The polarized rat hepatoma/human fibroblast hybrid cell line, WIF-B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF-B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190-kd membrane glycoprotein mediating adenosine triphosphate (ATP)-dependent transport of glucuronides, glutathione S-conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF-B cells was shown by reverse-transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789-bp fragment. Immunoblotting, using antibodies reacting with the amino-terminal or with the carboxyl-terminal sequence of rat Mrp2, detected the 190-kd glycoprotein in WIF-B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF-B cells with a membrane-permeable ester of the calcium-dependent fluorescent indicator, Fluo-3, was followed by Mrp2-mediated secretion of the amphiphilic anion, Fluo-3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP-dependent transport into Mrp2-containing plasma membrane vesicles in comparison with Mrp2-deficient vesicles established that Fluo-3 is transported by Mrp2 with a K m value of 3.7 mol/L. Our results indicate that the polarized WIF-B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo-3. (HEPATOLOGY 1998;28:1332-1340.)
The vectorial transport of cholephilic substances from blood into bile is brought about by a number of transporters in the sinusoidal 1 and the canalicular 2 membrane of the hepatocyte. Before excretion into bile, many of the cholephilic substances are intracellularly conjugated with glutathione, glucuronate, or sulfate. Excretion of these conjugates into bile is mediated by an adenosine triphosphate (ATP)dependent export pump, which has been cloned recently and termed canalicular multidrug-resistance protein (cMRP) 3 or canalicular multispecific organic anion transporter (cMOAT). [4][5][6] The term multidrug-resistance protein 2 (MRP2 for the human and Mrp2 for the rat ortholog) is used here for this 190-kd membrane glycoprotein because of similar substrate specificities 3,7-10 and related amino acid sequences 3-7 of MRP2 and MRP1. The latter is the first cloned member of the multidrug-resistance protein family. 11 In the rat, mrp2 mRNA was found mainly in liver, 3,4,6 but also at low levels in kidney and small intestines. 4,6 On the protein level, rat Mrp2 and human MRP2 were detected in the apical membrane domain of hepatocytes 3,7 and renal proximal tubule cells. 12 Before the molecular identification of the apical conjugate export pump, the function of this transporter was characterized in two mutant rat strains that are deficient in the transport of conjugated and amphiphilic anions across the canalicular membrane, the Groningen yellow/transportdeficient (GY/TR Ϫ ) Wistar rat [13][14][15] and the Eisai hyperbilirubinemic rat. 16,17 Hepatocytes from these two distinct rat strains are characterized by decreased mrp2 mRNA levels, a lack of the Mrp2 protein in apical membrane domains, and a loss of ATP-dependent membrane transport of anionic conjugates. 3,4,6,7,18 Transport function of Mrp2 has been frequently measured by ATP-dependent uptake of radiolabeled anionic compounds into inside-out-oriented canalicular membrane vesicles isolated from normal and mutant rat livers. 3,[18][19][20] The accumulation of fluorescent organic anions, such as glutathione-bimane and 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3oxo-3H-xanthen-9-yl)]-2-(2Ј-amino-5Ј-methyl-phenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid penta-ammonium salt (Fluo-3), was observed in intracellular vesicles of intact hepatocytes isolated from normal rat livers, but not in those from mutant rat livers lacking the Mrp2 transporter. [21][22][23][24] Fluo-3 is a penta-anionic compound widely used as an intracellular Ca 2ϩ indicator because of its large increase in fluorescence upon Ca 2ϩ binding. 25,26 The penta-acetoxymethyl ester of Fluo-3, 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2Ј-amino-5Ј-methyl-phenoxy)-Abbreviations: ATP, adenosine triphosphate; MRP2, human apical (canalicular) multidrug-resistance protein 2; Mrp2, rat apical (canalicular) multidrug-resistance protein 2; GY/TR Ϫ ,