Liu Huiping, Zhang Hong Yan, Zhu Xiangdong, Shao Zuohui, Yao Zhenhai
Department of Anesthesiology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Am J Physiol Heart Circ Physiol. 2002 Apr;282(4):H1380-6. doi: 10.1152/ajpheart.00348.2001.
The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels and the protein kinase C epsilon-isoform (PKC-epsilon) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 +/- 3% apoptosis (n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 +/- 4%, n = 8). The selective mitochondrial K(ATP) channel opener diazoxide (400 micromol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 +/- 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 micromol/l), a selective mitochondrial K(ATP) channel blocker, abolished preconditioning (42 +/- 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors Gö-6976 (0.1 micromol/l) or chelerythrine (4 micromol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-epsilon in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC delta-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 micromol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial K(ATP) channels blocks cardiocyte apoptosis via activating PKC-epsilon in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.
本研究的目的是确定预处理是否能阻止心肌细胞凋亡，并确定线粒体ATP敏感性钾（K(ATP)）通道和蛋白激酶Cε亚型（PKC-ε）在此效应中的作用。使用了10日龄鸡胚的心室肌细胞。在对照系列中，10小时的模拟缺血后再进行12小时的复氧导致42±3%的细胞凋亡（n = 8）。这些结果与DNA梯状条带和TdT介导的dUTP缺口末端标记（TUNEL）分析结果一致。在进行长时间模拟缺血之前，通过三个1分钟缺血周期并间隔5分钟复氧诱导预处理，可显著减轻凋亡过程（28±4%，n = 8）。在缺血前给予选择性线粒体K(ATP)通道开放剂二氮嗪（400 μmol/L），可模拟预处理效应以预防细胞凋亡（22±4%，n = 6）。用选择性线粒体K(ATP)通道阻滞剂5-羟基癸酸（100 μmol/L）预处理可消除预处理作用（42±2%，n = 6）。此外，在模拟缺血和复氧时给予特异性PKC抑制剂Gö-6976（0.1 μmol/L）或白屈菜红碱（4 μmol/L），可阻断预处理和二氮嗪的作用。此外，预处理和二氮嗪在模拟缺血前选择性激活颗粒部分中的PKC-ε，而对总部分、胞质部分和PKCδ亚型无影响。在模拟缺血和复氧期间添加特异性PKC激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯（0.2 μmol/L），可模拟预处理以阻断细胞凋亡。开放线粒体K(ATP)通道通过激活培养的心室肌细胞中的PKC-ε来阻止心肌细胞凋亡。通过这种信号转导，预处理可在缺血再灌注时阻断细胞凋亡并保护心脏功能。
