Mechanism of caspase-9 activation during hypoxia in the cerebral cortex of newborn piglets: The role of Src kinase

Abstract

We have previously shown that hypoxia results in increased activation of caspase-9 in the cerebral cortex of newborn piglets. The present study tests the hypothesis that the increased activation of caspase-9 during hypoxia is mediated by Src kinase. To test this hypothesis a highly selective Src kinase inhibitor PP2 [IC50 5nm] was administered to prevent caspase-9 activation during hypoxia. Cytosolic fraction from the cerebral cortical tissue was isolated and the activation of caspase-9 was documented by the expression of active caspase-9 and the activity of caspase-9 and caspase-3. Piglets were divided into: normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-treated with Src inhibitor (Hx-PP2). Hypoxia was induced by decreasing FiO2 to 0.07 for 60min. PP2 was administered (0.4mg/kg, i.v.) 30min prior to hypoxia. ATP and phosphocreatine (PCr) levels were determined to document cerebral tissue hypoxia. Activity of caspase-9 and caspase-3 were determined spectrofluorometrically using specific fluorogenic substrates. Expression of active caspase-9 was determined by Western blot using active caspase-9 antibody. Caspase-9 activity (nmoles/mgprotein/h) was 1.40±0.12 in Nx, 2.12±0.11 in Hx (p<0.05 vs Nx) and 1.61±0.14 in Hx-PP2 (p<0.05 vs Hx). Active caspase-9 expression (OD×mm2) was 42.3±8.3 in Nx, 78.9±11.0 in Hx (p<0.05 vs Nx) and 41.2±7.6 in Hx-PP2 (p<0.05 vs Hx). Caspase-3 activity (nmoles/mgprotein/h) was 4.11±0.1 in Nx, 6.51±0.1 in Hx (p<0.05 vs Nx) and 4.57±0.7 in Hx+PP2 (p<0.05 vs Hx). Active caspase-3 expression (OD×mm2) was 392.1±23.1 in Nx, 645.0±90.3 in Hx (p<0.05 vs Nx) and 329.7±51.5 in Hx-PP2 (p<0.05 vs Hx). The data show that pretreatment with Src kinase inhibitor prevents the hypoxia-induced increased expression of active caspase-9 and the activity of caspase-9. Src kinase inhibitor also prevented the hypoxia-induced increased activation of caspase-3, a consequence of caspase-9 activation. We conclude that the hypoxia-induced activation of caspase-9 is mediated by Src kinase. We propose Src kinase-dependent tyrosine phosphorylation (Tyr154) in the active site domain of caspase-9 is a potential mechanism of caspase-9 activation in the hypoxic brain.