The goals of this project are: 1) To localize and characterize bile acid transport systems in rat hepatocyte intracellular membranes. 2) To show that these transport systems are important in: a) normal hepatic bile acid transport and b) cholestasis. 1) Rat liver microsome, Golgi, lysosomes, endosomes, and multivesicular bodies will be isolated by sucrose density gradient centrifugation or free flow electrophoresis. Transport activity will be assayed using radiolabeled bile acids and a rapid filtration method. Criteria for carrier-mediated transport will be tested and possible driving forces will be evaluated. Membrane binding studies will be done. Carrier protein(s) will be identified by photoaffinity or inhibitor labeling. Data will be compared with known results for liver membranes. 2) The physiologic importance of intracellular transport systems will be confirmed: (i) To indicate multiple intracellular transport routes for bile acids, transit time analysis of biliary bile acid output will be done using isolated perfused livers. In these and subsequent studies perturbations will be used to modify the intracellular transmembrane route, i.e. low and high bile acid loads, temperature changes, inhibitors of microtubules and intracellular acidification. (ii) Intrahepatic compartmentation will be established by assaying bile acid contents of liver subfractions that are isolated rapidly using transport inhibitors to minimize bile acid redistribution during preparation. (iii) Bile acids will also be localized using anti-bile acid antibodies and immune=electronmicroscopy in liver slices and isolated hepatocyte couplets. Studies described above will use (a) normal livers and (b) livers from rat with cholestasis (induced by bile duct obstruction or estrogens) or phenobarbitone- induced endoplasmic reticulum proliferation. Collectively, these studies will identify hitherto unconfirmed intracellular pathways for bile acid transport that are important to liver function in health and cholestasis.
