NO-mediated activation of Src kinase during hypoxia in the cerebral cortex of newborn piglets

Abstract

The present study aims to investigate the mechanism of Src kinase activation during hypoxia and tests the hypothesis that the hypoxia-induced activation of Src kinase, as determined by Src kinase phosphorylation, in the cerebral cortical membranes of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor I (Hx-nNOSi) groups. Hypoxia was induced by decreasing FiO 2 to 0.06 for 1 h. nNOS inhibitor I (selectivity >2500 vs eNOS and >500 vs iNOS) was administered (0.4 mg/kg, i.v.) 30 min prior to hypoxia. Cortical membranes were isolated and phosphorylation of Src kinase was determined by Western blot analysis. Src kinase activity was determined by radioactive assay using immunopurified enzyme. Membrane proteins were separated by 12% SDS–PAGE and probed with anti-phospho (pTyr 418)-Src kinase antibody. Protein bands were detected, analyzed by densitometry and expressed as absorbance (OD × mm 2). Density (OD × mm 2) of phosphorylated Src kinase was 111.7 ± 21.1 in Nx, 234.5 ± 23.8 in Hx ( p < 0.05 vs Nx) and 104.7 ± 18.1 in Hx-nNOSi ( p < 0.05 vs Hx, p = NS vs Nx). Src kinase activity (pmol/mg protein/ h) was 2472 ± 75 in Nx, 4556 ± 358 in Hx ( p < 0.05 vs Nx) and 2259 ± 207 in Hx-nNOSi ( p < 0.05 vs Hx, p = NS vs Nx). The data show that pretreatment with nNOS inhibitor prevents the hypoxia-induced increase in tyrosine phosphorylation and the activity of Src kinase. We conclude that the mechanism of hypoxia-induced increased activation of Src kinase is mediated by nNOS derived NO. We propose that NO mediated inhibition of protein tyrosine phosphatases SH-PTP-1 and SH-PTP-2 leads to increased tyrosine phosphorylation and activation of Src kinase in the cerebral cortex of newborn piglets.