Y Kamimoto

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.

TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.

To study the effect of bile acids on P-glycoprotein-mediated drug transport, we performed experiments using multidrug resistant cells and rat canalicular membrane vesicles. Cellular accumulation and efflux of rhodamine 123 were measured in drug-resistant cells by means of computerized quantitative image analysis and fluorescence microscopy. ATP-dependent [3H]daunomycin transport was studied by means of rapid filtration in canalicular membrane vesicles prepared from normal rats. Doxorubicin-sensitive (PSI-2) and -resistant (PN1A) 3T3 cells and human-derived hepatocellular carcinoma doxorubicin-sensitive and -resistant cells were used. Taurochenodeoxycholate and glycochenodeoxycholate, taurolithocholate and ursodeoxycholate (50 to 200 mumol/L) inhibited rhodamine 123 and [3H]daunomycin transport in multidrug-resistant cells and canalicular membrane vesicles, respectively, whereas taurocholate, taurodeoxycholate and tauroursodeoxycholate did not. Primary and secondary unconjugated bile acids had no effect. These results reveal that taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate inhibit P-glycoprotein-mediated drug transport function in multidrug resistant cell lines and in canalicular membrane vesicles. These results suggest possible interaction between P-glycoprotein function and bile acids in cholestasis and after treatment of patients with ursodeoxycholic or chenodeoxycholic acid.