Delivoria-Papadopoulos Maria, Gorn Michael, Ashraf Qazi M, Mishra Om P
Department of Pediatrics, Drexel University College of Medicine, St. Christopher's Hospital for Children, Philadelphia, PA 19102, United States.
Neurosci Lett. 2007 Dec 18;429(2-3):115-9. doi: 10.1016/j.neulet.2007.09.072. Epub 2007 Oct 11.
In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study examines the mechanism of caspase-9 activation during hypoxia and tests the hypothesis that the ATP and cytochrome c-dependent activation of caspase-9 increases in the cytosol of the cerebral cortex of newborn piglets. Newborn piglets were divided into normoxic (Nx, n=4), and hypoxic (Hx, n=4) groups. Anesthetized, ventilated animals were exposed to an FiO(2) of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Cytosolic fraction was isolated and passed through a G25-Sephadex column to remove endogenous ATP and cytochrome c. Fractions were collected and protein determined by UV spectrophotometry at 280 nm. Eluted high-molecular weight samples from normoxic and hypoxic animals were divided into four subgroups: subgroup 1 (control), incubated without added ATP and cytochrome c; subgroup 2, incubated with added ATP; subgroup 3, incubated with added cytochrome c; and subgroup 4, incubated with added ATP and cytochrome c. The incubation was carried out at 37 degrees C for 30 min. Following incubation, the protein was separated by 12% SDS-PAGE and active caspase-9 was detected using specific active caspase-9 antibody. Protein bands were detected by enhanced chemiluminescence. Protein density was determined by imaging densitometry and expressed as absorbance (OD x mm(2)). ATP (mumol/g brain) level was 4.7 +/- 0.18 in normoxic, as compared to 1.53 +/- 0.16 in hypoxic (p < 0.05 vs. Nx). PCr (mumol/g brain) level was 4.03 +/- 0.11 in the normoxic and 1.1 +/- 0.3 in the hypoxic brain (p < 0.05 vs. Nx). In the normoxic preparations, active caspase-9 density increased by 9, 4 and 20% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. In the hypoxic preparations, active caspase-9 density increased by 30, 45 and 60% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. These results show that incubation with ATP, cytochrome c and ATP+cytochrome c result in a significantly increased activation of caspase-9 in the hypoxic group (p < 0.05). We conclude that the ATP and cytochrome c dependent activation of caspase-9 is increased during hypoxia. We propose that the ATP and cytochrome c sites of apoptotic protease activating factor I that mediate caspase-9 activation are modified during hypoxia.
在先前的研究中，我们已经表明，新生仔猪大脑皮层的胞质部分中，脑缺氧会导致起始半胱天冬酶-9和半胱天冬酶-3的活性增加。本研究探讨了缺氧过程中半胱天冬酶-9激活的机制，并验证了新生仔猪大脑皮层胞质中，半胱天冬酶-9依赖ATP和细胞色素c的激活增加这一假说。将新生仔猪分为常氧组（Nx，n = 4）和缺氧组（Hx，n = 4）。对麻醉并通气的动物分别给予0.21（Nx）或0.07（Hx）的吸入氧分数（FiO₂）60分钟。通过测定ATP和磷酸肌酸（PCr）水平，以生化方法记录脑组织缺氧情况。分离出胞质部分并通过G25-葡聚糖凝胶柱以去除内源性ATP和细胞色素c。收集各组分，并用紫外分光光度法在280nm处测定蛋白质含量。将常氧和缺氧动物洗脱的高分子量样品分为四个亚组：亚组1（对照），不添加ATP和细胞色素c进行孵育；亚组2，添加ATP进行孵育；亚组3，添加细胞色素c进行孵育；亚组4，添加ATP和细胞色素c进行孵育。孵育在37℃下进行30分钟。孵育后，通过12% SDS-PAGE分离蛋白质，并用特异性活性半胱天冬酶-9抗体检测活性半胱天冬酶-9。通过增强化学发光检测蛋白条带。通过图像密度测定法测定蛋白质密度，并表示为吸光度（OD×mm²）。常氧时ATP（μmol/g脑）水平为4.7±0.18，而缺氧时为1.53±0.16（与Nx相比，p < 0.05）。常氧时PCr（μmol/g脑）水平为4.03±0.11，缺氧脑为1.1±0.3（与Nx相比，p < 0.05）。在常氧制剂中，在ATP、细胞色素c和ATP +细胞色素c存在的情况下，活性半胱天冬酶-9密度分别增加了9%、4%和20%。在缺氧制剂中，在ATP、细胞色素c和ATP +细胞色素c存在的情况下，活性半胱天冬酶-9密度分别增加了30%、45%和60%。这些结果表明，在缺氧组中，与ATP、细胞色素c和ATP +细胞色素c孵育会导致半胱天冬酶-9的激活显著增加（p < 0.05）。我们得出结论，在缺氧过程中，半胱天冬酶-9依赖ATP和细胞色素c的激活增加。我们提出，介导半胱天冬酶-9激活的凋亡蛋白酶激活因子I的ATP和细胞色素c位点在缺氧过程中发生了改变。
