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Molecular Pharmacology

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Research ArticleArticle

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
Molecular Pharmacology October 1999, 56 (4) 824-833;
László Virág
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Gwen S. Scott
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Péter Antal-Szalmás
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Michael O’Connor
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Hiroshi Ohshima
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Csaba Szabó
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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.

Footnotes

  • Send reprint requests to: Dr. Csaba Szabó, Inotek Corporation, Suite 419E, 100 Cummings Ctr., Beverly, MA 01915. E-mail:szabocsaba{at}aol.com

  • This work was supported by National Institutes of Health Grant R01-GM58781 (C.S.).

  • Part of the current study was presented at the annual meeting of the European Shock Society in La Hulpe, Belgium, October 1998.

  • Abbreviations:
    PARS
    poly(ADP-ribose) synthase
    SSB
    single-strand breaks
    BAPTA-AM
    1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester
    Quin-2-AM
    8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetic acid-tetraacetoxymethyl ester
    DiOC6(3)
    3,3′-dihexyl-oxacarbocyanine iodide
    NAO
    nonyl-acridine orange
    DEVD
    Asp-Glu-Val-Asp
    AMC
    amino-4-methylcoumarine
    FITC
    fluorescein isothiocyanate
    TMB
    8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate
    BHQ
    2,5-di-(tert-butyl)-1,4-benzohydroquinone
    [Ca2+]i
    intracellular calcium concentration
    Br-A23187
    calcimycin
    PI
    propidium iodide
    TCA
    trichloroacetic acid
    • Received November 2, 1998.
    • Accepted July 13, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 56 (4)
Molecular Pharmacology
Vol. 56, Issue 4
1 Oct 1999
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Research ArticleArticle

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
Molecular Pharmacology October 1, 1999, 56 (4) 824-833;

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Research ArticleArticle

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
Molecular Pharmacology October 1, 1999, 56 (4) 824-833;
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