REDISTRIBUTION OF CANALICULAR ORGANIC ANION TRANSPORT ACTIVITY IN ISOLATED AND CULTURED RAT HEPATOCYTES

The hepatocanalicular transport of a large number of organic anions, s uch as bilirubin glucuronides and glutathione conjugates in the rat, i s mediated by an adenosine triphosphate (ATP)-dependent transport syst em, which is termed canalicular multispecific organic anion transporte r (cMOAT). This system is mainly de fined by its deficiency in mutant TR(-) rats. We have previously reported that in cultured hepatocytes t he fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. W e now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears w hen cells are incubated in suspension at 37 degrees C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activi ty coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appear ance of intracellular cMOAT and the disappearance of transport activit y from the plasma membrane were completely inhibited at temperatures b elow 20 degrees C. Residual cMOAT activity in 24-hour cultured hepatoc ytes could be further diminished by incubation of the cells with 1 mu mol/L monensin or 10 mmol/L methylamine. We conclude that after disrup tion of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37 degrees C. Evidence suggests that at lea st part of the transporters may recycle back to the plasma membrane af ter endocytosis. These observations may be relevant for the understand ing of regulation of canalicular transport.