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, Csaba Szabó

Research output: Article

74 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)824-833
Number of pages10
JournalMolecular pharmacology
Volume56
Issue number4
Publication statusPublished - okt. 29 1999

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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