Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity

Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperf usion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibi tion of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobi lization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 m u M), induces rapid (1 to 3 min) Ca2+ mobilization in thymocytes. Inhibitio n of this early calcium signaling by cell-permeable Ca2+ chelators [EGTA-ac etoxymethyl ester (AM), 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraaceti c acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy) methyl] 6-methox yquinoline- N, N, N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity a s measured by propidium iodide uptake. Intracellular Ca2+ chelators also in hibited DNA single-strand breakage and activation of poly(ADP-ribose) synth ase (PARS), which is a major mediator of cell necrosis in the current model . Intracellular Ca2+ chelators also protected PARS-deficient thymocytes fro m peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca 2+ dependent cytotoxic pathway. Chelation of intracellular Ca2+ blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, second ary superoxide production, and mitochondrial membrane damage. Peroxynitrite -induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatme nt in the wild-type cells but decreased in the PARS-deficient cells. Two ot her apoptotic parameters (phosphatidylserine exposure and caspase 3 activat ion) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficien t thymocytes. Our findings provide evidence for the pivotal role of an earl y Ca2+ signaling in peroxynitrite cytotoxicity.