Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1

Abstract

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.

Characterization of ABC Transporters in EpiAirway™, a Cellular Model of Normal Human Bronchial Epithelium

“…Lastly, MDR1 is a glycoprotein widely expressed on the apical membrane of different tissues. As for in vitro models of pulmonary epithelium, a clear dependence of the expression and function of MDR1 on the culture time and passage number has been established for polarized monolayers of Calu-3 cells [8,10,17]. Consistently, we observed only barely detectable levels of the transporter in Calu-3 cells after 8 d at ALI, while a much higher amount of the protein was evident at the apical side of "late" cultures.…”

“…To bypass this issue, in vitro models that more closely mimic the biology of normal airway epithelium in vivo have been employed. Among them, normal human bronchial epithelial cells (NHBE) appeared promising, since they can form a pseudostratified cell layer, including ciliated and goblet cells, that nearly resembles normal airways [8][9][10]; however, at present, there is no universally accepted protocol for the culture of these cells and transport data in NHBE layers appear poorly reproducible [8,10].…”

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar–Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography–Tandem Mass Spectrometry

“…Also, Mukherjee et al observed OCT3 and OCTN2 expressions in Calu‐3, whereas OCT2 could not be detected, which was similar with our data. On the contrary, MDR1 protein expressions were not observed in tested five immortalized lung cell lines except A549 in this study, whereas MDR1 expression in the lung has been previously reported by RT‐PCR, and MDR1 has been found to be functionally active in Calu‐3 cells and in ex vivo rodent lungs . Furthermore, PEPT2 was reported to be responsible for the uptake and transport Gly‐Sar in Calu‐3 monolayers, but this study showed no detection in PEPT2 protein expression.…”

Organic Cation Transporters in the Lung—Current and Emerging (Patho)Physiological and Pharmacological Concepts

“…However, there is no report, to our knowledge, that indicates ipratropium bromide as a substate of MRPs. OATs were undetectable in Calu-3 monolayers grown under similar condition [130]. Anions, including probenecid, have been proposed to inhibit OCTs [6] and thus might be involved in the interaction between probenecid and ipratropium bromide.…”