The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.
CitationExpression and function of the epithelial sodium channelsubunit in human respiratory epithelial cells in vitro. ABSTRACTUsing human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested, however, protein abundance was low and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor) did not show effects on short-circuit current (I SC ),suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilinstimulated increases in I SC that were effectively blocked by Evans blue, and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.
The expression and function of the epithelial sodium channel δ subunit (δ‐ENaC) as well as its contribution to ion and fluid transport in human respiratory epithelial cells. Expression of δ‐ENaC was investigated in human alveolar (A549) and bronchial (Calu‐3, 16HBE14o‐) epithelial cell lines on genomic and protein levels. Reverse transcriptase and real‐time PCR were carried out as well as analysis by Western blot and immuno‐confocal laser scanning microscopy (CLSM). Ussing chamber studies were performed with Calu‐3 monolayers using Evans blue at 10 – 50 μM. Delta‐ENaC expression was confirmed in all cell lines on mRNA level with higher abundance in A549 and 16HBE14o‐ than in Calu‐3 cell monolayers. Generally, mRNA levels of δ‐ENaC were weaker than that of α, β and γ ENaC subunits. By Western blot and CLSM δ‐ENaC protein was detected in all cell lines, with the highest expression found in Calu‐3 and 16HBE14o‐ cells. CLSM also revealed the δ‐ENaC subunit located in the apical aspect of cellular membranes. Evans blue exhibited a concentration‐dependant increase of ISC up to 650% at 50 μM. These results indicate that ä‐ENaC is functionally expressed in human respiratory epithelial cell monolayers. Further experiments will investigate its role in ion and fluid transport processes across the respiratory epithelial barrier. ES is funded by an IRSCET postgraduate scholarship.
The expression and function of the epithelial sodium channel δ subunit (δ‐ENaC) as well as its contribution to ion and fluid transport in human respiratory epithelial cells.Expression of δ‐ENaC was investigated in human alveolar (A549) and bronchial (Calu‐3, 16HBE14o‐) epithelial cell lines on genomic and protein levels. Reverse transcriptase and real‐time PCR were carried out as well as analysis by Western blot and immuno‐confocal laser scanning microscopy (CLSM). Ussing chamber studies were performed with Calu‐3 monolayers using Evans blue at 10 – 50 μM.Delta‐ENaC expression was confirmed in all cell lines on mRNA level with higher abundance in A549 and 16HBE14o‐ than in Calu‐3 cell monolayers. Generally, mRNA levels of δ‐ENaC were weaker than that of α, β and γ ENaC subunits. By Western blot and CLSM δ‐ENaC protein was detected in all cell lines, with the highest expression found in Calu‐3 and 16HBE14o‐ cells. CLSM also revealed the δ‐ENaC subunit located in the apical aspect of cellular membranes. Evans blue exhibited a concentration‐dependant increase of ISC up to 650% at 50 μM.These results indicate that ä‐ENaC is functionally expressed in human respiratory epithelial cell monolayers. Further experiments will investigate its role in ion and fluid transport processes across the respiratory epithelial barrier.ES is funded by an IRSCET postgraduate scholarship.
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