Ca2+-dependent cytotoxicity of H2O2 in L929 cells: the role of H2O2-induced Na+-influx.
We investigated the mechanism by which H2O2 mediates an increase in [Na+]i in L929 cells and the relevance of this Na+ load for H2O2-induced cell injury. [Na+]i increased early after exposure to H2O2 as monitored by fluorescence spectrophotometry of cells loaded with SBFI. The omission of Na+ from the incubation buffer significantly reduced H2O2-cytotoxicity. This protection could not be mimicked by inhibition of either the Na+/H+-antiporter, the Na+/HCO3- -cotransporter, or the Na+/K+/2Cl- -cotransporter by using Hoechst 694 (0.02 mM) or 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) (0.02 mM) or furosemide (1 mM) and bumetanide (0.5 mM). Only the blocker of the Na+/Ca2+-exchanger bepridil (0.2 mM) significantly reduced H2O2-cytotoxicity but without interfering with the increase in [Na+]i. H2O2 caused a rapid and sustained increase in [Ca2+]i, which was significantly reduced in bepridil pretreated cells and after replacing extracellular Na+ by choline. H2O2 was found to initiate a cellular uptake of unphysiological Ni2+ by using Newport Green diacetate as fluorescent dye. Our data suggest that H2O2 mediates Na+-influx across the plasma membrane rather unspecifically than through specific transporters. The protective effect of bepridil against H2O2-cytotoxicity occurs as a consequence of a reduced cellular Ca2+-uptake. We conclude that H2O2-mediated unspecific accumulation of Na+ seems to favor a Ca2+-influx into the cells, which takes place on the Na+/Ca2+-exchanger operating in reverse mode in exchange for Na+-efflux. Therefore, H2O2-induced cellular Na+ accumulation appears to play a permissive rather than a triggering role in H2O2-mediated cell injury.
Jussofie
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A
Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany.
Kirsch
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M
de Groot
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H
D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D015764:Bepridil / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity*; D051379:Mice; D009532:Nickel / Q000493:pharmacokinetics; D010042:Ouabain / Q000494:pharmacology; D010795:Phthalic Acids / Q000378:metabolism; D012964:Sodium / Q000493:pharmacokinetics*; D026941:Sodium Channel Blockers; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D005456:Fluorescent Dyes; D010795:Phthalic Acids; D026941:Sodium Channel Blockers; C103805:sodium binding benzofuran isophthalate acetoxymethyl ester; C049925:fura-2-am; D010042:Ouabain; D015764:Bepridil; D009532:Nickel; D012964:Sodium; D006861:Hydrogen Peroxide; D002118:Calcium; D016257:Fura-2