title: Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2',7'-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles.
abstract: The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.
authors: Department of Biology, Abo Akademi University, Abo/Turku, Finland.
authors: Małgorzata
authors: M
authors: Bobrowska-Hägerstrand
authors: Anna
authors: A
authors: Wróbel
authors: Błazej
authors: B
authors: Rychlik
authors: Ida
authors: I
authors: Ohman
authors: Henry
authors: H
authors: Hägerstrand
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001553:Benzbromarone / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D011339:Probenecid / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D018411:Spodoptera; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000255:Adenosine Triphosphate; D001553:Benzbromarone; D016572:Cyclosporine; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011339:Probenecid; D011422:Propionates; D011804:Quinolines; D014700:Verapamil; C473073:2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein; C059141:verlukast; C421109:multidrug resistance-associated protein 1