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 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 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.