Expression of the Apical Conjugate Export Pump, Mrp2, in the Polarized Hepatoma Cell Line, Wif–B

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Abstract
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 μmol/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.