Mechanism of tyrosine phosphorylation of procaspase-9 and Apaf-1 in cytosolic fractions of the cerebral cortex of newborn piglets during hypoxia.
Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia results in increased tyrosine phosphorylation of procaspase-9 and apoptotic protease activating factor-1 (Apaf-1) and the hypoxia-induced increased tyrosine phosphorylation of procaspase-9 and Apaf-1 is mediated by nitric oxide. To test this hypothesis, 15 newborn piglets were divided into three groups: normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic treated with nNOS inhibitor I (Hx+nNOS I 0.4mg/kg, i.v., 30min prior to hypoxia) [16]. The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Cytosolic fractions were isolated and tyrosine phosphorylated procaspase-9 and Apaf-1 were determined by immunoblotting using specific anti-procaspase-9, anti-Apaf-1 and anti-phosphotyrosine antibodies. ATP levels (mumoles/g brain) were 4.3+/-0.2 in the Nx and 1.4+/-0.3 in the Hx and 1.7+/-0.3 in Hx+nNOS I group (p<0.05 vs. Nx) groups. PCr levels (mumoles/g brain) were 3.8+/-0.3 in the Nx and 0.9+/-0.2 in the Hx and 1.0+/-0.4 in the Hx+nNOS I (p<0.05 vs. Nx) group. Density (ODxmm(2)) of tyrosine phosphorylatd procaspase-9 was 412+/-8 in the Nx, 1286+/-12 in the Hx (p<0.05 vs. Nx) and 421+/-10 in the Hx+nNOS I (p<0.05 vs. Hx) group. Density of tyrosine phosphorylated Apaf-1 was 11.72+/-1.11 in Nx, 24.50+/-2.33 in Hx (p<0.05 vs. Nx) and 16.63+/-1.57 in Hx+nNOS I (p<0.05 vs. Hx) group. We conclude that hypoxia results in increased tyrosine phosphorylation of procaspase-9 and Apaf-1 proteins in the cytosolic compartment and the hypoxia-induced increased tyrosine phosphorylation of procaspase-9 and Apaf-1 is mediated by nNOS derived nitric oxide. We propose that increased interaction between the tyrosine phosphorylated procaspase-9 and Apaf-1 molecules lead to increased activation of procaspase-9 to caspase-9 in the hypoxic brain that initiates programmed neuronal death.
Mishra
Om P
OP
Department of Pediatrics, Drexel University College of Medicine and St Christopher's Hospital for Children, Philadelphia, PA 19102, United States. omprakash.mishra@drexelmed.edu
Delivoria-Papadopoulos
Maria
M
D000818:Animals; D000831:Animals, Newborn; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D002534:Hypoxia, Brain / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism*
D053477:Apoptotic Protease-Activating Factor 1; D009569:Nitric Oxide; D014443:Tyrosine; D052248:Nitric Oxide Synthase Type I; D053453:Caspase 9