Expression and Function of ABC Transporters in Human Alveolar Epithelial Cells

Abstract

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolar^TM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

Keywords: ATP-binding cassette transporters; BCRP; MDR1; MRP1; alveolar epithelium.

Figure 1

Transepithelial electrical resistance (TEER) and cellular permeability (P_app) in EpiAlveolar™ and hAELVi. TEER values (panel (A)) and P_app of mannitol (panel (B)) and propranolol (panel (C)) were measured in cells cultured under air–liquid interface (ALI) conditions, as described in Materials and Methods. Bars are the mean ± SEM of 3 independent determinations (single dots). ** p < 0.01 with a two-tailed Student’s t-test.

Figure 2

Expression and activity of MDR1. Panel (A): The apical-to-basolateral (AB) and basolateral-to-apical (BA) fluxes of 1 µM Rhodamine123 were measured both in the absence (none) and in the presence of 10 µM PSC833, as indicated. Data obtained were employed to calculate the efflux ratio (ER) and uptake ratio (UR), as defined in Materials and Methods. Bars are the mean ± SEM of 3 independent determinations (single dots). Panel (B): The expression of MDR1 gene and protein were performed with RT-qPCR and Western blot analyses, respectively, as described in Materials and Methods. Data of mRNA expression are the mean ± SEM of 3 independent determinations (single dots). A representative Western blot is shown that, repeated twice, gave comparable results.

Figure 3

Expression and activity of ABCG2/BCRP. Panel (A): The apical-to-basolateral (AB) and basolateral-to-apical (BA) fluxes of 3 µM Bodipy™ FL Prazosin were monitored both in the absence (none) and in the presence of 20 µM Ko143 and 50 µM Febuxostat, as indicated. Data obtained were employed to calculate the efflux ratio (ER) and uptake ratio (UR), as defined in Materials and Methods. Bars are the mean ± SEM of 3 independent determinations (single dots). Panel (B): The expression of ABCG2/BCRP gene and protein were performed with RT-qPCR and Western blot analyses, respectively, as described in Materials and Methods. Data of mRNA expression are the mean ± SEM of 3 independent determinations (single dots), while a representative Western blot is shown that, repeated twice, gave comparable results.

Figure 4

Expression and activity of ABCCs/MRPs. Panel (A): The apical-to-basolateral (AB) and basolateral-to-apical (BA) fluxes of 0.5 µM ³H-estrone-3-sulfate were monitored both in the absence (none) and in the presence of 50 µM MK571, as indicated. Data obtained were employed to calculate the efflux ratio (ER) and uptake ratio (UR), as defined in Materials and Methods. Bars are the mean ± SEM of 3 independent determinations (single dots). * p < 0.05 vs. none with a two-tailed Student’s t-test. Panel (B): The expression of ABCC1/MRP1 and ABCC2/MRP2 genes and proteins were performed with RT-qPCR or and Western blot analyses, respectively, as described in Materials and Methods. Data of mRNA expression are the mean ± SEM of 3 independent determinations (single dots), while representative Western blots are shown that, repeated twice, gave comparable results. Panel (C): Immunolocalization of MRP1 in hAELVi and EpiAlveolar™. Confocal laser scanning microscopy of monolayers immunolabeled for MRP1 (green) is shown. Nuclei were stained with propidium iodide (red). Top, single XY scans of hAELVi (left) and EpiAlveolar™ (right) are shown; XZ sections of the planes are also shown in the rectangle images. Bottom, tridimensional reconstruction of acquired horizontal sections. Representative images from three independent experiments are shown. Bar = 20 µm.