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. [3H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [3H]17β-estradiol 17-β-d-glucuronide (E217βG), 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, withKm values of 16.9 ± 7.2 and 9.4 ± 1.2 μM, respectively. The uptake of [3H]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 [3H]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 E217βG 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.
Footnotes
-
Send reprint requests to: Hiroshi Suzuki, Ph.D., Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku. E-mail: suzuki{at}seizai.f.u-tokyo.ac.jp
-
↵1 This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “ABC proteins” (10044243) from the Ministry of Education, Science, and Culture of Japan.
- Abbreviations:
- BBMVs
- brush-border membrane vesicles
- ABC
- ATP-binding cassette
- MRP
- multidrug resistance-associated protein
- cMOAT
- canalicular multispecific organic anion transporter
- EHBR
- Eisai hyperbilirubinemic rat
- DNP-SG
- 2,4-dinitrophenyl-S-glutathione
- E217βG
- 17β-estradiol 17-β-d-glucuronide
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- 4-MU
- 4-methylumbelliferone
- Received June 1, 1999.
- Accepted September 15, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|