The amiloride-sensitive epithelial sodium channel (ENaC) is usually found in the apical membrane of epithelial cells but has also recently been described in vascular endothelium. Because little is known about the regulation and cell surface density of ENaC, we studied the influence of aldosterone, spironolactone, and amiloride on its abundance in the plasma membrane of human endothelial cells. Three different methods were applied, single ENaC molecule detection in the plasma membrane, quantification by Western blotting, and cell surface imaging using atomic force microscopy. We found that aldosterone increases the surface expression of ENaC molecules by 36% and the total cellular amount by 91%. The aldosterone receptor antagonist spironolactone prevents these effects completely. Acute application of amiloride to aldosterone-pretreated cells led to a decline of intracellular ENaC by 84%. We conclude that, in vascular endothelium, aldosterone induces ENaC expression and insertion into the plasma membrane. Upon functional blocking with amiloride, the channel disappears from the cell surface and from intracellular pools, indicating either rapid degradation and/or membrane pinch-off. This opens new perspectives in the regulation of ENaC expressed in the vascular endothelium.

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The epithelial sodium channel (ENaC) mediates the first step in Na+ reabsorption in epithelial cells such as kidney, colon, and airways and may consist of four homologous subunits (alpha, beta, gamma, delta). Predominantly, the alpha-subunit is expressed in these epithelia, and it usually forms functional channels with the beta- and gamma-subunits. The delta-subunit was first found in human brain and kidney, but the expression was also detected in human cell lines of lung, pancreatic, and colonic origin. When co-expressed with beta and gamma accessory subunits in heterologous systems, the two known isoforms of the delta-ENaC subunit (delta1 and delta2) can build amiloride-sensitive Na+ channels. In the present study we demonstrate the expression and function of the delta-subunit in human nasal epithelium (HNE). We cloned and sequenced the full-length cDNA of the delta-ENaC subunit and were able to show that in nasal tissue at least isoform 1 is expressed. Furthermore, we performed Western blot analyses and compared the cell surface expression of the delta-subunit with the classically expressed alpha-subunit by using immunofluorescence experiments. Thereby, we could show that the quantity of both subunits is almost similar. In addition, we show the functional expression of the delta-ENaC subunit with measurements in modified Ussing chambers, and demonstrate that in HNE a large portion of the Na+ transport is mediated by the delta-ENaC subunit. Therefore, we suppose that the delta-subunit may possess an important regulatory function and might interact with other ENaC subunits or members of the DEG/ENaC family in the human respiratory epithelium.

Cystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl(-) secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the alpha- and beta-hnENaC protein amount was increased in CF tissue the gamma-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF.

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC50 (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5′ RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC50 = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.