过程会受到氧自由基和过氧化氢（H2O2）的抑制。我们在此证明，另一种生物氧化剂——过氧亚硝酸盐（ONOO-），由超氧化物（O2-）和一氧化氮（NO）结合形成，能有效抑制脂质体中重组的纯化或重组高亲和力谷氨酸转运体对谷氨酸的摄取。ONOO-选择性降低转运的最大速率（Vmax）；其作用迅速（20秒内达到≥90%）、剂量依赖性（50μM时50%抑制），在ONOO-（或其产物）去除后仍持续存在，且对脂质体膜中脂质抗氧化剂维生素E的存在不敏感。因此，其作用可能依赖于ONOO-与谷氨酸转运体的直接相互作用。来自大鼠脑的三种不同重组谷氨酸转运体，即GLT1、GLAST和EAAC1，对ONOO表现出相同的敏感性。H2O2也抑制重组转运，且其作用在各方面均与ONOO-的作用相符；然而，这仅在5-10 mM H2O2且在高氧缓冲液中长时间暴露（10分钟）后才观察到。从NO供体释放的NO（高达10 mM）不会改变重组谷氨酸摄取，尽管在平行条件下它会促进突触体胞质部分中cGMP的形成。总体而言，我们的结果表明，谷氨酸转运体在其结构中含有保守位点，使其易受ONOO-和其他氧化剂的影响。
