Vol. 56, Issue 4, 824-833, October 1999

Requirement of Intracellular Calcium Mobilization for Peroxynitrite-Induced Poly(ADP-Ribose) Synthetase Activation and Cytotoxicity

László Virág, Gwen S. Scott, Péter Antal-Szalmás, Michael O'Connor, Hiroshi Ohshima, and Csaba Szabó

Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio (L.V., G.S.S., M.O., C.S. ); Department of Medical Chemistry (L.V.) and Third Department of Internal Medicine (P.A.), University Medical School of Debrecen, Debrecen, Hungary; International Agency for Research on Cancer, Unit of Endogenous Cancer Risk Factors, Lyon, France (H.O.); and Inotek Corporation, Beverly, Massachusetts (C.S.)

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 µM), induces rapid (1 to 3 min) Ca²⁺ mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca²⁺-dependent cytotoxic pathway. Chelation of intracellular Ca²⁺ 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 Ca²⁺ signaling in peroxynitrite cytotoxicity.