Fig 4. Expression and functionality of efflux transporters.

(A+B) Relative gene expression of the efflux transporters P-glycoprotein (Pgp) and breast cancer resistant protein (Bcrp) in mouse, rat, and porcine brain capillary endothelial cells (mBECs, rBECs, pBECs) cultured in mono-culture (black triangle) and co-culture with astrocytes (blue circle). (A) The expression of Pgp increases significantly in co-cultured mBECs, decreases significantly in co-cultured rBECs, but remains unchanged in pBECs. (B) The expression of Bcrp is highly consistent in the BECs showing a significant increase in the expression upon co-culturing. (A+B) Changes in gene expression between mono- and co-cultures for each species were analyzed using an unpaired t-test or non-parametric Mann-Whitney test, depending on the variance of the data. Data are shown as mean ± standard deviation (SD) (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001. (C) Immunofluorescent images showing green labeling of P-gp in the cell cytoplasm of BECs and also at the cell-cell borders in pBECs. Staining patterns are unaffected by the culturing condition. The nuclei are stained with DAPI (blue). Scale bar = 20μM. (D-F) The functionality of the efflux transporter P-gp was evaluated by measuring the apparent permeability (Papp) to [3H]-digoxin, a P-gp substrate, with and without the P-gp inhibitor zosuquidar (ZSQ) in the luminal to abluminal (L-A) and abluminal to luminal (A-L) direction. The difference between L-A and A-L transport, which is caused by an active P-gp efflux transporter, diminishes when P-gp is inhibited. This applies to all three species independent of the culturing condition. Data are shown as mean ± SD (n = 6). (G) The efflux ratio (ER) Papp(A-L)/Papp(L-A) calculated from Papp (D-F) shows ER above 2 for all species and culture conditions, which decreases below 2 when P-gp is inhibited, indicative of an active polarized efflux transport function in the BECs. Data are shown as mean ± SD (n = 2).