Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?
Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?
复制标题
DOI：
10.1023/a:1016192413308
复制
发表时间：
2002-06-01
影响因子：
3.7
通讯作者：
Borchardt, RT
中科院分区：
文献类型：
--
作者：
Tang, FX;Horie, K;Borchardt, RT
Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MRP2 gene (MDCK-MRP2) are a good model of the human intestinal mucosa.Methods. MRP2 expression in Caco-2 cells was compared with the expression of this efflux transporter in MDCK-wild type (MDCK-WT) and MDCK-MRP2 cells using Western blotting methods. The polarized efflux activities of MRP2 in the MDCK-MRP2, MDCK-WT, MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and Caco-2 cells were compared using vinblastine as a substrate. Apparent Michaelis-Menten constants (K-M, Vmax) for the efflux of vinblastine in Caco-2 and MDCK-MRP2 cells were determined in the presence of GF120918 (2 muM), which inhibits P-glycoprotein but does not affect MRP2. Apparent inhibitory constants (K-I) of known substrates/inhibitors of MRP2 were determined by measuring their effects on the efflux of vinblastine in these cell lines.Results. MDCK-MRP2 cells expressed higher levels of MRP2 than MDCK-WT and Caco-2 cells as measured by Western blotting technique. Two isoforms of MRP2 expressed in Caco-2 and MDCK cells migrated at molecular weights of 150 kD and 190 kD. In MDCK-MRP2 cells, the 150 kD isoform appeared to be overexpressed. MDCK-MRP2 cell monolayers exhibited higher polarized efflux of vinblastine than Caco-2 and MDCK-WT cell monolayers. K-M values for vinblastine in Caco-2 and MDCK-MRP2 cells were determined to be 71.8+/-11.6 and 137.3+/-33.6 muM, respectively, and V-max values were determined to be 0.54+/-0.03 and 2.45+/-0.31 pmolcm(-2)s(-1), respectively. Known substrates/inhibitors of MRP2 showed differences in their ability to inhibit vinblastine efflux in Caco-2 cells as compared to MDCK-MRP2 cells.Conclusions. These data suggest that MDCK-MRP2 cells overexpress only the 150 kD isoform of MRP2. The 190 kD isoform, which was also found in Caco-2 cells and MDCK-WT cells, was present in MDCK-MRP2 cells but not over expressed. The apparent kinetics constants and affinities of some MRP2 substrates were different in Caco-2 cells and MDCK-MRP2 cells. These differences in substrate activity could result from differences in the relative expression levels of the MRP2 isoforms present in Caco-2 cells and MDCK-MRP2 cells and/or differences in the partitioning of substrates in these two cell membrane bilayers.
