The long term objectives of this grant application are to characterize the basic transport mechanisms in hepatocytes and bile duct epithelial cells (cholangiocytes) that determine the secretion of bile and to define the alterations in mechanisms that result in the development of cholestatic liver disease.
The specific aims of this proposal are: A) To study the transcriptional and post-transcriptional regulation of those membrane transport systems that are important to the formation of bile and that are impaired in cholestatic liver injury. Several animal models of cholestasis (endotoxemia, estrogen administration and bile duct ligation) will be used to examine the transcriptional and post-transcriptional changes that occur with several cloned transporters that are determinants of bile secretion (ntcp.mrp2 and mdrl). Gel mobility shift assays will be used to examine the regulatory elements for the promoter region of ntcp. B) To determine the role that the transcytotic vesicular pathway plays in the establishment of cell polarity and canalicular membrane excretory capacity, the protein composition of microtubule associated vesicles isolated by Taxol polymerization will be assessed using Western blots and gold labeling to identify the canalicular transporters and regulatory (rab) proteins that traffic together. Isolated hepatocyte couplets will be used to study the effects of protein kinases, phosphatases and PI-kinases on the regulation of vesicle traffic between subapical and apical regions of the hepatocyte C) To determine the functional role of membrane transport systems in cholangiocytes that modify the final bile secretory product, novel bile duct units isolated from normal rats and mice and CFTR(-/-) and NHE-3(-/-) mice will be used to access transport mechanisms in cholangiocytes including signal transduction pathways and synergism between secretory agonists as well as the role of the NK2CL co-transporter in secretory events. Newly developed isolated perfused duct segments will also be used to directly assess bile duct luminal events in the intact epithelium.
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