We utilize live cell fluorescent, biochemical, genetic and molecular techniques to study mechanisms responsible for trafficking of ATP binding cassette transporters selectively to the apical domain of hepatocytes and other polarized cells. The goal is to identify components and regulation of these processes, their role in creating and maintaining hepatocyte polarity, and molecular defects responsible for inheritable and acquired bile secretory failure (cholestasis). To date, numerous specific proteins and lipids have been identified that are critical for trafficking, membrane fusion and recycling of the apical transporters. Genetic and pharmacologic manipulation of these factors results in defective biliary secretion (cholestasis). Our studies indicate a dependent relationship between cellular polarity and the apical trafficking systems. It is likely that acquired cholestasis, due to drugs, viruses, hyperalimentation and other factors, results from an intracellular traffic jam. Specific accomplishments during the past year are: (a) Development of a culture system (up to 80 days) for rat and human hepatocytes that retain gene expression and functional activity. Using these cells, we characterized development of polarity and its dependence on AMP kinase, the metabolic sensor of the cell. Formation of tight junctions, canalicular morphology and trafficking of apical ABC transporters paralelled AMPK activity as influenced by activators or inhibitors. These observations directly link cellular metabolism with polarity. (b) Requirement for two glycosylation sites for ABCB11 trafficking and a comparable mutation in an inheritable disease (c) ABC transporters traffic as cargo in endosomes which move along MTs and transfer to an actin-based system before fusion with the apical membrane. We established that the MT plus end of is at the apical region and that, using a specific inhibitor, dynamic microtubules are required for the trafficking process. Of interest is that several drugs and chemicals which produce cholestasis in man and animals have a structure similar to the dynamic MT specific inhibitor.(d) Hepatic endothelial cells are heavily fenestrated which, in asence of a basement membrane, provides direct contact between hepatocytes and plasma. Fenestrae measure approx. 140 nm diameter and are dynamic structures. Caveolin-1 has been proposed to participate in their formation. We observed abnormal fenestral number and shape in endothelial cells form mice in which cav-1 had been knocked out. This finding provides the most direct evidence thus far for a role of cav-1 in fenestral formation and perhaps function.
