Oldenburg Olaf, Yang Xi-Ming, Krieg Thomas, Garlid Keith D, Cohen Michael V, Grover Gary J, Downey James M
Department of Physiology, University of South Alabama, College of Medicine, Mobile, AL 36688, USA.
J Mol Cell Cardiol. 2003 Sep;35(9):1035-42. doi: 10.1016/s0022-2828(03)00151-2.
We have recently proposed that opening of mitochondrial K(ATP) channels (mitoK(ATP)) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective K(ATP) channel opener P1075 also opens mitoK(ATP) channels and may be cardioprotective. We purified mitoK(ATP) channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoK(ATP) channels from each of these tissues were opened by P1075 with EC(50) values of 60-90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a K(ATP)-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker Red was oxidized. P1075 (100 microM) led to a 44 +/- 9% increase in ROS generation (P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 microM diazoxide, a selective mitoK(ATP) channel opener (49 +/- 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 microM glibenclamide (GLI), a non-selective K(ATP) blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoK(ATP) blocker (-6 +/- 14% and +4 +/- 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 +/- 8.1% of the area at risk vs. 31.5 +/- 5.6%, P < 0.05). GLI (5 microM) as well as 5-hydroxydecanoate (200 microM) completely blocked P1075's anti-infarct effect (31.7 +/- 9.5% and 27.7 +/- 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoK(ATP) channels and protects the ischemic rabbit heart in a mitoK(ATP)-dependent manner.
我们最近提出，线粒体ATP敏感性钾通道（mitoK(ATP)）的开放通过促使线粒体产生活性氧（ROS）而成为预处理（PC）的触发因素。关于公认的肌膜选择性ATP敏感性钾通道开放剂P1075是否也能开放mitoK(ATP)通道以及是否具有心脏保护作用，目前存在争议。我们从兔心脏、大鼠心脏或大鼠脑中纯化了mitoK(ATP)通道，并将这些蛋白质重组到脂质体中。来自这些组织的mitoK(ATP)通道均被P1075开放，其半数有效浓度（EC(50)）值为60 - 90 nM。接下来，我们测试了P1075是否能以ATP敏感性钾通道（K(ATP)）依赖的方式使兔心肌细胞产生活性氧。通过还原型线粒体追踪红荧光被氧化时荧光的出现来监测线粒体活性氧的产生。P1075（100 microM）使活性氧生成增加了44±9%（与未处理细胞相比，P < 0.001），这与50 microM二氮嗪（一种选择性mitoK(ATP)通道开放剂）所引起的增加相似（49±9%，与未处理细胞相比，P < 0.001）。P1075在浓度为150 nM时效果同样显著。P1075诱导的活性氧生成增加被50 microM格列本脲（GLI，一种非选择性K(ATP)阻滞剂）和1 mM 5 - 羟基癸酸（一种高度选择性mitoK(ATP)阻滞剂）阻断（分别为 - 6±14%和 + 4±12%；P = 无显著性差异）。在离体兔心脏中，与对照动物相比，P1075（150 nM）显著减小了梗死面积（梗死面积占危险区域的10.6±8.1%，而对照为31.5±5.6%，P < 0.05）。GLI（5 microM）以及5 - 羟基癸酸（200 microM）完全阻断了P1075的抗梗死作用（梗死率分别为31.7±9.5%和27.7±4.6%；与未处理心脏相比，P = 无显著性差异）。这些数据提供了强有力的证据，表明P1075确实能开放mitoK(ATP)通道，并以mitoK(ATP)依赖的方式保护缺血的兔心脏。
