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1.  Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition 
Biochimica et biophysica acta  2011;1808(7):1818-1826.
External Na+ self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- and external Na+ concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δβγ more potently than αβγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (βΔV348 and γH233R) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αβS520Cγ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αβγ and δβγ ENaC channels by relieving self-inhibition.
doi:10.1016/j.bbamem.2011.03.004
PMCID: PMC3091966  PMID: 21419751
2.  K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport 
Respiratory Research  2010;11(1):65.
Background
Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.
Methods
Verapamil-induced depression of heterologously expressed human αβγ ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM.
Results
The rate of in vivo AFC was reduced significantly (40.6 ± 6.3% of control, P < 0.05, n = 12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways.
Conclusions
Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.
doi:10.1186/1465-9921-11-65
PMCID: PMC2889873  PMID: 20507598
3.  Genetic Variation of αENaC Influences Lung Diffusion During Exercise in Humans 
Respiratory physiology & neurobiology  2011;179(2-3):212-218.
Exercise, decompensated heart failure, and exposure to high altitude have been shown to cause symptoms of pulmonary edema in some, but not all, subjects, suggesting a genetic component to this response. Epithelial Na+ Channels (ENaC) regulate Na+ and fluid reabsorption in the alveolar airspace in the lung. An increase in number and/or activity of ENaC has been shown to increase lung fluid clearance. Previous work has demonstrated common functional genetic variants of the α-subunit of ENaC, including an A→T substitution at amino acid 663 (αA663T). We sought to determine the influence of the T663 variant of αENaC on lung diffusion at rest and at peak exercise in healthy humans. Thirty healthy subjects were recruited for study and grouped according to their SCNN1A genotype [n= 17vs.13, age=25±7vs.30±10yrs., BMI= 23±4vs.25±4kg/m2, V̇O2peak= 95±30vs.100±31%pred., mean±SD, for AA (homozygous for αA663) vs. AT/TT groups (at least one αT663), respectively]. Measures of the diffusing capacity of the lungs for carbon monoxide (DLCO), the diffusing capacity of the lungs for nitric oxide (DLNO), alveolar volume (VA), and alveolar-capillary membrane conductance (DM) were taken at rest and at peak exercise. Subjects expressing the AA polymorphism of ENaC showed a significantly greater percent increase in DLCO and DLNO, and a significantly greater decrease in systemic vascular resistance from rest to peak exercise than those with the AT/TT variant (DLCO=51±12vs.36±17%, DLNO=51±24vs.32±25%, SVR=−67±3vs.−50±8%, p<0.05). The AA ENaC group also tended to have a greater percent increase in DLCO/VA from rest to peak exercise, although this did not reach statistical significance (49±26vs.33±26%, p=0.08). These results demonstrate that genetic variation of the α-subunit of ENaC at amino acid 663 influences lung diffusion at peak exercise in healthy humans, suggesting differences in alveolar Na+ and, therefore, fluid handling. These findings could be important in determining who may be susceptible to pulmonary edema in response to various clinical or environmental conditions.
doi:10.1016/j.resp.2011.08.007
PMCID: PMC3215907  PMID: 21893217
DLCO; DLNO; polymorphism; lung fluid balance; epithelial sodium channel
4.  Genetic Variation of the Alpha Subunit of the Epithelial Na+ Channels Influences Exhaled Na+ in Healthy Humans 
Respiratory physiology & neurobiology  2011;179(2-3):205-211.
Epithelial Na+ Channels (ENaC) are located on alveolar cells and are important in β2-adrenergic receptor-mediated lung fluid clearance through the removal of Na+ from the alveolar airspace. Previous work has demonstrated that genetic variation of the alpha subunit of ENaC at amino acid 663 is important in channel function: cells with the genotype resulting in alanine at amino acid 663 (A663) demonstrate attenuated function when compared to genotypes with at least one allele encoding threonine (T663, AT/TT). We sought to determine the influence of genetic variation at position 663 of ENaC on exhaled Na+ in healthy humans. Exhaled Na+ was measured in 18 AA and 13 AT/TT subjects (age=27±8 vs. 30±10yrs., ht.=174±12 vs. 171±10cm., wt=68±12 vs. 73±14kg., BMI=22±3 vs. 25±4kg/m2, mean±SD, for AA and AT/TT, respectively). Measurements were made at baseline and at 30, 60 and 90 minutes following the administration of a nebulized β2-agonist (albuterol sulfate, 2.5mg diluted in 3ml normal saline). The AA group had a higher baseline level of exhaled Na+ and a greater response to β2-agonist stimulation (baseline= 3.1±1.8 vs. 2.3±1.5mmol/l; 30min-post= 2.1±0.7 vs. 2.2±0.8mmol/l; 60min-post= 2.0±0.5 vs. 2.3±1.0mmol/l; 90min-post= 1.8±0.8 vs. 2.6±1.5mmol/l, mean±SD, for AA and AT/TT, respectively, p<0.05). The results are consistent with the notion that genetic variation of ENaC influences β2-adrenergic receptor stimulated Na+ clearance in the lungs, as there was a significant reduction in exhaled Na+ over time in the AA group.
