Bile acid secretion and direct targeting of mdr1-green fluorescent proteinfrom Golgi to the canalicular membrane in polarized WIF-B cells

The bile canalicular membrane contains several ATP-dependent transporters t hat are involved in biliary secretion. Canalicular transporters are synthes ized in ER, modified in Golgi and transported to the apical plasma membrane . However, the route and regulation of intracellular trafficking of ATP-dep endent transporters have not been elucidated, In the present study, we gene rated a translational fusion of mdr1 and green fluorescent protein and inve stigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i,e, rhodamine-123) into the bile canaliculi, Canalicular secretion of flu orescein isothiocyanate-glycocholate was stimulated by taurocholate and a d ecapeptide activator of phosphoinositide 3-kinase and was decreased by wort mannin, WIF-B9 cells were transiently and stably transfected with a mdr1-GFP constr uct, Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majo rity of which traveled directly to the canalicular membrane. Recycling betw een the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basalateral plasma membrane. At 15 degree s C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluoresc ence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin, In these studies as well, no mdr 1-GFP fluorescence was observed at any time in basolateral membranes or oth er intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the ca nalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependen t transporter of organic cations, As in normal hepatocyes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of md r1 in WIF-B cells, WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters;