Viráģ L, Scott G S, Antal-Szalmás P, O'Connor M, Ohshima H, Szabó C
Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio, USA.
Mol Pharmacol. 1999 Oct;56(4):824-33.
Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.
过氧亚硝酸盐是在休克、缺血再灌注和炎症过程中产生的一种细胞毒性氧化剂。介导过氧亚硝酸盐细胞毒性作用的细胞事件包括聚（ADP-核糖）合成酶的激活、线粒体呼吸的抑制以及半胱天冬酶-3的激活。本研究的目的是探讨细胞内钙动员在过氧亚硝酸盐诱导的坏死和凋亡性细胞死亡中的作用。过氧亚硝酸盐在低浓度、与病理生理相关的浓度（20微摩尔）下，可诱导胸腺细胞快速（1至3分钟）的Ca(2+)动员。细胞可渗透的Ca(2+)螯合剂[乙二醇双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸-乙酰氧甲酯（EGTA-AM）、1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸-乙酰氧甲酯（BAPTA-AM）、8-氨基-2-[(2-氨基-5-甲基苯氧基)甲基]-6-甲氧基喹啉-N,N,N',N'-四乙酸-四乙酰氧甲酯]对这种早期钙信号的抑制消除了通过碘化丙啶摄取测量的细胞毒性。细胞内Ca(2+)螯合剂还抑制了DNA单链断裂和聚（ADP-核糖）合成酶（PARS）的激活，在当前模型中，PARS是细胞坏死的主要介质。细胞内Ca(2+)螯合剂还保护PARS缺陷型胸腺细胞免受过氧亚硝酸盐的细胞毒性，为一条不依赖PARS、依赖Ca(2+)的细胞毒性途径提供了证据。细胞内Ca(2+)的螯合阻断了过氧亚硝酸盐诱导的线粒体膜电位降低、继发性超氧化物产生和线粒体膜损伤。在野生型细胞中，BAPTA-AM预处理后过氧亚硝酸盐诱导的核小体间DNA裂解增加，但在PARS缺陷型细胞中减少。在野生型和PARS缺陷型胸腺细胞中，BAPTA-AM均抑制了另外两个凋亡参数（磷脂酰丝氨酸暴露和半胱天冬酶3激活）。我们的研究结果为早期Ca(2+)信号在过氧亚硝酸盐细胞毒性中的关键作用提供了证据。
