Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane

Background & Aims: Circumstantial evidence suggests a regulation of biliary secretion by transporter insertion and retrieval into and from the canalicular membrane. This study was undertaken to provide direct evidence for such a process. Methods: Osmosensitivity of the subcellular localization of the mrp2 gene-encoded conjugate export pump (MRP2) was studied by immunofluorescence and confocal laser scanning microscopy of isolated hepatocyte aggregates and in perfused rat liver. Results: MRP2 was localized largely in membranes of the pseudocanaliculi formed by isolated hepatocyte aggregates during hypo-osmotic exposure, whereas after hyperosmotic exposure MRP2 was also detectable in intracellular vesicles. In perfused liver, the EAG15 antibody specific for rat MRP2 and the ZO-1 antibody specific for tight junctions produced immunostaining of the canalicular membrane. However, the relative amount of MRP2 increased significantly in the pericanalicular region with increasing perfusate osmolarity, as shown by confocal microscopy of intracellular vesicles containing MRP2 (but not ZO-1) and by computed densitometry. The osmodependent distribution of MRP2 between the canalicular membrane and intracellular, pericanalicular vesicles occurred within 30 minutes and was fully reversible. Conclusions: The findings provide direct evidence for an osmosensitive dynamic insertion and retrieval of the canalicular MRP2 transporter into and out of the canalicular membrane.