Department of Pediatrics, Drexel University College of Medicine and St Christopher's Hospital for Children, Philadelphia, PA 19102, United States.
Neurosci Lett. 2010 Aug 9;480(1):35-9. doi: 10.1016/j.neulet.2010.05.081. Epub 2010 Jun 4.
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.
先前的研究表明，脑缺氧会导致新生仔猪大脑皮质胞质部分的半胱天冬酶-9 活性增加。本研究检验了这样一个假设，即缺氧会导致原半胱天冬酶-9 和凋亡蛋白酶激活因子-1（Apaf-1）的酪氨酸磷酸化增加，而缺氧诱导的原半胱天冬酶-9 和 Apaf-1 的酪氨酸磷酸化是由一氧化氮介导的。为了验证这一假设，将 15 只新生仔猪分为三组：常氧组（Nx，n=5）、缺氧组（Hx，n=5）和缺氧并用 nNOS 抑制剂 I 处理组（Hx+nNOS I 0.4mg/kg，静脉注射，在缺氧前 30 分钟）[16]。缺氧仔猪暴露于 FiO(2)为 0.06 的环境中 1 小时。通过 ATP 和磷酸肌酸（PCr）水平来记录组织缺氧。使用特定的抗原半胱天冬酶-9、抗 Apaf-1 和抗磷酸酪氨酸抗体通过免疫印迹法分离胞质部分，并测定酪氨酸磷酸化的原半胱天冬酶-9 和 Apaf-1。ATP 水平（每克脑的毫摩尔）在 Nx 组为 4.3+/-0.2，在 Hx 组为 1.4+/-0.3，在 Hx+nNOS I 组为 1.7+/-0.3（p<0.05 与 Nx 组相比）。PCr 水平（每克脑的毫摩尔）在 Nx 组为 3.8+/-0.3，在 Hx 组为 0.9+/-0.2，在 Hx+nNOS I 组为 1.0+/-0.4（p<0.05 与 Nx 组相比）。酪氨酸磷酸化的原半胱天冬酶-9 的密度（ODxmm(2)）在 Nx 组为 412+/-8，在 Hx 组为 1286+/-12（p<0.05 与 Nx 组相比），在 Hx+nNOS I 组为 421+/-10（p<0.05 与 Hx 组相比）。酪氨酸磷酸化的 Apaf-1 的密度在 Nx 组为 11.72+/-1.11，在 Hx 组为 24.50+/-2.33（p<0.05 与 Nx 组相比），在 Hx+nNOS I 组为 16.63+/-1.57（p<0.05 与 Hx 组相比）。我们得出结论，缺氧会导致原半胱天冬酶-9 和 Apaf-1 蛋白在胞质部分的酪氨酸磷酸化增加，而缺氧诱导的原半胱天冬酶-9 和 Apaf-1 的酪氨酸磷酸化是由 nNOS 衍生的一氧化氮介导的。我们提出，增加酪氨酸磷酸化的原半胱天冬酶-9 和 Apaf-1 分子之间的相互作用会导致缺氧脑中原半胱天冬酶-9 向 caspase-9 的激活增加，从而引发程序性神经元死亡。
