Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?
OBJECTIVE
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 microM), 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 microM. respectively, and Vmax 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.
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
Fuxing
F
Tang
Kazutoshi
K
Horie
Ronald T
RT
Borchardt
D000818:Animals; D018938:Caco-2 Cells / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism* / Q000502:physiology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection / Q000379:methods*; D014747:Vinblastine / Q000493:pharmacokinetics
C000715087:ABCC2 protein, human; C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014747:Vinblastine