Table 1.
The transporter inhibitors used in this study along with [3H]pentamidine.
All inhibitors were used in the presence of 0.05% DMSO and used at the published concentration ranges where they affect transporter activity.
Fig 1.
The effect of ATP depletion and self-inhibition on [3H]pentamidine accumulation was determined in hCMEC/D3s (A) and bEnd.3s (B). Significant differences compared to control was observed—*p<0.05, **p<0.01, ***p<0.001. All data expressed as mean ± SEM, n = 4 passages of cells with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.
Fig 2.
The effect of P-gp substrates and inhibitors on the [3H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with P-gp substrate dexamethasone (200 μM) or P-gp inhibitor haloperidol (40 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n = 4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0,
Fig 3.
The effect of BCRP and MRP inhibitors on [3H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with BCRP inhibitor ko143 (1 μM), BCRP substrate pheophorbide A (1 μM) or MRP inhibitor MK571 (10 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n = 4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.
Fig 4.
Influence of OCT 1, 2, and 3 transporters on [3H]pentamidine accumulation were studied on hCMEC/D3(A) and bEnd.3 (B) cell lines. Cells were incubated with amantadine (500 μM, OCT1 and 2 inhibitor), prazosin (100 μM, OCT1 and 3 inhibitor), N-methylnicotinamide (100 μM, OCT2 inhibitor) or corticosterone (50 μM, OCT3 inhibitor). Significant differences were observed compared to control—*p<0.05, **p<0.01, ***p<0.001. All data expressed as mean ± SEM, n = 3–4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed by two-way ANOVA with SigmaPlot 13.0.
Fig 5.
The effect of OCTN1 and OCTN2 substrates on the [3H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with OCTN1 substrate ergothioneine (20 μM) or OCTN2 substrate L-carnitine (5 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n = 4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.
Fig 6.
The effect MATE1, MATE2 and PMAT inhibitors on the [3H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with MATE1 inhibitor famotidine (1 μM), MATE2 inhibitor nifekalant (3 μM) or PMAT inhibitor lopinavir (2 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n =.4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.
Fig 7.
MTT assay was carried out to assess the cytotoxic effects of compounds used by incubating the compounds on confluent monolayers of hCMEC/D3 (A) and bEnd.3 (B) cells. Cells grown in 96-well plates were exposed to the inhibitor containing buffer for 120 minutes. Results are expressed as percentage viability of cells ± SEM compared to control where cells were incubated with accumulation buffer alone. A positive control of 1% Triton X-100 was also included. Significant differences compared to control was observed—*p<0.05, **p<0.01, ***p<0.001. All data expressed as mean ± SEM, n = 6 wells. Data were analysed using one-way ANOVA with Sigma Plot 13.0.
Fig 8.
P-gp expression in confluent hCMEC/D3 cells and bEnd.3 cells.
Confocal microscopy image of P-gp expression in confluent hCMEC/D3 cells (A) and bEnd.3 cells (B) was detected using anti-P-gp antibody (Abcam, ab170904) diluted at 1:200. Western blot band of P-gp was detected at 170 kD after probing with anti-P-gp antibody (Abcam, ab170904) diluted at 1:2000. Tubulin (55 kD) was used as a loading control. (C)—Lane 1 –hCMEC/D3 passage 28, Lane 2—hCMEC/D3 passage 33, Lane 3- bEnd.3 passage 18, Lane 4 –bEnd.3 passage 23, and MDCK-hMDR (positive control from the same gel).
Fig 9.
BCRP expression in confluent hCMEC/D3 cells and bEnd.3 cells.
Confocal microscopy image of BCRP expression in confluent hCMEC/D3 cells (A) and bEnd.3 cells (B) was not detected in the plasma membranes when using anti-BCRP antibody (New England Biolabs, 4477S diluted at 1:200)—Western blot band of BCRP was detected at 143 kD which corresponds to the dimer version of BCRP in positive control (C). No band was observed at 70 kD (monomer version of BCRP). Tubulin (55 kD) was used as a loading control. Lane 1 –hCMEC/D3 passage 28, Lane 2- hCMEC/D3 passage 33, Lane 3- bEnd.3 passage 18, Lane 4- bEnd.3 passage 23, Lane 5 –brain endothelial cells isolated from mouse (positive control).
Fig 10.
Confocal microscopy image of MRP4 expression in confluent hCMEC/D3 cells (A) and bEnd.3 cells (B). No fluorescent signals were detected when using MRP4 antibody [M4I-10] from Abcam at 1:200 dilution and goat anti-rat Alexa Fluor® 488 secondary antibody (Abcam, ab181448) at 1:200 dilution.
Fig 11.
OCT expression in confluent bEnd.3 cells and hCMEC/D3 cells.
A) OCT1 expression observed in brain endothelial cells isolated from mouse (positive control) at 61 kD when using anti-OCT1 antibody (Abcam, ab55916) at 1:750 dilution. B) OCT1 bands were also observed at 61kD in 2,3,5 and 6. C) OCT2 expression was observed in all cell lines at ~ 95 kD when using anti-OCT2 antibody (Abcam, ab170871) at 1:2000 dilution in accordance to the datasheet. D) OCT3 expression was observed in all cell lines at 61 kD when using anti-OCT3 antibody (Abcam, ab183071) at 1:600 dilution. Tubulin expression was used as a loading control in A and D at 55 kD when using anti-tubulin antibody (Millipore Limited, UK) at 1:10000 dilution. GAPDH expression was used as a loading control in B and C at 37 kD when using anti-GAPDH antibody (Abcam, ab9485) at 1:2500 dilution. 1 –Brain endothelial cells isolated from mouse (positive control), 2—bEnd.3 passage 18, 3 –bEnd.3 passage 19, 4 –bEnd.3 passage 23, 5—hCMEC/D3 passage 28, 6—hCMEC/D3 passage 33.
Fig 12.
Expression of OCT1 was detected by Transmission Electron Microscopy and immunogold labelling.
hCMEC/D3 (p28) and bEnd.3 cells (p23) were grown to confluency on transwell polycarbonate membrane and stained for OCT1 using the method described. Gold nanoparticles on the membranes (arrows) were counted in the 60 sequential images acquired at 11500 x magnification. Significantly increased expression of OCT1 was found on the luminal membrane compared to the abluminal membrane. ***p<0.001 using student’s t-test.