Ogbi Mourad, Johnson John A
Department of Pharmacology and Toxicology, School of Medicine and the Program in Regenerative Medicine, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2300, USA.
Biochem J. 2006 Jan 1;393(Pt 1):191-9. doi: 10.1042/BJ20050757.
We have previously identified a phorbol ester-induced PKCepsilon (protein kinase Cepsilon) interaction with the ( approximately 18 kDa) COIV [CO (cytochrome c oxidase) subunit IV] in NCMs (neonatal cardiac myocytes). Since PKCepsilon has been implicated as a key mediator of cardiac PC (preconditioning), we examined whether hypoxic PC could induce PKCepsilon-COIV interactions. Similar to our recent study with phorbol esters [Ogbi, Chew, Pohl, Stuchlik, Ogbi and Johnson (2004) Biochem. J. 382, 923-932], we observed a time-dependent increase in the in vitro phosphorylation of an approx. 18 kDa protein in particulate cell fractions isolated from NCMs subjected to 1-60 min of hypoxia. Introduction of a PKCepsilon-selective translocation inhibitor into cells attenuated this in vitro phosphorylation. Furthermore, when mitochondria isolated from NCMs exposed to 30 min of hypoxia were subjected to immunoprecipitation analyses using PKCepsilon-selective antisera, we observed an 11.1-fold increase in PKCepsilon-COIV co-precipitation. In addition, we observed up to 4-fold increases in CO activity after brief NCM hypoxia exposures that were also attenuated by introducing a PKCepsilon-selective translocation inhibitor into the cells. Finally, in Western-blot analyses, we observed a >2-fold PC-induced protection of COIV levels after 9 h index hypoxia. Our studies suggest that a PKCepsilon-COIV interaction and an enhancement of CO activity occur in NCM hypoxic PC. We therefore propose novel mechanisms of PKCepsilon-mediated PC involving enhanced energetics, decreased mitochondrial reactive oxygen species production and the preservation of COIV levels.
我们之前已经确定，在新生心肌细胞（NCMs）中，佛波酯可诱导蛋白激酶Cε（PKCε）与（约18 kDa的）细胞色素c氧化酶亚基IV（COIV）相互作用。由于PKCε被认为是心脏预处理（PC）的关键介质，我们研究了低氧预处理是否能诱导PKCε-COIV相互作用。与我们最近关于佛波酯的研究[Ogbi、Chew、Pohl、Stuchlik、Ogbi和Johnson（2004年），《生物化学杂志》382卷，923 - 932页]相似，我们观察到，在经历1 - 60分钟低氧的NCMs分离出的颗粒细胞组分中，一种约18 kDa蛋白质的体外磷酸化呈时间依赖性增加。向细胞中引入PKCε选择性转位抑制剂可减弱这种体外磷酸化。此外，当对暴露于30分钟低氧的NCMs分离出的线粒体进行使用PKCε选择性抗血清的免疫沉淀分析时，我们观察到PKCε-COIV共沉淀增加了11.1倍。另外，我们观察到，在对NCMs进行短暂低氧暴露后，细胞色素c氧化酶（CO）活性增加了4倍，而向细胞中引入PKCε选择性转位抑制剂也可减弱这种增加。最后，在蛋白质免疫印迹分析中，我们观察到在9小时的低氧预处理后，预处理诱导COIV水平的保护作用增加了2倍以上。我们的研究表明，在NCMs低氧预处理中发生了PKCε-COIV相互作用以及CO活性增强。因此，我们提出了PKCε介导的预处理的新机制，包括能量增强、线粒体活性氧产生减少以及COIV水平的维持。
