Another Facet of Nitric Oxide: Reduction of Toxic Zinc Influx Through Voltage-Gated Channels

The neurotoxicity of zinc, released from nerve terminals during global ischemia, may contribute to the delayed death of certain selectively vulnerable neuronal populations. A likely first event in zinc-induced neuronal death appears to be its permeation across the plasma membrane, largely through voltage- and agonist-gated calcium channels. Considering the possibility that cellular Zn2+ overload might be lethal for reasons similar to cellular calcium overload, we tested the hypothesis that Zn2+ neurotoxicity might be mediated by activation of neuronal nitric oxide synthase (NOS), an event implicated in the pathogenesis of excitotoxic neuronal death. However, physiologically relevant concentrations of zinc (30–100 nM) had no effect on NOS activity, while 100–300 μM Zn2+ actually inhibited NOS activity in solution. The addition of extracellular Zn2+ did not affect NOS activity in cultured murine neocortical neurons, assessed by measuring cyclic guanosine 5’-monophosphate (cGMP) levels, and the concurrent addition of NOS inhibitors did not alter Zn2+-induced neuronal death (cultures were exposed to 300–500 μM Zn2+ for 5 min under depolarizing conditions; neuronal degeneration was assessed 24 h later). Rather, addition of the nitric oxide (NO) precursor, l-arginine, or the diazeniumdiolate (NONOate) NO donors (DEA/NO) or 1-propanamine,3-(2-hydroxy-2-nitroso-l-propyihydrazino) NONOate (PAPA/NO) markedly reduced Zn2+-induced neuronal death and produced a dose-dependent block of high K+-stimulated cellular 45Ca2+ uptake. The oxidizing agents thimerosal and 2,2’-dithiodipyridine (DTDP) also reduced K+-stimulated cellular 45Ca2+ uptake, while alkylation of thiols by pretreatment with N-ethylmaleimide (NEM) blocked the reduction of 45Ca2+ uptake by NO donors. These results suggest that Zn2+-induced neuronal death is not mediated by the activation of NOS; rather, any available NO may attenuate Zn2+ neurotoxicity, in part through a down-modulation of Zn2+ entry through voltage-gated Ca2+ channels.