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 apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.
Highlights
MATERIALS AND METHODSMultidrug resistance-associated protein-1 (MRP1, 190 kDa), a member of the ATP binding cassette (ABC) superfamily of transporters, is encoded by the ABCC1 gene (Cole, 2014a)
Inhibition of MRP1 was observed to worsen cigarette smoke extract (CSE)-induced cytotoxicity in vitro and pre-clinical and clinical data suggest that changes in abundance or function (Budulac et al, 2010) of the transporter are associated with occurrence and severity of chronic obstructive pulmonary disease (COPD)
Semi-quantitative real-time PCR analysis revealed ABCC1 mRNA expression at similar levels in freshly isolated alveolar type 2 epithelial (AT2) cells and cells differentiated into an AT1-like phenotype (Figure 1A)
Summary
MATERIALS AND METHODSMultidrug resistance-associated protein-1 (MRP1, 190 kDa), a member of the ATP binding cassette (ABC) superfamily of transporters, is encoded by the ABCC1 gene (Cole, 2014a). Glucuronate, and sulfate conjugates of drugs and endogenous molecules (Cole, 2014a,b). We have become interested in pulmonary MRP1 for two reasons, its impact on inhaled drugs disposition and its potential role as a target in the treatment of chronic obstructive pulmonary disease (COPD). Inhibition of MRP1 was observed to worsen cigarette smoke extract (CSE)-induced cytotoxicity in vitro (van der Deen et al, 2007) and pre-clinical and clinical data suggest that changes in abundance (van der Deen et al, 2006; Wu et al, 2019) or function (Budulac et al, 2010) of the transporter are associated with occurrence and severity of COPD. Recent in vivo data from our group showed that pulmonary distribution and clearance of the MRP1 substrate S(6-(7-[11C] methylpurinyl)) glutathione ([11C]MPG), measured with positron emission tomography (PET), was significantly dependent on MRP1 abundance (Mairinger et al, 2020)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
