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 gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiolalkylating agent N-ethyltnaleimide (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. (c) 2006 Elsevier Inc. All rights reserved.