The objectives of this proposal are to: investigate the structure-function relations in bile acid induced increase in biliary permeability; and to define the structural requirements of the major bile acid transport mechanism - the sodium-dependent taurocholate uptake site - in the hepatocyte plasma membrane. The effect of different bile acids upon biliary permeability will be studied in Sprague-Dawley rats with choledocho-caval shunts. In this model, the common duct is connected via an exteriorized PE-50 cannula to the superior vena cava. This yields high stable bile acid fluxes for up to 48 hours. The model is especially suitable because small amounts of any bile acid suffice to produce a high stable bile acid flux through the liver at a rate not exceeding the maximum secretory rate. The model avoids the hazard of biliary failure from bile acid infusion above the maximum secretory rate, and avoids the prohibitive expense of constant infusions of costly pure bile acids. Permeability will be assessed one, two, and six hours after substitution of .25 to 1.0 pool of test bile acid for endogenous bile acid by determining the bile:plasma ratios of infused 14C sucrose and, in some cases, by electron microscopic detection of intravenously infused lanthanum in the canalicular spaces. Bile acid biotransformation will be monitored by high-pressure liquid chromatrography. Definition of the effects of various bile acids on biliary permeability will expand our knowledge of mechanisms of bile acid cholestasis, and should also help clarify the relationship between changes in biliary permeability and cholestasis in general. The proposal will continue to characterize the structural specificity of the sodium-dependent taurocholate uptake site using the principle of competitive inhibitiion of taurocholate uptake in freshly isolated rat hepatocytes. Most attention will be given to the effect of nonsterol chain length, the effect of hydroxy configuration (Alpha or Beta), and the effect of sterol nucleus substituent size upon ability of a compound to inhibit uptake of 5MuM 14C taurocholate into isolated hepatocytes. These investigations employ a number of custom-synthesized bile acid analogues. Such knowledge will help us not only understand differences in enterohepatic handling of certain bile acids but will also provide principles for construction of an affinity column suitable for isolation of putative taurocholate binding and/or transport proteins from solubilized liver plasma membrane.
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