Free Radicals, PKC Delta Signal ACh Preconditioning

描述（由申请人提供）：单阶段或多阶段预处理 冠状动脉长时间缺血前短暂缺血再灌注 闭塞，减少心肌梗死面积和细胞凋亡。乙酰胆碱（ACh） 在体外和体内模拟预处理效应。细胞凋亡一直是 在许多与缺血再灌注相关的心脏疾病中发现 损伤这个提议的目的是阐明细胞内信号传导 ACh阻断心肌细胞凋亡和保护心脏功能的途径 功能蛋白激酶C（PKC）和线粒体ATP钾通道是信号传导途径， 预处理和ACh的组分。乙酰胆碱产生氧自由基， 与缺血-再灌注中的心脏保护相关。ACh影响冠状动脉 内皮细胞，循环血液元素，并激活一系列信号 心肌细胞中的转导级联，如激活PKC δ亚型。这 对心脏保护的作用仍不清楚。我们假设 打开线粒体KATP通道释放氧自由基， PKC δ亚型。通过这一信号级联，ACh阻断了心肌细胞的增殖， 在缺血-再灌注中保护心脏功能。为了验证这一 假设，我们将直接测量新生大鼠氧自由基产生 心肌细胞我们将使用重组腺病毒载体， 心肌细胞是一种携带全长cDNA的活性PKC 6亚型， （AdCMVPKCepsilon-FL）或显性负突变失活PKCS（AdCMVPKC delta-DN）。这些重组构建体对心肌细胞功能的影响， 细胞凋亡、梗死和氧化应激将在体外和 体内大鼠缺血-再灌注模型。对联合国作用的理解 ACh阻断心肌细胞凋亡的特异性信号通路， 通过体细胞基因转移对途径进行局部调节 可以为许多临床疾病的管理提供新的疗法。

DESCRIPTION (provided by applicant): Preconditioning single or multiple periods of brief ischemia and reperfusion before a prolonged period of coronary.artery occlusion, reduces myocardial infarct size and apoptosis. Acetylcholine (ACh) mimics preconditioning effects in vitro and in vivo. Apoptosis has been identified in many cardiac conditions associated with ischemia-reperfusion injury. The goal of this proposal is to elucidate the intracellular signaling pathways by which ACh blocks cardiocyte apoptosis and preserves cardiac function. Protein kinase C (PKC) and mitochondrial K ATP channels are signaling components of preconditioning and ACh. ACh generates oxygen radicals that correlate with cardioprotection in ischemia-reperfusion. ACh affects coronary endothelium, circulating blood elements, and activates a series of signal transduction cascades in cardiocytes such as activating PKCdelta isoform. Which effect accounts for cardioprotection remains unclear. We hypothesize that opening mitochondrial K ATP channels releases oxygen radicals that activate PKCdelta isoform. Through this signaling cascade, ACh blocks cardioeyte apoptosis and preserves cardiac function in ischemia-reperfusion. To test this hypothesis, we will measure oxygen radical production directly in neonatal rat cardiomyocytes. We will use recombinant adenoviral vectors to overexpress in cardiocytes an active PKC6 isoform carrying its full length of cDNA (AdCMVPKCepsilon-FL) or a dominant negative mutant inactive PKCS (AdCMVPKC delta-DN). The effects of these recombinant constructs on cardiocyte function, apoptosis, infarction, and oxidant stress will be examined in both in vitro and in vivo rat models of ischemia-reperfusion. Understanding of the role of specific signaling pathways by which ACh blocks cardiocyte apoptosis and development of local modulation of the pathways through somatic gene transfer may provide novel therapies for the management of many clinical disorders.