Inducible nitric oxide synthase up-regulation and mitochondrial glutathione depletion mediate cyanide-induced necrosis in mesencephalic cells.

We have previously shown in rat primary cultured mesencephalic cells that cyanide induces a high level of oxidative stress and necrotic death. To evaluate the mechanism of the cytotoxicity, the effects of cyanide on intracellular glutathione (GSH) pools and inducible nitric oxide synthase (iNOS)-mediated reactive nitrogen species (RNS) generation were studied. Cyanide rapidly depleted intracellular GSH. Restoration of GSH blocked cell death, whereas depletion of GSH by synthesis inhibition increased the necrosis. Selective depletion of mitochondrial GSH (mtGSH) increased oxidative stress and enhanced cell death, whereas the cytoplasmic pool was not critical to cell survival. These actions were accompanied by increased iNOS expression as determined by Western blot analysis, RT-PCR and immunohistochemistry. Up-regulation of iNOS led to increased generation of NO as reflected by elevated nitrite levels (an end product of NO metabolism). It was determined by use of a selective inhibitor that up-regulation of iNOS expression was transcriptionally regulated by activation of nuclear factor-kappaB, a redox-sensitive transcription factor. It was concluded that, in cyanide-mediated neurotoxicity, mtGSH is a vital component of the cellular antioxidant defense, and its depletion can lead to oxidative stress-mediated iNOS up-regulation, thus enhancing RNS generation and necrosis.