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. 2014 Sep;16(5):1046-55.
doi: 10.1208/s12248-014-9628-1. Epub 2014 Jun 17.

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

Hans Christian Helms  1 , Maria Hersom, Louise Borella Kuhlmann, Lasina Badolo, Carsten Uhd Nielsen, Birger Brodin
Affiliations

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

Hans Christian Helms et al. AAPS J. 2014 Sep.

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.

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Figures

Fig. 1
Fig. 1
Immunocytochemical characterization of in vitro blood–brain barrier models at day 6 of co-culture. Upper row Endothelial cells were stained with Alexa-488 phalloidin to visualize filamentous actin (a), with an antibody against von Willebrand’s factor (b) or an antibody against glial fibrillary acidic protein (c) (green). Cell nuclei were counterstained with propidium iodide (red). Lower row Astrocytes stained with Alexa-488 phalloidin (d), an antibody against von Willebrand’s factor (e) or an antibody against glial fibrillary acidic protein (f) (green). Bars = 20 μm
Fig. 2
Fig. 2
Change in (TEER of co-cultured monolayers as a function of experimental time. TEER was measured at 37°C to avoid cooling of the cells prior to each measurement. The co-cultures were either incubated with their culture medium (solid square) or in HBSS with 10 mM HEPES and 0.5% BSA (solid circle). The first data point in both series corresponds to initial TEER-measurement in the culture media at 37°C. The medium was replaced with HBSS in one treatment group after the initial TEER measurement and both groups were incubated 15 min before the onset of stirring. The stirring was started at time zero. Data are averages ± SEM (n = 3–4, N = 2–3)
Fig. 3
Fig. 3
Mannitol permeability as a function of TEER. Apparent mannitol permeabilities obtained from 14C–D-mannitol (20 μM) flux experiments across single filter inserts plotted as a function of the transendothelial electrical resistance measured across the corresponding filter inserts at room temperature prior to the experiment
Fig. 4
Fig. 4
Transendothelial transport of 3H-digoxin (0.03 μM) (a), 3H-estrone-3-sulphate (b) (0.02 μM) and 3H-etoposide (c) (2.64 μM) across the co-culture model as a function of time, in the luminal-to-abluminal (solid triangle) and abluminal-to-luminal direction (solid circle) without or with inhibitors (luminal-to-abluminal, open triangle; abluminal-to-luminal, open circle), verapamil (50 μM) (a), Ko 143 (0.5 μM) (b) or zosuquidar + reversan (0.5 and 5 μM, respectively) (c). Data are means ± SEM (n = 3–9, N = 2–3)
Fig. 5
Fig. 5
Efflux ratios derived from the apparent substrate permeabilities. Inhibitors were applied in concentrations of 50 μM (verapamil), 5 μM (reversan) or 0.5 μM (Ko 143 and zosuquidar). Data are means ± SEM (n = 3–25, N = 2–3).*P < 0.05, ****P < 0.0001
Fig. 6
Fig. 6
Abluminal-to-luminal transport of 3H-digoxin (0.03 μM) (a), 3H-estrone-3-sulphate (b) (0.02 μM) and 3H-etoposide (c) (2.64 μM) across the co-culture model in the presence of different inhibitors. Inhibitors were applied in concentrations of 50 μM (verapamil and MK 571), 5 μM (reversan) or 0.5 μM (Ko 143 and zosuquidar). Data are standardized against the substrate permeability without inhibitors performed within the same experiment and shown as means ± SEM (n = 3–4, N = 2–3). **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 7
Fig. 7
Immunocytochemical staining of P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein-1 (Mrp-1) in bovine brain endothelial cells: Freshly isolated bovine brain capillaries (a–c) and endothelial cells after 6 days of co-culture (d–i) were incubated with antibodies against P-glycoprotein (a, d, g), breast cancer resistance protein (b, e, h) or multidrug resistance-associated protein-1 (c, f, i) followed by incubation with species-specific secondary antibodies labelled with Alexa-488 (green). All samples were incubated with propidium iodide to visualize cell nuclei (red). Bars = 20 μm (a–f) or 5 μm (g–i)
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