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

• Published in: Molecular pharmacology 1999-10, Vol.56 (4), p.824
• Main Authors: László Virág, Gwen S. Scott, Péter Antal-Szalmás, Michael O’Connor, Hiroshi Ohshima, Csaba SzabÃ
• Format: Article
• Language: English
• Subjects: Animals; Biological Transport - drug effects; Calcium - metabolism; Caspase 3; Caspases - metabolism; Cell Division - drug effects; Chelating Agents - pharmacology; DNA Damage; DNA Fragmentation - drug effects; Drug Interactions; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Enzyme Activation; Female; In Vitro Techniques; Male; Mice; Mitochondria - drug effects; Mitochondria - physiology; Nitrates - pharmacology; Nitrates - toxicity; Oxidants - pharmacology; Phosphatidylserines - metabolism; Poly(ADP-ribose) Polymerases - metabolism; Thymus Gland - drug effects; Thymus Gland - metabolism
• ISSN: 0026-895X; 1521-0111

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) 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.