Kim M-Y, Kim M J, Yoon I S, Ahn J H, Lee S H, Baik E J, Moon C-H, Jung Y-S
Department of Physiology, School of Medicine, Ajou University, Suwon, South Korea.
Br J Pharmacol. 2006 Dec;149(8):1059-70. doi: 10.1038/sj.bjp.0706922. Epub 2006 Oct 16.
Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection.
In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay.
Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD.
Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.
二氮嗪是一种著名的线粒体ATP敏感性钾通道（mitoK(ATP)）开放剂，已被证明可通过mitoK(ATP)通道和蛋白激酶C（PKC）对缺血性损伤发挥心脏保护作用。我们旨在阐明PKC亚型的作用以及PKC亚型与mitoK(ATP)通道在二氮嗪诱导的心脏保护中的关系。
在H9c2细胞和新生大鼠心肌细胞中，通过蛋白质印迹法和激酶测定检测PKC-ε的激活情况。通过共聚焦显微镜测量黄素蛋白荧光、线粒体Ca(2+)和线粒体膜电位。通过TUNEL测定法确定细胞死亡情况。
二氮嗪（100μM）诱导PKC-ε从胞质向线粒体部分转位。用εV1-2或显性负性突变体PKC-ε（PKC-εKR）特异性阻断PKC-ε可消除二氮嗪的抗凋亡作用。εV1-2或PKC-εKR转染可抑制二氮嗪诱导的黄素蛋白氧化。用5-羟基癸酸（5-HD）处理不影响二氮嗪诱导的PKC-ε转位和激活。野生型PKC-ε转染模拟了二氮嗪的黄素蛋白氧化作用，且该作用被εV1-2或5-HD完全阻断。二氮嗪可防止缺氧诱导的线粒体Ca(2+)增加、线粒体去极化和细胞色素c释放，二氮嗪的所有这些作用均被εV1-2或5-HD阻断。
二氮嗪诱导PKC-ε亚型特异性转位，作为mitoK(ATP)通道的上游信号分子，通过抑制线粒体死亡途径使心肌细胞对缺氧损伤具有抗性。
