Hypoxia/ischaemia is known to trigger neuronal death, but the role of
neuronal nitric oxide synthase (nNOS) in this process is controversial.
Nitric oxide (NO) inhibits
cytochrome oxidase in competition with
oxygen. We tested whether NO derived from nNOS synergises with
hypoxia to induce neuronal death by inhibiting mitochondrial
cytochrome oxidase. Sixteen hours of
hypoxia (2%
oxygen) plus
deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added
glutamate or by added
azide (an uncompetitive inhibitor of
cytochrome oxidase), indicating that nNOS does not act downstream of
glutamate or
cytochrome oxidase.
Hypoxia plus
deoxyglucose-induced
glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this.
Hypoxia inhibited
cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting
cytochrome oxidase in competition with
oxygen. These data indicate that
hypoxia synergises with NO from nNOS to induce neuronal death via
cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/
stroke-induced neuronal death in vivo.