Mitochondrial glutathione status during Ca2+ ionophore-induced injury to isolated hepatocytes.
In this study the Ca2+ ionophore, A23187, was used to determine the effects of disrupted Ca2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca2+ and A23187 to induce accumulation or loss of cellular Ca2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca2+, the antioxidants alpha-tocopherol and diphenyl-p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca2+. In the presence of extracellular Ca2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca2+. Leupeptin, an inhibitor of Ca2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of cellular Ca2+ homeostasis.
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010655:Phenylenediamines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism
D010655:Phenylenediamines; D013438:Sulfhydryl Compounds; D000001:Calcimycin; C004845:N,N'-diphenyl-4-phenylenediamine; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D002118:Calcium