Gong Kai-zheng, Zhang Zhen-gang, Li Ai-hua, Huang Yi-feng, Bu Ping, Dong Feng, Liu Jian
Department of Cardiology, First People's Hospital of Yangzhou, Yangzhou 225001, China.
Chin Med J (Engl). 2004 Mar;117(3):395-400.
The activation of extracellular signal-regulated kinase1/2 (ERK1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mKATP) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mKATP and ERK1/2 in the delayed protection mechanism of anoxic preconditioning (APC).
An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopropionylglycine (MPG), and PD98059] were used to modulate mKATP and ERK1/2 activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mKATP opener).
Cell viability and SOD activity in the APC [(81.9 +/- 11.4)%, (13.6 +/- 3.7) U/L] and diazoxide [(79.2 +/- 12.4)%, (16.5 +/- 4.6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2 +/- 7.3)%, (8.8 +/- 2.8) U/L] group (all P < 0.01). LDH activity in the APC group [(101.9 +/- 18.9) U/L] and diazoxide group [(97.5 +/- 17.7) U/L] was significantly lower than in the A/R group [(250.5 +/- 43.6) U/L] (all P < 0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mKATP blocker) and by MPG (a free radical scavenger). In addition, the ERK1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide.
mKATP may mediate ERK1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.
细胞外信号调节激酶1/2（ERK1/2）的激活已被证明是缺血预处理（IPC）反应中的重要信号通路。最近，一些研究表明线粒体ATP敏感性钾通道（mKATP）作为IPC急性和延迟保护的触发因素及终效应器发挥关键作用。因此，本研究旨在阐明mKATP与ERK1/2在缺氧预处理（APC）延迟保护机制中的关系。
使用培养的新生大鼠心肌细胞建立APC模型。采用药理试剂[二氮嗪、5-羟基癸酸（5-HD）、2-巯基丙酰甘氨酸（MPG）和PD98059]调节mKATP和ERK1/2的激活。通过测量细胞超氧化物歧化酶（SOD）活性、细胞活力和乳酸脱氢酶（LDH）释放来评估细胞损伤。从缺氧或二氮嗪（一种mKATP开放剂）预处理开始后的不同时间点，测定细胞活性氧（ROS）的产生和ERK1/2的激活情况。
APC组[（81.9±11.4）%，（13.6±3.7）U/L]和二氮嗪组[（79.2±12.4）%，（16.5±4.6）U/L]的细胞活力和SOD活性显著高于缺氧/复氧（A/R）组[（42.2±7.3）%，（8.8±2.8）U/L]（均P<0.01）。APC组[（101.9±18.9）U/L]和二氮嗪组[（97.5±17.7）U/L]的LDH活性显著低于A/R组[（250.5±43.6）U/L]（均P<0.01）。APC和二氮嗪均同时促进细胞内ROS生成和ERK1/2的快速激活。但5-HP（一种mKATP阻滞剂）和MPG（一种自由基清除剂）可显著减弱APC和二氮嗪的作用。此外，ERK1/2抑制剂PD98059也消除了二氮嗪诱导的细胞保护作用。
mKATP可能通过产生活性氧介导缺氧预处理期间的ERK1/2激活，进而触发大鼠心肌细胞中APC的延迟保护作用。
