or superoxide, can generate reactive nitrogen oxide spe-AS was found to be cytotoxic to Chinese hamster V79 cies (RNOS) 2 , which can oxidize nitrate, or nitrosate, lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-other biomolecules (2, 3). RNOS are proposed to medi-(CN 6 ) 30 ), which converts NO 0 to NO, afforded dramatic ate various toxic and cytotoxic mechanisms. In conprotection against AS-mediated cytotoxicity. Treat-trast, NO can protect cells from damage caused by reacment of V79 cells with L-buthionine sulfoximine to re-tive oxygen species (ROS) (4-6), as a result of different duce intracellular glutathione markedly enhanced AS chemical reactions than those involving RNOS (7-10).cytotoxicity, which suggests that GSH is critical for NO participates in chemical reactions associated cellular protection against the toxicity of NO 0 . Fur-with both oxidative and nitrosative stress which can ther experiments showed that low molecular weight have deleterious consequences in vivo. Studies suggest transition metal complexes associated with the forma-that there exists a balance between NO and two of tion of reactive oxygen species are not involved in AS-the major reactive nitrogen oxide species, dinitrogen mediated cytotoxicity since metal chelators had no ef-trioxide (N 2 O 3 ) and peroxynitrite (ONOO 0 ) (11-13) fect. However, under aerobic conditions, AS was more that can determine the toxicological outcome under toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA 2 Abbreviations used: RNOS, reactive nitrogen oxide species; AS, double strand breaks in whole cells, and this effect was Angelis's salt; DETAPAC, diethylenetriaminepentaacetic acid; DF, desferrioxime; TPH, tempol-H; NADPH, b-nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; GSH, intra-1 To whom correspondence and reprint requests should be ad-cellular glutathione; ER, enhancement ratio; ESR, electron spin resonance. dressed. Fax: (301) 480-2238. 66

The nitroxyl anion (NO. ), respectively, can lead to oxidation, hydroxylation, nitration, and nitrosation of biomolecules (1). Although a substantial literature exists on the putative biological effects of other RNOS, few studies have focused on nitroxyl (NO Ϫ ), the one electron reduction product of NO. Several reports suggest that NO Ϫ (or its conjugate acid, HNO) can be generated from chemical reactions that occur in vivo (2, 3) including oxidation of L-arginine by tetrahydrobiopterin-free nitric oxide synthase (NOS) (4 -6) and decomposition of Snitrosothiols (7, 8). Taken together, these studies indicate that the chemistry of NO Ϫ is an essential component of the redox chemistry of NO in biological systems.Angeli's salt (AS) is the most commonly used synthetic donor in the study of NO Ϫ effects under biological conditions (9). At physiological pH and temperature, AS spontaneously decomposes to HNO and nitrite with a half-life of 2.5 min,The cytotoxic effects of AS are several orders of magnitude greater than those of other RNOS and are comparable to alkylhydroperoxides (10), suggesting that NO Ϫ formation in vivo could have deleterious consequences. In a myocardial ischemiareperfusion model, treatment with AS markedly increased infarct area (11). In contrast, NO, either from a donor or from oxidation of NO Ϫ in the presence of an electron acceptor, afforded protection in the same model. In the present report, the chemistry of AS is compared with that of ONOO Ϫ and NO/N 2 O 3 to gain insight into the biological mechanisms in which the chemistry of NO Ϫ could be involved. MATERIALS AND METHODSAngeli's salt (Na 2 N 2 O 3 ) was synthesized as described previously (10). The NONOate, DEA/NO (NaEt 2 NN(O)NO), was a generous gift from Dr. Joseph Saavedra (National Cancer Institute, Frederick, MD). Stock solutions (ϳ10 mM) of AS and DEA/NO were prepared in 10 mM NaOH and stored at Ϫ20°C (12). Peroxynitrite was synthesized by mixing solutions of 0.5 M NO 2 Ϫ in 0.5 M HCl and 0.5 M hydrogen peroxide (H 2 O 2 ) followed by rapid quenching in 1 M NaOH, as described previously (13). The resulting basic solution was exposed to MnO 2 to remove excess H 2 O 2 , which was reduced to Ͻ1% per mol of ONOO Ϫ . After filtering, aliquots were stored at Ϫ20°C for less than 2 weeks. Directly prior to use, the concentrations of these RNOS donors in 10 mM NaOH were

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Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptormediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex, and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO' equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H, O, was not altered by the presence of DedNO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2 + , whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by PapdNO and DedNO. However, addition of ascorbate (400 pM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by DedNO and PapdNO without altering their neuroprotective properties or their effects on 45Ca*+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level. Key Words: Nitric oxide donors-NMDA-induced neurotoxicity-Murine mixed cortical cell cultureNONOate-type nitric oxide donors-4-Nitrosothiols.