Requirement of Intracellular Calcium Mobilization for Peroxynitrite-Induced Poly(ADP-Ribose) Synthetase Activation and Cytotoxicity
- László Virág1,2,
- Gwen S. Scott1,
- Péter Antal-Szalmás3,
- Michael O’Connor1,
- Hiroshi Ohshima4 and
- Csaba Szabó1,5
- 1Division of Critical Care Medicine, Children’s Hospital Medical Center, Cincinnati, Ohio (L.V., G.S.S., M.O., C.S. ); 2Department of Medical Chemistry (L.V.) and 3Third Department of Internal Medicine (P.A.), University Medical School of Debrecen, Debrecen, Hungary;4International Agency for Research on Cancer, Unit of Endogenous Cancer Risk Factors, Lyon, France (H.O.); and 5Inotek Corporation, Beverly, Massachusetts (C.S.)
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
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Send reprint requests to: Dr. Csaba Szabó, Inotek Corporation, Suite 419E, 100 Cummings Ctr., Beverly, MA 01915. E-mail:szabocsaba{at}aol.com
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This work was supported by National Institutes of Health Grant R01-GM58781 (C.S.).
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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
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- Received November 2, 1998.
- Accepted July 13, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
