PARS and peroxynitrite induced cell death

描述（由申请人提供）：过氧亚硝酸盐是一种有效的一氧化氮衍生氧化剂，是炎症、缺血再灌注损伤和休克的重要介质。过氧亚硝酸盐可引起细胞凋亡和高浓度的坏死细胞死亡。后一过程涉及核酶聚（adp -核糖）合成酶/聚合酶（PARS或PARP）的激活，该过程在DNA损伤的反应中被激活，并通过其底物NAD和ATP的消耗导致坏死。在之前的资助周期中，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，并证明了PARS在这一过程中的核心作用。在其他机制中，我们已经获得的数据表明，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。在目前的后续项目中，我们将进一步详细研究过氧亚硝酸盐细胞毒性的一些关键方面。具体目标：#1。探讨钙信号在过氧亚硝酸盐诱导细胞毒性中的作用机制。我们的初步数据表明，过氧亚硝酸盐触发Cai++的快速上升，抑制其（通过细胞渗透性Cai++螯合剂）减少过氧亚硝酸盐引起的PARS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。探讨细胞内钙信号在过氧亚硝酸盐诱导的DNA单链断裂和PARS激活中的作用。我们将研究过氧亚硝酸盐是直接还是通过钙依赖的线粒体形成次生自由基导致DNA单链断裂，以及钙信号如何参与PARS激活。# 3。建立蛋白激酶C （PKC）在过氧亚硝酸盐诱导的细胞毒性调控中的作用，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PKC同工酶参与过氧亚硝酸盐诱导的细胞死亡和PARS活性的调节。#。4目的探讨Bel家族蛋白在过氧亚硝酸盐诱导细胞死亡过程中钙稳态调控中的作用。这里提出的研究具有广泛的病理生理学相关性。

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.