Bile is the principal pathway for cholesterol elimination from the body, and abnormalities in bile secretion may result in gallstone formation, cholestasis, biliary cirrhosis, hypercholesterolemia and fat malabsorption. The applicant has proposed physicochemical and molecular biological experiments to delineate the hepatocellular mechanisms whereby bile salts promote biliary secretion of cholesterol together with highly specific phosphatidylcholines. Preliminary studies in model systems suggest that bile salts in submicellar concentrations promote transfer of biliary phosphatidylcholine molecules from smooth endoplasmic reticulum to canalicular plasma membranes via a specific phosphatidylcholine transfer protein (PC-TP). To test the hypothesis that PC-TP plays a key role phosphatidylcholine selection and transport in vivo, bile salt activation of this protein will be examined employing native smooth endoplasmic reticulum and canalicular plasma membrane vesicles. Potential regulation of molecular expression of hepatic PC-TP by bile salts will be explored by measuring steady state cytosolic protein and mRNA levels as well as gene transcription rates. A putative transfer protein for biliary phosphatidylcholines necessitates independent cytosolic transfer of biliary cholesterol, hypothesized herein to be via hepatic sterol carrier protein 2 (SCP2). The influence of submicellar bile salts on inter-membrane ferrying of cholesterol molecules by this protein will be studied in vitro. As with PC-TP, molecular regulation of SCP2 by bile salts as well as potential overexpression in cholesterol gallstone disease will be investigated. Both phosphatidylcholine and cholesterol molecules translocate across canalicular plasma membranes for biliary secretion. Whereas this may occur passively for cholesterol, a specific bilayer translocase is required for phosphatidylcholine. In preliminary experiments, the principal investigator has demonstrated functional activity in hepatocyte membranes of an ATP-independent long-chain phosphatidylcholine translocase that is distinct from the ATP-dependent phosphatidylcholine translocating activity of the multidrug resistance gene (mdr2) product, P-glycoprotein. The responsible protein will be isolated from canalicular plasma membrane vesicles by immobilized artificial membrane affinity chromatography and then characterized physical-chemically and biochemically. Following secretion into bile canaliculi, bile salts are believed to promote biliary phosphatidylcholine and cholesterol secretion as vesicles. The hypothesis that vesiculation of phosphatidylcholine and cholesterol molecules results from physical-chemical partitioning of dilute bile salts into exoplasmic leaflets of canalicular membranes will be tested using an automated Langmuir-Pockels surface balance and quasielastic light scattering spectroscopy. These studies should, in part, elucidate biliary lipid secretion at a fundamental cellular level and potentially lead to early interventions in cholelithiasis and cholestasis as well as new strategies for management of hypercholesterolemia.
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