Selo Mohammed Ali, Delmas Anne-Sophie, Springer Lisa, Zoufal Viktoria, Sake Johannes A, Clerkin Caoimhe G, Huwer Hanno, Schneider-Daum Nicole, Lehr Claus-Michael, Nickel Sabrina, Langer Oliver, Ehrhardt Carsten
School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
Faculty of Pharmacy, University of Kufa, Al-Najaf, Iraq.
Front Bioeng Biotechnol. 2020 Sep 8;8:1030. doi: 10.3389/fbioe.2020.01030. eCollection 2020.
Multidrug resistance-associated protein-1 (MRP1/) 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 . 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, 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 , 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 model to study MRP1 in distal lung epithelium.
多药耐药相关蛋白1（MRP1/）在人肺组织中高表达。最近的研究表明，无论是经肺部给药还是全身给药后，它都会显著影响其底物在肺部的处置。为了更好地理解其中涉及的分子机制，我们研究了原代培养的新鲜分离的人肺泡Ⅱ型上皮细胞（AT2）和Ⅰ型样上皮细胞（AT1样）以及NCI-H441细胞系中MRP1的表达、亚细胞定位和活性。此外，还研究了香烟烟雾提取物（CSE）和一系列吸入药物对MRP1丰度和活性的影响。通过q-PCR和免疫印迹法测量来自不同供体的AT2和AT1样细胞以及NCI-H441细胞系多个传代中的MRP1表达水平。通过共聚焦激光扫描显微镜和细胞表面蛋白生物素化研究转运蛋白的亚细胞定位。使用MRP1底物5(6)-羧基荧光素[CF；由5(6)-羧基荧光素二乙酸酯（CFDA）在细胞内形成]，通过双向转运和外排实验评估AT1样和NCI-H441细胞单层中MRP1的活性。此外，还研究了CSE以及几种支气管扩张剂和吸入性糖皮质激素对MRP1丰度和CF外排的影响。从AT2分化为AT1样表型时，MRP1蛋白丰度增加，然而基因水平保持不变。NCI-H441细胞中的MRP1丰度与AT1样细胞中的相当。在两种细胞类型的基底外侧膜中均主要检测到该转运蛋白，这与CF的基底外侧净外排一致。同样，双向转运研究显示CF从顶端到基底外侧的净转运，其对MRP1抑制剂MK-571敏感。布地奈德、二丙酸倍氯米松、硫酸沙丁胺醇和CSE以浓度依赖性方式降低CF外排。有趣的是，CSE增加MRP1丰度，而布地奈德、二丙酸倍氯米松、硫酸沙丁胺醇没有这种作用。CSE和吸入药物可降低MRP1活性，这意味着该转运蛋白是慢性阻塞性肺疾病（COPD）治疗中的潜在药物靶点。此外，人AT1样细胞和NCI-H441细胞中MRP1的表达水平、定位和活性相当。因此，该细胞系可作为研究远端肺上皮中MRP1的有用替代模型。
