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

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 N^G-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.