Involvement of Na+ and Ca2+ channel activation and resultant nitric oxide synthesis in glutamate-mediated hypoxic injury in rat cerebrocortical slices

The role of Na+ and Ca2+ channels in glutamate-mediated hypoxic injury was investigated in slices of the rat cerebral cortex. Hypoxic injury was deter mined by mitochondrial reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl tetrazolium bromide after exposure of brain slices to 30-min of hypoxia/glu cose deprivation followed by 3-h of reoxygenation. Endogenous glutamate rel ease was markedly elevated during hypoxia/glucose deprivation, but it retur ned almost to basal level during reoxygenation. Hypoxic injury was prevente d by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Combined treatment wit h omega-conotoxin GVIA, omega-agatoxin IVA, and tetrodotoxin reversed the h ypoxic injury, although none of these agents alone or nifedipine was effect ive. Moreover, a novel Na+/Ca2+ channel blocker NS-7 [4-(4-fluorophenyl)-2- methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] significantly in hibited the hypoxic injury. Several inhibitors of nitric oxide synthase als o blocked the hypoxic injury. Consistently, nitric oxide synthesis, as esti mated from cyclic GMP formation in the extracellular fluids, was enhanced d uring hypoxia/glucose deprivation. NS-7 and other Na+ and Ca2+ channel bloc kers suppressed the enhancement of nitric oxide synthesis, although these c ompounds alone, or in combination, did not reduce hypoxic glutamate release . These findings suggest that hypoxic injury in rat cerebrocortical slices is triggered by glutamate and subsequent enhancement of nitric oxide synthe sis through activation of both Na+ and Ca2+ channels. Thus, the simultaneou s blockade of both Na+ channel as well as N-type and P/Q-type Ca2+ channels is required to sufficiently reverse the hypoxic injury. (C) 2000 Elsevier Science Inc. All rights reserved.