Bile salt secretion into the bile canaliculus is rate limiting and immature at birth and develops during postnatal life. Therefore, the overall objective of this grant is to define the cellular and molecular mechanisms involved in the ontogenic expression of the canalicular bile acid transport system. The canalicular bile acid transport protein will be isolated and purified from triton solubilized canalicular membranes using affinity chromatography and preparative SDS gel electrophoresis. Purified proteins will then be used to generate polyclonal and monoclonal antibodies. Specific antibodies directed against this protein will be used to inhibit bile acid transport by membrane vesicles and to follow the ontogenic expression of the carrier protein on the plasma membrane domains by immunochemical quantitation and immunocytochemistry. Further evidence of the identity of the 100 kDa protein as a bile acid carrier will be demonstrated by functional reconstitution of its transport activity into synthetic proteoliposomes. The timing and intracellular events associated with sorting of the 100 kDa bile acid transport protein to the canalicular membrane will be studied through the technique of pulse chase and immunoprecipitation. Precursor forms of the canalicular bile acid transport protein isolated from various subcellular domains during development will be characterized by endoglycosidase digestion and subsequent immunochemical analysis. A liver CDNA library will be screened using a polyclonal antibody directed against the transport protein or synthetic oligonucleotide probes prepared according to partial sequencing of the transport protein. Hydropathy analysis of the amino acid sequence deduced from the CDNA will provide insight into the functional organization of the transport protein within the canalicular membrane. Hepatoma cell lines lacking the 100 kDa protein on the plasma membrane will be transfected with the CDNA to demonstrate functional activity. CDNA probes specific for the 100 kDa protein will be used in hybridization studies to determine if ontogenic regulation of this transport system occurs by transcriptional or translational mechanisms. Nuclear transcription assays will then be performed to confirm the results obtained from Northern and slot blot analyses. Hormonal induction of this protein and its mRNAs during ontogeny will be studied. CDNA probes will also be used for in situ hybridization studies to determine the predominant localization for the expression of the transporter gene within the hepatic lobule in the adult animal and during development. Knowledge of the mechanisms involved in the ontogenic expression of the canalicular bile acid transport protein will be important for developing new strategies for the diagnoses and treatment of previously undefined cholestatic liver diseases in children.
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