title: Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity.
abstract: 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.
authors.affiliation: Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio, USA.
authors.forename: L
authors.initials: L
authors.lastname: Viráģ
authors.forename: G S
authors.initials: GS
authors.lastname: Scott
authors.forename: P
authors.initials: P
authors.lastname: Antal-Szalmás
authors.forename: M
authors.initials: M
authors.lastname: O'Connor
authors.forename: H
authors.initials: H
authors.lastname: Ohshima
authors.forename: C
authors.initials: C
authors.lastname: Szabó
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009566:Nitrates / Q000494:pharmacology* / Q000633:toxicity; D016877:Oxidants / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013950:Thymus Gland / Q000187:drug effects / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D009566:Nitrates; D016877:Oxidants; D010718:Phosphatidylserines; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C039642:peroxynitric acid; D004533:Egtazic Acid; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D002118:Calcium