Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro

Abstract

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.

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

“…The analysis was not carried out in freshly isolated AT2 cells due to their unpolarized phenotype. Apical or basolateral membrane proteins of confluent, Transwell-grown cell monolayers were labeled with sulpho-NHS-biotin (Thermo Fisher Scientific), isolated and analyzed as described in a previously published protocol ( Schwagerus et al, 2015 ).…”

Transient receptor potential vanilloid 1 (TRPV1)‐independent actions of capsaicin on cellular excitability and ion transport

“…In subsequent studies, capsazepine was shown to activate currents in ENaCδ1βγ‐ and ENaCδ2βγ‐expressing oocytes with EC 50 values of 6.3 and 11.9 µM, respectively 83 . In another study, capsazepine activated Na + currents in NCI‐H441 cells overexpressing ENaCδ but inhibited these currents in wild‐type cells 84 . However, high concentrations of capsazepine (250 µM) have been reported to inhibit currents in Xenopus oocytes expressing the rat ENaCαβγ channel 85 .…”

“…83 In another study, capsazepine activated Na + currents in NCI-H441 cells overexpressing ENaCδ but inhibited these currents in wild-type cells. 84 However, high concentrations of capsazepine (250 µM) have been reported to inhibit currents in Xenopus oocytes expressing the rat ENaCαβγ channel. 85 In summary, these results indicate that while capsaicin inhibits VGSCs, capsazepine inhibits ENaCs in a TRPV1-independent manner.…”

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro

“…Cell surface proteins were labelled with biotin, isolated and analysed according to a previously published protocol (34). Briefly, Transwell-grown cell monolayers were washed with ice-cold PBS and incubated for 20 min at 4°C with Pierce sulfo-NHS-biotin (Thermo Nuclear and cytoplasmic protein concentrations were determined by Pierce BCA protein assay kit and equal amounts of proteins were used for immunoblotting as described above.…”