PARS and peroxynitrite induced cell death

说明(申请人提供)：过氧亚硝酸盐是一种有效的一氧化氮衍生的氧化剂，也是炎症、缺血再灌注损伤和休克的重要介质。过氧亚硝酸根既可引起细胞凋亡，也可在高浓度时引起坏死细胞死亡。后一个过程涉及核酶多聚(ADP-核糖)合成酶/聚合酶(PARP)的激活，该酶在DNA损伤时被激活，并通过耗尽其底物NAD从而导致ATP而导致坏死。在之前的资金周期中，我们已经确定了过氧亚硝酸盐介导的细胞死亡的几个关键方面，并展示了PARs在这一过程中的核心作用。在其他机制中，我们已经获得了数据，表明在过氧亚硝酸盐诱导的细胞毒性过程中，细胞钙稳态紊乱的作用。在目前的继续项目中，我们将进一步详细地研究过氧亚硝酸盐细胞毒性的一些关键方面。具体目的：#1.阐明钙信号在过氧亚硝酸盐诱导的细胞毒性中起作用的机制。我们的初步数据表明，过氧亚硝酸根触发了CaI++的快速升高，其抑制作用(通过细胞通透性的CaI+螯合剂)降低了过氧亚硝酸根引起的PARS的激活。我们建议进行研究，以确定过氧亚硝酸盐中毒过程中CaI++动员的来源。2.确定细胞内钙信号在过氧亚硝酸盐诱导的DNA单链断裂和PARS激活中的调节作用。我们将研究过氧亚硝酸根是否直接或通过次级自由基的钙依赖的线粒体形成导致DNA单链断裂，以及钙信号如何参与PARS的激活。#3.确定蛋白激酶C(PKC)在调节过氧亚硝酸盐诱导的细胞毒性中的作用，特别是在PARS激活方面。我们的初步数据表明，在胸腺细胞中，过氧亚硝酸盐诱导的PARS激活被PKC激活剂抑制。我们希望研究过氧亚硝酸盐是否激活PKC，以及哪些PKC同工酶参与了过氧亚硝酸盐诱导的细胞死亡和PARS活性的调节。#.4确定Bel家族蛋白在过氧亚硝酸盐诱导的细胞死亡过程中调节钙稳态的作用。本文提出的研究具有广泛的病理生理学意义。

DESCRIPTION (provided by applicant): Peroxynitrite is a potent NO-derived oxidant and important mediator of inflammation, ischemia- reperfusion injury and shock. Peroxynitrite can cause both apoptotic and at higher concentrations necrotic cell death. This latter process involves the activation of the nuclear enzyme poly (ADP-ribose) synthetase/polymerase (PARS or PARP) that becomes activated in response to DNA damage and contributes to necrosis via depletion of its substrate NAD and consequently of ATP. In the previous funding cycle, we have identified several key aspects of peroxynitrite-mediated cell death, and demonstrated the central role of PARS in this process. Among other mechanisms, we have obtained data demonstrating the role of disturbances in cellular calcium homeostasis during peroxynitrite-induced cytotoxicity. In the current continuation project, we will investigate, in further detail, some of the key aspects of peroxynitrite cytotoxicity. Specific aims: #1. To delineate the mechanisms by which calcium signaling plays a role in the peroxynitrite- induced cytotoxicity. Our preliminary data indicate that peroxynitrite triggers a rapid) rise of Cai++ , the inhibition of which (by cell- permeable Cai++ chelators) reduces peroxynitrite-elicited PARS activation. We propose studies, which will identify the sources of Cai++ mobilization during peroxynitrite toxicity. #2. To establish the role of intracellular calcium signals in the regulation of peroxynitrite-induced DNA single strand breakage and PARS activation. We will investigate whether peroxynitrite directly or through the calcium dependent mitochondrial formation of secondary radicals causes DNA single strand breakage and how calcium signals are involved in PARS activation. #3. To establish the role of protein kinase C (PKC) in the regulation of peroxynitrite-induced cytotoxicity with special regard to PARS activation. Our preliminary data indicate that in thymocytes, peroxynitrite-induced PARS activation is inhibited by PKC activators. We wish to investigate whether peroxynitrite activates PKC and what PKC isozymes are involved in the regulation of of peroxynitrite induced cell death and of PARS activity. #.4 To establish the role of Bel family proteins in the regulation of calcium homeostasis during peroxynitrite-induced cell death. The studies proposed here have broad pathophysiological relevance.