Tobacco Smoke and Inhaled Drugs Alter Expression and Activity of Multidrug Resistance-Associated Protein-1 (MRP1) in Human Distal Lung Epithelial Cells in vitro

Abstract

Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apicalto-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased

Prediction and enrichment analyses of the Homo sapiens-Drosophila melanogaster COPD-related orthologs: potential for modeling of human COPD genomic responses with the fruit fly

“…Of note, the Nrf2 agonists sulforaphane and compound 7 were both found to reverse defects in opsonic phagocytosis of Streptococcus pneumoniae and nontypeable Hemophilus influenzae by COPD alveolar macrophages (AMs) (32). Furthermore, sulforaphane was also reported to reverse the negative effect of CS extract on MRP1 activity (33). The latter finding enhances our computational predictions regarding the contribution of the Drosophila ABC transporters in the induction of COPDrelated phenotypes.…”

Effect of Corticosteroids on Peptide Transporter 2 Function and Induction of Innate Immune Response by Bacterial Peptides in Alveolar Epithelial Cells

“…Though the expression of PEPT2 mRNA was decreased by corticosteroid treatment, no apparent change in PEPT2 protein expression was observed under the present treatment conditions, as confirmed by Western blotting and by CLSM after immunostaining. Recently, Selo et al 26) examined the effect of inhaled compounds, including BUD and cigarette smoke extract (CSE), on multidrug resistance-associated protein-1 (MRP1; an efflux transporter) using human alveolar type II and type Ilike epithelial cells in primary culture and H441 cells. In that study, they first compared the mRNA and protein expression of MRP1 in type II and type I-like epithelial cells and found that MRP1 protein expression was significantly higher in type I-like cells than in type II cells, while the mRNA expression level was similar between these cells.…”

Alveolus Lung-on-a-Chip Platform: A Proposal

“…Adenocarcinomic alveolar A549 and NCI-H441 cell lines have commonly been used as a model for alveolar epithelium in different studies and seem to have a good ability to model lung injury and repair [22][23][24][25]. Furthermore, bronchial epithelial cell lines such as BEAS-2B are usually co-cultured with the epithelial cell line A549 to model airway epithelial cell injury in a cigarette smoke model [26], allergic airway inflammation [27], acute respiratory distress syndrome [28] and lung cancer [29].…”