title: An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1.
abstract: Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.
authors.affiliation: Department of Pharmacy, The Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Helms
authors.forename: Maria
authors.initials: M
authors.lastname: Hersom
authors.forename: Louise Borella
authors.initials: LB
authors.lastname: Kuhlmann
authors.forename: Lasina
authors.initials: L
authors.lastname: Badolo
authors.forename: Carsten Uhd
authors.initials: CU
authors.lastname: Nielsen
authors.forename: Birger
authors.initials: B
authors.lastname: Brodin
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002450:Cell Communication; D016764:Cell Polarity; D002478:Cells, Cultured; D018920:Coculture Techniques; D004077:Digoxin / Q000378:metabolism; D004553:Electric Conductivity; D042783:Endothelial Cells / Q000378:metabolism*; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D005047:Etoposide / Q000378:metabolism; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010539:Permeability; D010641:Phenotype; D051381:Rats
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D004077:Digoxin; D004970:Estrone; D005047:Etoposide; D008353:Mannitol; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C489249:Abcg2 protein, rat; C095122:multidrug resistance protein 3; C017296:estrone sulfate; C421109:multidrug resistance-associated protein 1