Bobrowska-Hägerstrand Małgorzata, Wróbel Anna, Rychlik Błazej, Ohman Ida, Hägerstrand Henry
Department of Biology, Abo Akademi University, Abo/Turku, Finland.
Mol Membr Biol. 2007 Sep-Dec;24(5-6):485-95. doi: 10.1080/09687680701383069.
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.
人红细胞膜中存在人多药耐药蛋白1（MRP1/ABCC1）已得到充分证实。在本研究中，引入了流式细胞术监测，以确定MRP1是红细胞膜中2',7'-双（3-羧丙基）-5-（和-6）-羧基荧光素（BCPCF）的主要转运蛋白，并促进对MRP1介导转运的抑制和动力学研究。研究了BCPCF在ATP依赖下转运至人红细胞内翻囊泡，以及作为对照转运至表达MRP1的Sf9细胞膜内翻囊泡的情况。MRP1特异性单克隆抗体QCRL-3和MRP1抑制剂MK-571均强烈降低了BCPCF进入红细胞和表达MRP1的Sf9细胞膜内翻囊泡的摄取。环孢素A、维拉帕米、苯溴马隆和丙磺舒在红细胞膜囊泡中的抑制谱是MRP1介导转运的典型特征。此外，在不存在和存在选定抑制剂（MK-571、环孢素A、苯溴马隆和维拉帕米）的情况下，测定了BCPCF转运至红细胞膜内翻囊泡的动力学常数K(m)和V(max)。所呈现的结果确定MRP1是人红细胞膜中BCPCF的主要转运蛋白，并首次表明可通过流式细胞术监测荧光底物向内翻囊泡的主动转运。
