Intracellular depletion of ATP and a decrease in intracellular pH are prominent features of anoxic and other types of cell injury, Although the effects of ATP depletion have drawn considerable attention, there is a lack of basic information regarding the effects of intracellular pH (pHi) in cell damage. Preliminary experiments by us in rat hepatocytes demonstrated that an acidic pHi developed during ATP depletion and protected against the onset of cell death. The overall objective of this proposal is to extend this observation by testing the hypothesis that (1) intracellular acidosis develops in all liver cells during anoxic, oxidative and hypoxic stress, and that (ii) an acidic pHi exerts a protective effect by inhibition of a specific, identifiable mechanism responsible for lethal cell injury.
The specific aims are:1) to establish individual models for cell injury in hepatocytes, endothelial, Kupffer and bile duct epithelial cells isolated from rat liver and to determine the influence of intracellular and extracellular pH on cell killing; 2) to determine pHi during basal conditions and cell injury; 3) to determine the contribution of plasma membrane transporters to pHi homeostasis during basal conditions and cell injury; 4) to determine the pH dependence of possible causative mechanisms responsible for cell death; and 5) to determine the effect of pHi on specific cell functions. These studies will employ cultures and suspensions of liver cells and utilize novel biochemical and fluorescent imaging techniques. Employing single, cultured cells, fluorescent probes and multiparameter digitized video microscopy, pHi, cytosolic free Ca+2, mitochondrial membrane potential, loss of cell viability, organic anion transport, rates of fluid phase and receptor-mediated endocytosis, and changes in cytosolic free Ca+2 mediated by hormonal stimulation will be determined during basal conditions and during cell injury. In dispersed cell suspensions, rates of cell killing, proteolysis, phospholipid hydrolysis, ATP depletion, intracellular H2O2 generation, lipid peroxidation and thiol depletion will be measured and the effect of pHi on these various processes evaluated. The results will provide fundamental new insight into the role of pHi in cell injury, permit identification of common and dissimiliar mechanisms culminating in cell death, and provide an understanding of hepatic dysfunction during liver injury. In addition, the results may lead to the identification of treatment modalities effective in the preservation of liver tissue during anoxic, oxidative and hypoxic insults.
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