Graduate Thesis Or Dissertation
 

Mitochondrial glutathione and the effect of perturbed calcium homeostasis on rat hepatocyte thiols

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  • Recent studies have demonstrated that perturbations of intracellular thiol and calcium homeostasis may be important events in the early development of cell injury by toxic chemicals. Incubation of isolated rat hepatocytes in a calcium free medium, severely depleted intracellular Ca²⁺ levels and resulted in the loss of both cytosolic and mitochondrial glutathione (GSH), which preceded cell injury. Elevation of endogeneous a-tocopherol levels, by supplementing the hepatocyte suspension with vitamin E-succinate, inhibited the loss of GSH and reversed cell injury. Increased levels of GSH in the presence of vitamin E-succinate were induced by an apparent a-tocopherol-mediated effect on GSH biosynthesis, indicating a close relationship between these two important cellular antioxidant systems. Perturbation of intracellular calcium homeostasis in hepatocytes by the administration of A23187, a calcium ionophore, in the presence of different concentrations of extracellular Ca²⁺ , revealed a striking correlation between the loss of mitochondrial GSH and cell injury. The loss of mitochondrial GSH was preceded by a loss of cytosolic GSH, whereas protein thiol levels were not significantly affected until after the depletion of non-protein thiols. Lipid peroxidation did not have a clear association with cell injury, induced by A23187 in the presence of extracellular Ca ²⁺ . In the absence of extracellular Ca²⁺, antioxidants prevented A23187- induced cell injury and loss of mitochondrial GSH and thus dissociated the mobilization of intracellular Ca²⁺ from the expression of toxicity. In the presence of extracellular Ca²⁺, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. These results demonstrate that the level of mitochondrial GSH is critical for cell survival during calcium ionophore-induced perturbation of cellular calcium homeostasis. Incubation of isolated mitochondria with t-butylhydroperoxide revealed that oxidized glutathione is not transported out of hepatocyte mitochondria, as a response to oxidative stress. These results indicate greater susceptibility of protein thiols in mitochondria to oxidation than in the rest of the cell, and may explain the protective role of mitochondrial GSH in cell injury.
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