Abstract
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 sulfhydryl-sensitive source of pathogenic calcium in the cytosol of mammalian cells.
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Abbreviations
- ER:
-
endoplasmic reticulum
- InsP3:
-
inositol 1,4,5-trisphosphate
- BAPTA-AM:
-
1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester
- BSO:
-
L-buthionine-[S,R]-sulfoximine
- LDH:
-
lactate dehydrogenase
- KRH:
-
Krebs–Ringer–HEPES buffer
- Glc:
-
glucose
- BCECF:
-
2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
- TMRM:
-
tetramethylrhodamine methyl ester
- ROIs:
-
regions of interest
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Acknowledgements
We thank Ole Petersen and Sergio Grinstein for helpful discussions. We also thank Rafael Burgos for his aid with fluorimetry measurements and Steffen Haertel for his aid with image analysis. This work was funded by Fondecyt 1020648. Institutional support to the Centro de Estudios Científicos (CECS) from Empresas CMPC is gratefully acknowledged. CECS is a Millennium Science Institute and is funded in part by grants from Fundación Andes and the Tinker Foundation.
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Castro, J., Bittner, C., Humeres, A. et al. A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death. Cell Death Differ 11, 468–478 (2004). https://doi.org/10.1038/sj.cdd.4401372
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DOI: https://doi.org/10.1038/sj.cdd.4401372
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