Xu Zhelong, Ji Xiang, Boysen Philip G
Department of Anesthesiology, University of North Carolina, Chapel Hill, NC 27599-7010, USA.
Am J Physiol Heart Circ Physiol. 2004 Apr;286(4):H1433-40. doi: 10.1152/ajpheart.00882.2003. Epub 2003 Dec 4.
We examined whether cGMP-dependent protein kinase (PKG) and mitochondrial ATP-sensitive potassium (K(ATP)) channels are involved in S-nitroso-N-acetyl penicillamine (SNAP)-induced reactive oxygen species (ROS) generation. SNAP significantly increased ROS generation in cardiomyocytes. This increase was suppressed by both 5-hydroxydecanoate (5-HD) and glibenclamide. Direct opening of mitochondrial K(ATP) channels with diazoxide led to ROS generation. The increased ROS generation was reversed by N-(2-mercaptopropionyl)glycine (MPG), a scavenger of ROS. Myxothiazol partially suppressed the ROS generation. KT-5823, an inhibitor of PKG, prevented ROS generation, indicating that PKG is required for ROS generation. In addition, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), an activator of PKG, induced ROS generation. The effect of 8-BrcGMP was reversed by either 5-HD or MPG. YC-1, an activator of guanylyl cyclase, also increased ROS production, which was reversed by 5-HD. Neither LY-294002 nor wortmannin, the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), affected SNAP's action. In a whole heart study, SNAP significantly reduced infarct size. The anti-infarct effect of SNAP was abrogated by either MPG or 5-HD. This effect was also blocked by PD-98059, an ERK inhibitor, but not by LY-294002. A Western blotting study showed that SNAP significantly enhanced phosphorylation of ERK, which was reversed by MPG. These results suggest that SNAP-induced ROS generation is mediated by activation of PKG and mitochondrial K(ATP) channels and that opening of mitochondrial K(ATP) channels is the downstream event of PKG activation. ROS and mitochondrial K(ATP) channels participate in the anti-infarct effect of SNAP. Moreover, phosphorylation of ERK is the downstream signaling event of ROS and plays a role in the cardioprotection of SNAP.
我们研究了环磷酸鸟苷依赖性蛋白激酶（PKG）和线粒体ATP敏感性钾通道（K(ATP)）是否参与了亚硝基谷胱甘肽（SNAP）诱导的活性氧（ROS）生成。SNAP显著增加了心肌细胞中的ROS生成。5-羟基癸酸（5-HD）和格列本脲均可抑制这种增加。用二氮嗪直接开放线粒体K(ATP)通道可导致ROS生成。增加的ROS生成可被ROS清除剂N-(2-巯基丙酰基)甘氨酸（MPG）逆转。黏噻唑部分抑制了ROS生成。PKG抑制剂KT-5823可阻止ROS生成，表明PKG是ROS生成所必需的。此外，PKG激活剂8-溴鸟苷3',5'-环一磷酸（8-BrcGMP）可诱导ROS生成。8-BrcGMP的作用可被5-HD或MPG逆转。鸟苷酸环化酶激活剂YC-1也增加了ROS生成，这一作用可被5-HD逆转。磷脂酰肌醇3激酶（PI3激酶）抑制剂LY-294002和渥曼青霉素均不影响SNAP的作用。在一项全心研究中，SNAP显著减小了梗死面积。MPG或5-HD均可消除SNAP的抗梗死作用。ERK抑制剂PD-98059也可阻断这一作用，但LY-294002不能。蛋白质印迹研究表明，SNAP显著增强了ERK的磷酸化，这一作用可被MPG逆转。这些结果表明，SNAP诱导的ROS生成是由PKG和线粒体K(ATP)通道的激活介导的，线粒体K(ATP)通道的开放是PKG激活的下游事件。ROS和线粒体K(ATP)通道参与了SNAP的抗梗死作用。此外，ERK的磷酸化是ROS的下游信号事件，在SNAP的心脏保护作用中发挥作用。
