Hepatic Expression of Multidrug Resistance-Associated Protein-Like Proteins Maintained in Eisai Hyperbilirubinemic Rats
- Tomoko Hirohashi1,
- Hiroshi Suzuki1,
- Kousei Ito1,
- Kotaro Ogawa1,
- Kazuhiko Kume2,
- Takao Shimizu2 and
- Yuichi Sugiyama1
- 1Graduate School of Pharmaceutical Sciences (T.H., H.S., K.I., K.O., Y.S.) and 2Graduate School of Medicine (K.K., T.S.) The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Abstract
The biliary excretion of several organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rats (EHBR). In addition, using a kinetic study with isolated canalicular membrane vesicles, we recently suggested the presence of ATP-dependent organic anion transporter(s) other than cMOAT in EHBR [Pharm Res (NY) 12:1746–1755 (1995);J Pharmacol Exp Ther 282:866–872 (1997)]. The aim of this study is to provide a molecular basis for the presence of multiplicity in the biliary excretion of organic anions in rats. Based on the homology with human multidrug resistance-associated protein (hMRP), two cDNA fragments encoding the carboxyl-terminal ATP-binding cassette region were amplified by reverse transcription-polymerase chain reaction from EHBR liver. These fragments exhibited approximately 70% amino acid identity with hMRP and rat cMOAT;, therefore, they were designated MRP-like proteins (MLP-1 and MLP-2). The cloned full length cDNA of MLP-1 and -2 from the Sprague-Dawley (SD) rat liver and colon cDNA library was composed of 1502 and 1523 amino acids, respectively, had the characteristics of ATP-binding cassette transporters, and exhibited homology with hMRP and rat cMOAT. Northern blot analysis indicated that MLP-1 is expressed predominantly in the liver in both SD rats and EHBR, whereas hepatic expression of MLP-2 was observed only in EHBR. In addition, MLP-2 was markedly induced by ligation of the bile duct in SD rat liver. In both SD rats and EHBR, MLP-2 was expressed predominantly in the duodenum, jejunum, and colon. These findings suggest that MLP-1 and MLP-2 might be novel members of the MRP family responsible for the excretion of organic anions from these epithelial cells, and that MLP-2 is an inducible one.
Footnotes
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Send reprint requests to: Yuichi Sugiyama, Ph.D., Professor, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail:bxg05433{at}niftyserve.or.jp
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This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, and the Core Research for Evolutional Sciences and Tecnology of Japan Sciences and Technology Corporation.
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↵1 The sequences reported in this paper have been submitted to the GenBank with the accession numbers AB010466 (MLP-1) and AB010467(MLP-2).
- Abbreviations:
- cMOAT
- canalicular multispecific organic anion transporter
- hMRP
- human multidrug resistance-associated protein
- MLP
- multidrug resistance-associated protein-like protein
- ABC
- ATP-binding cassette
- DNP-SG
- S-(2,4-dinitrophenyl) glutathione
- E3040
- 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole
- CMVs
- canalicular membrane vesicles
- SD
- Sprague-Dawley
- EHBR
- Eisai hyperbilirubinemic rats
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- bp
- base pair(s)
- kb
- kilobase pair(s)
- SSC
- standard saline citrate
- SDS
- sodium dodecyl sulfate
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
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- Received December 9, 1997.
- Accepted March 10, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
