Nitric oxide reduces Ca2+ and Zn2+ influx through voltage-gated Ca2+ channels and reduces Zn2+ neurotoxicity

Abstract The translocation of synaptic Zn 2+ from nerve terminals into selectively vulnerable neurons may contribute to the death of these neurons after global ischemia. We hypothesized that cellular Zn 2+ overload might be lethal for reasons similar to cellular Ca 2+ overload and tested the hypothesis that Zn 2+ neurotoxicity might be mediated by the activation of nitric oxide synthase. Although Zn 2+ (30–300 μM) altered nitric oxide synthase activity in cerebellar extracts in solution, it did not affect nitric oxide synthase activity in cultured murine neocortical neurons. Cultured neurons exposed to 300–500 μM Zn 2+ for 5 min under depolarizing conditions developed widespread degeneration over the next 24 h that was unaffected by the concurrent addition of the nitric oxide synthase inhibitor N G -nitro- l -arginine. Furthermore, Zn 2+ neurotoxicity was attenuated when nitric oxide synthase activity in the cultures was induced by exposure to cytokines, exogenous nitric oxide was added or nitric oxide production was pharmacologically enhanced. The unexpected protective effect of nitric oxide against Zn 2+ toxicity may be explained, at least in part, by reduction of toxic Zn 2+ entry. Exposure to nitric oxide donors reduced Ba 2+ current through high-voltage activated calcium channels, as well as K + -stimulated neuronal uptake of 45 Ca 2+ or 65 Zn 2+ . The oxidizing agents thimerosal and 2,2′-dithiodipyridine also reduced K + -stimulated cellular 45 Ca 2+ uptake, while akylation of thiols by pretreatment with N- ethylmaleimide blocked the reduction of 45 Ca 2+ uptake by a nitric oxide donor. The results suggest that Zn 2+ -induced neuronal death is not mediated by the activation of nitric oxide synthase; rather, available nitric oxide may attenuate Zn 2+ neurotoxicity by reducing Zn 2+ entry through voltage-gated Ca 2+ channels, perhaps by oxidizing key thiol groups.