title: Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione.
abstract: Multidrug resistance proteins (MRPs) are ATP-dependent export pumps that mediate the export of organic anions. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are all able to facilitate the efflux of anionic conjugates including glutathione (GSH), glucuronide and sulfate conjugates of xenobiotics and endogenous molecules. Earlier studies showed that ABCC4 functions as an ATP-driven export pump for cyclic AMP and cyclic GMP, as well as estradiol-17-beta-D-glucuronide. However, it was unclear if other conjugated metabolites can be transported by ABCC4. Hence in this study, a fluorescent substrate, bimane-glutathione (bimane-GS) was used to further examine the transport activity of ABCC4. Using cells stably overexpressing ABCC4, this study shows that ABCC4 can facilitate the efflux of the glutathione conjugate, bimane-glutathione. Bimane-glutathione efflux increased with time and >85% of the conjugate was exported after 15min. This transport was abolished in the presence of 2.5microM carbonylcyanide m-chlorophenylhydrasone (CCCP), an uncoupler of oxidative phosphorylation. Inhibition was also observed with known inhibitors of MRP transporters including benzbromarone, verapamil and indomethacin. In addition, 100microM methotrexate, an ABCC4 substrate or 100microM 6-thioguanine (6-TG), a compound whose monophosphate metabolite is an ABCC4 substrate, reduced efflux by >40%. A concentration-dependent inhibition of bimane-glutathione efflux was observed with 1-chloro-2,4-dinitrobenzene (CDNB) which is metabolized intracellularly to the glutathione conjugate, 2,4-dinitrophenyl-glutathione (DNP-GS). The determination that ABCC4 can mediate the transport of glucuronide and glutathione conjugates indicates that ABCC4 may play a role in the cellular extrusion of Phase II detoxification metabolites.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD7, 8 Medical Drive, S117597, Singapore, Singapore.
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.forename: Liqi
authors.initials: L
authors.lastname: Lai
authors.forename: Hui Ching
authors.initials: HC
authors.lastname: Yeo
authors.forename: Boon Cher
authors.initials: BC
authors.lastname: Goh
authors.forename: Theresa M C
authors.initials: TM
authors.lastname: Tan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000838:Anions; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001553:Benzbromarone / Q000494:pharmacology; D001692:Biological Transport; D001693:Biological Transport, Active; D001643:Bridged Bicyclo Compounds / Q000493:pharmacokinetics*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004351:Drug Resistance; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D013866:Thioguanine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000255:Adenosine Triphosphate; D000838:Anions; D000964:Antimetabolites, Antineoplastic; D001553:Benzbromarone; D001643:Bridged Bicyclo Compounds; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000242:Cyclic AMP; D006152:Cyclic GMP; D016861:Cyclooxygenase Inhibitors; D004137:Dinitrochlorobenzene; D004958:Estradiol; D005978:Glutathione; D007213:Indomethacin; D008727:Methotrexate; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010100:Oxygen; D013866:Thioguanine; D014700:Verapamil; C042630:1-chloro-2,4-dinitrobenzene-glutathione conjugate; C000715087:ABCC2 protein, human; C073492:ABCC4 protein, human; C080120:glutathione-bimane; C025483:estradiol-17 beta-glucuronide; C030190:S-(2,4-dinitrophenyl)glutathione