Ca2+-dependent cytotoxicity of H2O2 in L929 cells: The role of H2O2-induced Na+-influx

  • Jussofie A
  • Kirsch M
  • De Groot H
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Abstract

We investigated the mechanism by which H2O2mediates 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 H2O2as 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). H2O2caused a rapid and sustained increase in [Ca2+](i), which was significantly reduced in bepridil pretreated cells and after replacing extracellular Na+by choline. H2O2was found to initiate a cellular uptake of unphysiological Ni2+by using Newport Green diacetate as fluorescent dye. Our data suggest that H2O2mediates 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.

Author-supplied keywords

  • Ca2+
  • Cytotoxicity
  • Hydrogen peroxide
  • Intracellular Na+

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Authors

  • Astrid Jussofie

  • Michael Kirsch

  • Herbert De Groot

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