The long-term objectives of this proposal are a better understanding of the hepatocellular damage that occasionally follows anesthesia with the volatile anesthetics. A new experimental approach is proposed that relates hepatocellular damage by halothane, enflurane and isoflurane to disturbances in intracellular Ca++ homeostasis. Changes in intracellular Ca++ concentration have been associated with cell damage in a number of model systems of drug toxicity. Current theories of the consequences of increasing intracellular Ca++ predict that such increases lead to cellular responses which ultimately are lethal to the cell. The first hypothesis we will examine is that the volatile anesthetics alter the Ca++ homeostasis of hepatocytes. We have determined that halothane, enflurane and isoflurane increase both phosphorylase a content and pholpholipase A2 activity in hepatocytes and in the presence of the volatile anesthetics 1) adrenergic receptor activity will be determined, 2) the bioluminescent indictor, aequorin, and the fluorescent indicator, fura-2, will be used to quantitate the changes of intracellular Ca++ concentration, 3) 45Ca++ will be used to measure Ca++ fluxes and intracellular Ca++ pools. A second hypothesis we will examine is that physiological factors may alter the effects of the volatile anesthetics on intracellular Ca++ homeostasis. Phenobarbital pretreatment to induce microsomal enzyme and hypoxia during treatment with the volatile anesthetics are required to produce hepatotoxicity in rats. The effect of microsomal enzyme induction using either phenobarbital or PCN singly and combined with the volatile anesthetics, will be studied on 1) adrenergic receptor activity and CA++ fluxes and intracellular Ca++ pools, 2) phosphokinase stimulation of cytosolic Ca++ increase, 3) effects on reaction directly related to increase cytosolic Ca++ which include the conversion on xanthine dehydrogenase to xanthine oxidase by Ca++-sensitive protease, the formation of hydroxyl radicals from xanthine oxidase-generated hydrogen peroxide and the oxidation of glutathione. All studies will be conducted in whole rat hepatocytes or subcellular fractions.
