Tyrosine phosphorylation of neuronal nitric oxide synthase (nNOS) during hypoxia in the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study aims to investigate the mechanism of activation of nNOS during hypoxia and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of nNOS in the cerebral cortical membranes of newborn piglets is mediated by nNOS-derived nitric oxide (NO). Fifteen newborn piglets were divided into normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-pretreated with nNOS inhibitor I (Hx-nNOSi) groups. Hypoxia was induced by an FiO(2) of 0.07 for 60 min. nNOS inhibitor I (selectivity>2500 vs endothelial NOS and >500 vs inducible NOS) was administered (0.4 mg/kg, i.v.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation of nNOS determined by Western blot. Membrane protein was immunoprecipitated with nNOS antibody, separated on 12% SDS-PAGE and blotted with anti-phosphotyrosine antibody. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance (OD x mm(2)). Density (OD x mm(2)) of tyrosine phosphorylated nNOS was 51.66+/-14.11 in Nx, 118.39+/-14.17 in Hx (p<0.05 vs Nx) and 45.56+/-10.34 in Hx-nNOSi (p<0.05 vs Hx, p=NS vs Nx). The results demonstrate that pretreatment with nNOS inhibitor prevents the hypoxia-induced increased tyrosine phosphorylation of nNOS. We conclude that the mechanism of hypoxia-induced increased tyrosine phosphorylation of nNOS is mediated by nNOS-derived NO.