Bile secretory function is immature at birth and develops during postnatal life. Therefore the overall objective of this competitive grant renewal is to continue with our studies defining the development of the hepatocyte transport mechanisms involved in bile formation. The ontogeny of inorganic ion transporters on the basolateral (multispecific ion exchanger) and on the canalicular (Cl-/HCO3- exchange) domains will be studied using highly purified plasma membrane vesicles. The kinetic and developmental properties of a basolateral Na+/HCO3- cotransport system will also be defined. The basolateral and canalicular bile acid transport proteins will be isolated from the plasma membrane using their developmental appearance as a strategy for identification. The proteins isolated from solubilized membranes by SDS-polyacrylamide gel electrophoresis and by affinity chromatography will be used to generate polyclonal and monoclonal antibodies. Specific antibodies against the proteins will be used to inhibit bile acid transport by membrane vesicles and isolated hepatocytes and to follow the ontogenic expression of the carrier proteins on the plasma membrane domains by immunochemical quantitation and immunofluorescence microscopy. Monoclonal antibodies will be used to probe the structure and function of the transport proteins (including binding sites for substrates and inhibitors of the carrier). Further evidence of the identity of the 48 kDa protein as a bile acid carrier will result from functional reconstitution of its transport activity into synthetic proteoliposomes. A size-selected rat liver cDNA library prepared in the plasmid pBR322 will be screened using synthetic oligonucleotide probes prepared according to partial sequencing of the basolateral 48 kDa and canalicular 100 kDa bile acid transport proteins. cDNA probes specific for the 48 kDa and 100 kDa proteins will be used in hybridization studies to determine if ontogenic regulation of these transport systems occurs by transcriptional or translational mechanisms. Possible hormonal induction of these proteins and their mRNAs will be studied. Knowledge of the ontogenic development of hepatocyte transport mechanisms and the regulation of the bile acid transport proteins will be important in developing new strategies for the diagnosis and treatment of cholestatic liver disease in the newborn.
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| Li, Hai; Chen, Frank; Shang, Quan et al. (2005) FXR-activating ligands inhibit rabbit ASBT expression via FXR-SHP-FTF cascade. Am J Physiol Gastrointest Liver Physiol 288:G60-6 |
| Anwer, M Sawkat; Gillin, Henry; Mukhopadhyay, Sunil et al. (2005) Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp. J Biol Chem 280:33687-92 |
| Geier, Andreas; Dietrich, Christoph G; Voigt, Sebastian et al. (2005) Cytokine-dependent regulation of hepatic organic anion transporter gene transactivators in mouse liver. Am J Physiol Gastrointest Liver Physiol 289:G831-41 |
| Balasubramaniyan, N; Shahid, Mohammad; Suchy, Frederick J et al. (2005) Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development. Am J Physiol Gastrointest Liver Physiol 288:G251-60 |
| Dubrac, Sandrine; Lear, Steven R; Ananthanarayanan, Meena et al. (2005) Role of CYP27A in cholesterol and bile acid metabolism. J Lipid Res 46:76-85 |
| Chen, Frank; Ananthanarayanan, M; Emre, Sukru et al. (2004) Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity. Gastroenterology 126:756-64 |
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