Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2).
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.
Hirohashi
T
T
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.
Suzuki
H
H
Chu
X Y
XY
Tamai
I
I
Tsuji
A
A
Sugiyama
Y
Y
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D015152:Blotting, Northern; D018938:Caco-2 Cells; D003570:Cytidine Triphosphate / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004958:Estradiol / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008871:Microvilli / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides / Q000378:metabolism*; D009994:Osmolar Concentration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014544:Uridine Triphosphate / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003570:Cytidine Triphosphate; D004958:Estradiol; D020719:Glucuronides; D005978:Glutathione; D006160:Guanosine Triphosphate; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; D014544:Uridine Triphosphate; D014638:Vanadates; C030190:S-(2,4-dinitrophenyl)glutathione