Mechanism of tyrosine phosphorylation of procaspase-9 and Apaf-1 in cytosolic fractions of the cerebral cortex of newborn piglets during hypoxia

NEUROSCIENCE LETTERS

卷:480

Article

Mishra, Om P.¹ 

[1] Drexel Univ, Coll Med, Dept Pediat, Philadelphia, PA 19102 USA

关键词: Tyrosine phosphorylation;    nNOS;    NO;    Procaspase-9;    Apaf-1;    Hypoxia;    Newborn;   

DOI  :  10.1016/j.neulet.2010.05.081

来源: Elsevier

【 摘 要 】

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 0.4 mg/kg, i.v., 30 min prior to hypoxia) [16]. The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1 h. 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 (mu moles/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 (mu moles/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 (OD x mm(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. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

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