OSMODEPENDENT DYNAMIC LOCALIZATION OF THE MULTIDRUG-RESISTANCE PROTEIN-2 IN THE RAT HEPATOCYTE CANALICULAR MEMBRANE

Background & Aims: Circumstantial evidence suggests a regulation of bi liary secretion by transporter insertion and retrieval into and from t he canalicular membrane. This study was undertaken to provide direct e vidence for such a process. Methods: Osmosensitivity of the subcellula r localization of the mrp2 gene-encoded conjugate export pump (MRP2) w as studied by immunofluorescence and confocal laser scanning microscop y of isolated hepatocyte aggregates and in perfused rat liver. Results : MRP2 was localized largely in membranes of the pseudocanaliculi form ed by isolated hepatocyte aggregates during hypo-osmotic exposure, whe reas after hyperosmotic exposure MRP2 was also detectable in intracell ular vesicles. In perfused liver, the EAG15 antibody specific for rat MRP2 and the ZO-1 antibody specific for tight junctions produced immun ostaining of the canalicular membrane. However, the relative amount of MRP2 increased significantly in the pericanalicular region with incre asing perfusate osmolarity, as shown by confocal microscopy of intrace llular vesicles containing MRP2 (but not ZO-1) and by computed densito metry. The osmodependent distribution of MRP2 between the canalicular membrane and intracellular, pericanalicular vesicles occurred within 3 0 minutes and was fully reversible. Conclusions: The findings provide direct evidence for an osmosensitive dynamic insertion and retrieval o f the canalicular MRP2 transporter into and out of the canalicular mem brane.