MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.
The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.
Cantz
T
T
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany.
Nies
A T
AT
Brom
M
M
Hofmann
A F
AF
Keppler
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D
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D000814:Aniline Compounds / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*; D001647:Bile Acids and Salts / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D006801:Humans; D015151:Immunoblotting; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000378:metabolism; D014966:Xanthenes / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*
D018435:ATP Binding Cassette Transporter, Subfamily B; D000814:Aniline Compounds; D001647:Bile Acids and Salts; D016572:Cyclosporine; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D014966:Xanthenes; C000715087:ABCC2 protein, human; C059715:Fluo-3; C421109:multidrug resistance-associated protein 1