title: Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers.
abstract: In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km=69.41 ± 22.3 μM and Vmax=14.03 ± 2.27 nmol/(cm(2).h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP.
authors: Epithelial Research Group, Institute of Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK.
authors: Jamie A
authors: JA
authors: Wright
authors: Iain S
authors: IS
authors: Haslam
authors: Tanya
authors: T
authors: Coleman
authors: Nicholas L
authors: NL
authors: Simmons
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D004032:Diet; D054659:Diketopiperazines; D004285:Dogs; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects; D006576:Heterocyclic Compounds, 4 or More Rings; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009582:Nitrofurantoin / Q000378:metabolism*; D011224:Prazosin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: C541506:3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino(1',2'-1,6)pyrido(3,4-b)indol-3-yl)propionic acid tert-butyl ester; C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D054659:Diketopiperazines; D006576:Heterocyclic Compounds, 4 or More Rings; D009363:Neoplasm Proteins; D009582:Nitrofurantoin; D014700:Verapamil; D000241:Adenosine; D011224:Prazosin