Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B.
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 Km value of 3.7 micromol/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.
Nies
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Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Cantz
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Brom
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Leier
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Keppler
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D006822:Hybrid Cells; D015151:Immunoblotting; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D014407:Tumor Cells, Cultured; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D027321:Anion Transport Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D014966:Xanthenes; C059715:Fluo-3; D000255:Adenosine Triphosphate