Mitochondrial glutathione status during Ca 2+ ionophore-induced injury to isolated hepatocytes

Abstract

In this study the Ca 2+ ionophore, A23187, was used to determine the effects of disrupted Ca 2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca 2+ and A23187 to induce accumulation or loss of cellular Ca 2+. 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 Ca 2+. 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 Ca 2+, the antioxidants α-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 Ca 2+. In the presence of extracellular Ca 2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca 2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca 2+. Leupeptin, an inhibitor of Ca 2+-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 Ca 2+ homeostasis.