A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

Oxidative stress releases intracellular calcium, which plays a pathogenic role in mammalian cell death. Here we report a search for the source of oxidative calcium in HeLa cells based on confocal epifluorescence microscopy. H₂O₂ caused a rapid increase in cytosolic calcium, which was followed by mitochondrial Ca²⁺ loading. Combined mitochondrial uncoupling with full depletion of thapsigargin-sensitive stores abrogated inositol 1,4,5-trisphosphate-mediated calcium release but failed to inhibit H₂O₂-induced calcium release, observation that was confirmed in MDCK cells. Prevention of peroxide-induced acidification with a pH clamp was also ineffective, discarding a role for endosomal/lysosomal Ca²⁺/H⁺ exchange. Lysosomal integrity was not affected by H₂O₂. Mature human erythrocytes also reacted to peroxide by releasing intracellular calcium, thus directly demonstrating the cytosolic source. Glutathione depletion markedly sensitized cells to H₂O₂, an effect opposite to that achieved by DTT. Iron chelation was ineffective. In summary, our results show the existence of a previously unrecognized sulfhydrylsensitive source of pathogenic calcium in the cytosol of mammalian cells.