Mishra Om P, Delivoria-Papadopoulos Maria
Department of Pediatrics, Drexel University College of Medicine and St Christopher's Hospital for Children, Philadelphia, PA 19129, USA.
Neurosci Lett. 2006 Jun 19;401(1-2):81-5. doi: 10.1016/j.neulet.2006.02.070. Epub 2006 Mar 20.
Previous studies have shown that cerebral hypoxia results in increased activity of caspase-9, a key initiator of programmed cell death. We have also shown increased nitric oxide (NO) free radical generation during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced increase in caspase-9 activity in the cerebral cortex of newborn piglets is mediated by NO derived from neuronal nitric oxide synthase (nNOS). To test this hypothesis, cytosolic caspase-9 activity was determined in 15 newborn piglets divided into three groups: normoxic (Nx, n=5), hypoxic (Hx, n=5), and Hx pretreated with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, 1mg/kg, i.p., 1h prior to hypoxia (Hx+7NI, n=5). The hypoxic piglets were exposed to an FiO(2) of 0.06 for 1h. Tissue hypoxia was documented by ATP and phosphocreatinine (PCr) levels. The cytosolic fraction was obtained from the cerebral cortical tissue following centrifugation at 100,000 x g for 1h and caspase-9 activity was assayed using Ac-Leu-Glu-His-Asp-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-9. Caspase-9 activity was determined spectroflourometrically at 460 nm using 380 nm as excitation wavelength. ATP levels (micromol/g brain) were 4.35+/-0.21 in the Nx 1.43+/-0.28 in the Hx (p<0.05 versus Nx), and 1.73+/-0.33 in the Hx+7-NINA group (p<0.05 versus Nx, p=NS versus Hx). PCr levels (micromol/g brain) were 3.80+/-0.26 in the Nx, 0.96+/-0.20 in the Hx (p<0.05 versus Nx), and 1.09+/-0.39 in the Hx+7 NINA group (p<0.05 versus Nx, p=NS versus Hx). Cytosolic caspase-9 activity (nmol/mg protein/h), increased from 1.27+/-0.15 in the Nx to 2.13+/-0.14 in the Hx (p<0.05 versus Nx) compared to 1.10+/-0.21 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). Caspase-3 activity (nmol/mg protein/h) also increased from 9.39+/-0.73 in Nx to 18.94+/-3.64 in Hx (p<0.05 versus Nx) compared to 8.04+/-1.05 in the Hx+7-NINA group (p<0.05 versus Hx, p=NS versus Nx). The data show that administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increase in caspase-9 activity that leads to increase in caspase-3 activity. Since nNOS inhibition blocked the increase in caspase-9 activity during hypoxia, we conclude that hypoxia-induced increase in caspase-9 activity is mediated by nNOS derived NO. We propose that the NO generated during hypoxia leads to activation of caspase-9 and results in initiation of caspase-cascade-dependent hypoxic neuronal death.
先前的研究表明，脑缺氧会导致半胱天冬酶 - 9（程序性细胞死亡的关键启动因子）的活性增加。我们还发现新生仔猪大脑皮层在缺氧期间一氧化氮（NO）自由基生成增加。本研究检验了这样一个假设：新生仔猪大脑皮层中缺氧诱导的半胱天冬酶 - 9活性增加是由神经元型一氧化氮合酶（nNOS）产生的NO介导的。为了验证这一假设，对15只新生仔猪进行分组并测定胞质半胱天冬酶 - 9的活性，分为三组：常氧组（Nx，n = 5）、缺氧组（Hx，n = 5）和在缺氧前1小时腹腔注射1mg/kg选择性nNOS抑制剂7 - 硝基吲唑钠盐（7 - NINA）的缺氧预处理组（Hx + 7NI，n = 5）。将缺氧仔猪暴露于0.06的吸入氧分数（FiO₂）下1小时。通过ATP和磷酸肌酸（PCr）水平记录组织缺氧情况。通过在100,000×g离心1小时从大脑皮层组织中获得胞质部分，并用Ac - Leu - Glu - His - Asp - 氨基 - 4 - 甲基香豆素（一种半胱天冬酶 - 9的特异性荧光底物）测定半胱天冬酶 - 9的活性。使用380nm作为激发波长，在460nm处通过荧光分光光度法测定半胱天冬酶 - 9的活性。ATP水平（微摩尔/克脑）在Nx组为4.35±0.21，在Hx组为1.43±0.28（与Nx组相比，p < 0.05），在Hx + 7 - NINA组为1.73±0.33（与Nx组相比，p < 0.05，与Hx组相比，p = 无显著差异）。PCr水平（微摩尔/克脑）在Nx组为3.80±0.26，在Hx组为0.96±0.20（与Nx组相比，p < 0.05），在Hx + 7 - NINA组为1.09±0.39（与Nx组相比，p < 0.05，与Hx组相比，p = 无显著差异）。胞质半胱天冬酶 - 9活性（纳摩尔/毫克蛋白/小时）从Nx组的1.27±0.15增加到Hx组的2.13±0.14（与Nx组相比，p < 0.05），而在Hx + 7 - NINA组为1.10±0.21（与Hx组相比，p < 0.05，与Nx组相比，p = 无显著差异）。半胱天冬酶 - 3活性（纳摩尔/毫克蛋白/小时）也从Nx组的9.39±0.73增加到Hx组的18.94±3.64（与Nx组相比，p < 0.05），而在Hx + 7 - NINA组为8.04±1.05（与Hx组相比，p < 0.05，与Nx组相比，p = 无显著差异）。数据表明，给予nNOS抑制剂7 - NINA可防止缺氧诱导的半胱天冬酶 - 9活性增加，而这种增加会导致半胱天冬酶 - 3活性增加。由于nNOS抑制阻断了缺氧期间半胱天冬酶 - 9活性的增加，我们得出结论，缺氧诱导的半胱天冬酶 - 9活性增加是由nNOS产生的NO介导的。我们提出，缺氧期间产生的NO导致半胱天冬酶 - 9活化，并引发半胱天冬酶级联依赖性缺氧神经元死亡。
