Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation Herman Wolosker, Marcelo Reis, *Jamjl Assreuy, and Leopoldo de Meis Departamentos de Bioqulmica Mddica and de *Farmacologia, instituto de Ciencias Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Abstract: Nitric oxide (NO; including N0, NO~, and NO-) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S-nitrosogluthathione and S- nitroso- N-acetylpenicillamine, were used. The primary target of NO is the H~-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxy- hemoglobin (12 tiM) and, to a much lesser extent, methe- moglobin protected the vacuolar H + -ATPase from inhibi- tion. Inhibition of H~ pumping by NO was reversed by addition of 0.5 mM dithiothreitol. The results indicate that the vacuolar H + -ATPase from synaptic vesicles is inhib- ited by NO by a mechanism that involves S-nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neuro- transmission. Key Words: Nitric oxide—Synaptic vesi- des — Glutamate — Neurotransmitter uptake —V - ATP - ase—H + -ATPase. J. Neurochem. 66, 1943—1948 (1996). Nitric oxide (No; including N0, NO ~ and NO ~) is an intercellular messenger molecule that is synthe- sized from L-argirnne in several tissues by a reaction catalyzed by NO synthases (Ignarro, 1990; Moncada et al., 1991; Bredt and Snyder, 1994; Dawson and Snyder, 1994). In the CNS, NO has been shown to be implicated in numerous physiological and pathological conditions, such as learning, synaptic plasticity, and glutamate neurotoxicity (Bohme et al., 1991; Dawson et al., 1991; O’Dell et al., 1991; Schuman and Madi- son, 1991; Shibuki and Okada, 1991; Daniel et al., 1993; Bredt and Snyder, 1994; Dawson and Snyder, 1994; Haley and Schuman, 1994). Once produced in the postsynaptic region, NO can readily diffuse to the presynaptic terminal, thus providing a retrograde con- trol of neurotransmission. The effects of NO in neuro- transmitter release, however, are still controversial. Depending on the experimental approach used, either an increase (Bohme et al., 1991; O’Dell et al., 1991; Schuman and Madison, 1991; Haley et al., 1992; Zhuo et al., 1993; Meffert et al., 1994) or a decrease (Shi- buki and Okada, 1991; Daniel et al., 1993; Boulton et al., 1994; Lindgren and Laird, 1994; Kamisaki et al., 1995; Sun et al., 1995) in neurotransmitter release was observed. Glutamate is the major excitatory neurotransmitter found in the CNS and is released into the synaptic cleft by synaptic vesicle exocytosis (Jahn and Sudhof, 1994). After synaptic vesicle recycling, glutamate from the cytosol is accumulated into the vesicles by a carrier-mediated transport driven by an electrochemi- cal proton gradient (A 1zH ~) generated by a vacuolar H~-ATPase(Disbrow et al., 1982; Naito and Ueda, 1983, 1985; Maycox et al., 1989; Cidon and Sihra, 1989). The vacuolar H~-ATPases of different tissues are highly sensitive to sulfhydryl reagents such as N- ethylmaleimide and are specifically inhibited by bafi- lomycin A1 (Cidon and Nelson, 1986; Bowman et al., 1988; Cidon and Sihra, 1989). Recently, it has been proposed that oxidation of critical sulfhydryl groups of the enzyme may be involved in the regulation of the vacuolar H + -ATPase in vivo (Feng and Forgac, 1992, 1994; Dschida and Bowman, 1995). However, as far as we know, no physiological molecules have yet been reported to be involved in this regulation. In the present study, we tested the effects of different NO donors on the kinetics of glutamate uptake into synaptic vesicles. We found that NO inhibits glutamate uptake by inactivation of the vacuolar H -ATPase present in the synaptic vesicles. The results suggest that NO directly interacts with the vacuolar H ± - ATPase, promoting S-nitrosylation of critical sulfhy- Received October 31, 1995; revised manuscript received Decem- ber 21, 1995; accepted December 21, 1995. Address correspondence and reprint requests to Dr. H. Wolosker at Departamento de BioquImica Médica, instituto de Ciencias Bio- médicas, Universidade Federal do Rio de Janeiro, Cidade Universitá- na, Rio de Janeiro 21910-590, Brazil. Abbreviations used.’ L~iH electrochemical proton gradient; ~W, transmembrane potential gradient; ~pH, transmembrane proton gra- dient; dcGMP, N 2,03’-dibutyryl guanosine 3’,5’-cyclic monophos- phoric acid; DTT, dithiothreitol; FCCP, carbonyl cyanide p-(trifluo- romethoxy ) phenylhydrazone; GSNO, S-nitrosogluthathione; MOPS, 3- (N-morpholino )propanesulfonic acid; NO, nitric oxide, including N0, NO and NO ; SNAP, S-nitroso-N-acetylpenicillainine. 1943