The long-term objective of this proposal is to understand the mechanism of canalicular bile formation and drug-induced cholestasis. In order to achieve this objective the mechanism of hepatic electrolyte transport and the functional importance of biliary membane permeability in hepatic bile formation will be studied. The research plan will focus on the following hypotheses: 1) a sinusoidal sodium/hydrogen exchange coupled with a canalicular chloride/bicarbonate exchange may be involved in hepatic bicarbonate transport. 2) Hepatic uptake of bile acid may involve an exchange mechanism involving intercellular bicarbonate. 3) Amino acid reabsorption may be involved in fluid reabsorption at the level of canaliculi and/or interlobular ducts. 4) Bile acids at cholestatic doses may increase paracellular permeability by increasing intracellular calcium. Studies will be conducted using isolated perfused rat livers, isolated hepatocytes and plasma membrane vesicles. Established techniques will be used to study hepatic transport of sodium, chloride, calcium and bile acids. Electrolyte substitution and pharmacological agents (amiloride, FCCF, DIDS, NAP-taurine) known to modify specific transport systems will be used to investigate the role of sodium/hydrogen exchange in hepatic bicarbonate secretion and the possible presence of a bile acid/bicarbonate exchange mechanism. Hepatic bicarbonate transport will be further studied in the isolated guinea pig liver which is characterized by high bicarbonate secretion. Intracellular pH will be determined from the equilibrium distribution of radiolabelled dimethyloxazolidinedione and methylamine. A fluorescence technique using Quin-2 will be used to determine the effect of bile acid on intracellular calcium in isolated hepatocytes, and biliary permeability will be determined using standard probes and methods. In addition, new permeability markers will be synthesized in an effort to better estimate the relative contribution of transcellular and paracellular pathways in hepatic bile formation. Apart from increasing our understanding of the mechanism of bile formation and cholestasis, these studies, if successful, will further define the mechanism of hepatic electrolyte transport which is of considerable relevance to a variety of other hepatocellular functions.
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