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Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells
T Hirohashi1, H Suzuki, X Y Chu
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.
Abstract:
Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides. To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes. [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively. The uptake of [(3)H]DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP. An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of [(3)H]DNP-SG to some extent. Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5. Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5. Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached. Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells. Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2.
Insights
Multidrug resistance-associated proteins (MRPs) in Caco-2 cells facilitate organic anion transport. The study demonstrates ATP-dependent uptake of DNP-SG and E217bG, suggesting cMOAT/MRP2
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Organic anions are actively transported across intestinal epithelial cells.
- The multidrug resistance-associated protein (MRP) family plays a role in intestinal efflux.
Purpose of the Study:
- To investigate the function and expression of MRP proteins in intestinal organic anion transport.
- To examine the role of MRPs in the efflux of organic anions using Caco-2 cells.
Main Methods:
- Utilized Caco-2 cells, a human adenocarcinoma cell line mimicking enterocytes.
- Investigated uptake of [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) into brush-border membrane vesicles (BBMVs).
- Employed ATP-dependent transport assays, ATPase inhibitor studies, reverse-transcriptase polymerase chain reaction (RT-PCR), and Northern blot analysis.
Main Results:
- Demonstrated ATP-dependent uptake of DNP-SG and E(2)17betaG in Caco-2 BBMVs.
- RT-PCR detected MRP1, MRP3, and MRP5; Northern blot showed extensive expression of cMOAT/MRP2 and MRP3.
- cMOAT/MRP2 was continuously expressed, while MRP3 expression followed confluency; MRP1 and MRP5 showed minimal expression.
Conclusions:
- Caco-2 cells possess ATP-dependent transport systems for DNP-SG and E(2)17betaG.
- The observed transport activity in BBMVs likely involves cMOAT/MRP2, potentially localized to the brush border.
- MRP3 may be involved in basolateral transport, contributing to overall organic anion handling in intestinal cells.