Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen–glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca2+]i) response in the rat hippocampal neurons. The levels of NO and [Ca2+]i in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca2+]i responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca2+]i response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca2+]i responses to hypoxia and OGD, indicating an involvement of L-type Ca2+ channels in the NO-mediated mechanisms. In addition, the [Ca2+]i responses were attenuated by ODQ or KT5823, inhibitors of the cGMP–PKG pathway, and by acivicin, an inhibitor of γ-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca2+ currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca2+ currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca2+ current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca2+ via the hippocampal L-type Ca2+ channel by S-nitrosylation during an initial phase of energy deprivation.