The translocation of synaptic Zn2+ from nerve terminals into selectively vulnerable neurons may contribute to the death of these neurons after global ischemia. We hypothesized that cellular Zn2+ overload might be lethal for reasons similar to cellular Ca2+ overload and tested the hypothesis that Zn2+ neurotoxicity might be mediated by the activation of nitric oxide synthase. Although Zn2+ (30-300μM) 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 300-500μM Zn2+ for 5min under depolarizing conditions developed widespread degeneration over the next 24h that was unaffected by the concurrent addition of the nitric oxide synthase inhibitor NG-nitro-L-arginine. Furthermore, Zn2+ 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 Zn2+ toxicity may be explained, at least in part, by reduction of toxic Zn2+ entry. Exposure to nitric oxide donors reduced Ba2+ current through high-voltage activated calcium channels, as well as K+-stimulated neuronal uptake of 45Ca2+ or 65Zn2+. The oxidizing agents thimerosal and 2,2′-dithiodipyridine also reduced K+-stimulated cellular 45Ca2+ uptake, while akylation of thiols by pretreatment with N-ethylmaleimide blocked the reduction of 45Ca2+ uptake by a nitric oxide donor.The results suggest that Zn2+-induced neuronal death is not mediated by the activation of nitric oxide synthase; rather, available nitric oxide may attenuate Zn2+ neurotoxicity by reducing Zn2+ entry through voltage-gated Ca2+ channels, perhaps by oxidizing key thiol groups.
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