Cyclic GMP-dependent protein kinase Iα attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte
Cyclic GMP-dependent protein kinase Iα attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte
复制标题
DOI：
10.1074/jbc.m606142200
复制
发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Kukreja, Rakesh C.
中科院分区：
文献类型：
--
作者：
Das, Anindita;Smolenski, Albert;Kukreja, Rakesh C.
环gmp依赖性蛋白激酶蛋白激酶G (PKG) I α和PKGI β是心血管系统中cGMP信号传导的主要介质。PKGI α存在于心脏中，尽管其在防止缺血/再灌注损伤中的作用尚不清楚。我们研究了PKGI α对心肌细胞模拟缺血（SI）和再氧化（RO）后坏死和凋亡的直接作用。用含有hPKGI α或催化失活突变体hPKGI α K390A的腺病毒载体感染成年大鼠心肌细胞。24 h后，细胞接受90 min的SI和2h的RO进行坏死（台盼蓝排除和乳酸脱氢酶释放）或18 h的RO进行凋亡研究。为了评估K-ATP通道的作用，在SI前分别用5-羟基乙酸酯（100 μ M）、HMR1098 （30 μ M）或格列本脲（50 μ M）处理细胞亚组，分别是线粒体、肌层或两种K-ATP通道的阻滞剂。SI-RO对照组心肌细胞坏死率为33.7 +/- 1.6%，经PKGI α处理后减少至18.6 +/- 0.8%（平均+/- S.E, n = 7, p < 0.001）。PKGI α使SI-RO对照组17.9 +/- 1.3%的总肌细胞凋亡率降至6.0 +/- 0.6%（平均+/-标准差，n = 7, p < 0.001）。此外，PKGI α抑制肌细胞SI-RO后caspase-3的激活。感染失活PKGI α K390A突变体的肌细胞没有表现出保护作用。PKGI α增强Akt、ERK1/2和JNK的磷酸化，增加bcl -2、诱导一氧化氮合酶、内皮一氧化氮合酶的表达，降低Bax的表达。5-羟基癸酸酯和格列本脲可消除PKGI α介导的对坏死和细胞凋亡的保护作用。然而，HMR1098没有效果。一种活性氧清除剂，以及磷脂酰肌醇3-激酶、ERK、JNK1和NOS的抑制剂，也阻断PKGI α介导的对坏死和凋亡的保护。这些结果表明，线粒体KATP通道的打开和活性氧的产生，与Akt、ERK和JNK的磷酸化以及NOS和Bcl-2的表达增加有关，在PKGI α的保护作用中发挥了重要作用。
Cyclic GMP-dependent protein kinases protein kinase G (PKG) I alpha and PKGI beta are major mediators of cGMP signaling in the cardiovascular system. PKGI alpha is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGI alpha against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGI alpha or catalytically inactive mutant hPKGI alpha K390A. After 24 h, the cells were subjected to 90 min of SI and 2h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of K-ATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 mu M), HMR1098 (30 mu M), or glibenclamide (50 mu M), the respective blockers of mitochondrial, sarcolemmal, or both types of K-ATP channels prior to SI. The necrosis observed in 33.7 +/- 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 +/- 0.8% by PKGI alpha (mean +/- S.E., n = 7, p < 0.001). Theapoptosisobservedin 17.9 +/- 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 +/- 0.6% by PKGI alpha (mean +/- S.E., n = 7, p < 0.001). In addition, PKGI alpha inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGI alpha K390A mutant showed no protection. PKGI alpha enhanced phosphorylation of Akt, ERK1/2, and JNK, increasedBcl-2, induciblenitric-oxidesynthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGI alpha-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGI alpha-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGI alpha.