doi:10.1016/j.resp.2011.08.008
PMCID: PMC3215916  PMID: 21889619
Breath Condensate; Airway Surface Fluid; Beta-agonist; SCNN1A; ADRB2; Gene
5.  ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 
EMBO Molecular Medicine  2010;2(1):26-37.
Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to β2-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by β2-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.
doi:10.1002/emmm.200900050
PMCID: PMC3377187  PMID: 20043279
β-agonist; elastase; prostasin; sodium channel; alveolar epithelium
6.  Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury 
Respiratory Research  2012;13(1):29.
Background
Stimulation of epithelial sodium channel (ENaC) increases Na+ transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo.
Methods
A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting.
Results
In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.
Conclusions
Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.
doi:10.1186/1465-9921-13-29
PMCID: PMC3362779  PMID: 22458687
Alveolar fluid clearance; Akt; Epithelial sodium channel; Insulin; Phosphatidylinositol 3-kinase; Acute lung injury
7.  Regulation of Alveolar Epithelial Na+ Channels by ERK1/2 in Chlorine-Breathing Mice 
The mechanisms by which the exposure of mice to Cl2 decreases vectorial Na+ transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na+ channels (ENaCs) after exposure to Cl2, and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (Iamil) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na+ channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl2, the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl2 increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased Iamil and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl2 decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in Iamil and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl2 activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.
doi:10.1165/rcmb.2011-0309OC
PMCID: PMC3326429  PMID: 21997487
lung slices; patch clamp; radicals
8.  α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo 
A variety of studies have shown that Na+ reabsorption across epithelial cells depends on the protease–antiprotease balance. Herein, we investigate the mechanisms by which α1-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na+ channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 μM) decreased ENaC currents across Xenopus laevis oocytes injected with human α,β,γ-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO−), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO−-treated A1AT (1 μM) in C57BL/6 mice also decreased Na+-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO−-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na+-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.
doi:10.1165/rcmb.2008-0384OC
PMCID: PMC2742747  PMID: 19131639
alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaC
9.  Essential structural features of TNF-α lectin-like domain derived peptides for activation of amiloride-sensitive sodium current in A549 cells 
Journal of medicinal chemistry  2010;53(22):8021-8029.
The amiloride-sensitive epithelial sodium channel (ENaC) plays a prominent role in sodium uptake from alveolar fluid, and is the major component in alveolar fluid clearance in normal and diseased lungs. The lectin-like domain of TNF-α has been shown to activate amiloride-sensitive sodium uptake in type II alveolar epithelial cells. Therefore, several synthetic peptides that mimic the lectin-like domain of TNF-α (TIP) were synthesised and their ability to enhance sodium current through ENaC was studied in A549 cells with the patch clamp technique. Our data suggest that a free positively-charged N-terminal amino group on residue 1 and/or a free negatively-charged carboxyl group on residue 17 of the TIP peptide is essential for the ENaC-activating effect. Ventilation strategies apart, no standard treatment exists for pulmonary permeability oedema. Therefore, novel therapies activating sodium uptake from the alveolar fluid via ENaC could improve clinical outcome.
doi:10.1021/jm100767p
PMCID: PMC2996585  PMID: 20979368
10.  AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers 
Transepithelial transport of Na+ across the lung epithelium via amiloride-sensitive Na+ channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na+ conductance (GNa+) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to GNa+: a 5-pS highly Na+ selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to −38 mV and 0 mV to −35 mV. Amiloride at 1 μM inhibited HSC activity and 56% of short-circuit current (Isc), whereas 10 μM amiloride partially reduced NSC activity and inhibited a further 30% of Isc. Neither conductance was associated with CNG channels as there was no effect of 10 μM pimoside on Isc, HSC, or NSC activity, and 8-bromo-cGMP (0.3–0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na+-permeable channels by a mechanism that likely reduces channel open probability.
doi:10.1152/ajplung.90353.2008
PMCID: PMC2584878  PMID: 18723760
5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMP-activated protein kinase; ENaC
11.  Respiratory distress and perinatal lethality in Nedd4-2-deficient mice 
Nature Communications  2011;2:287-.
The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2−/− animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2−/− animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.
In vitro studies have suggested that the ubiquitin ligase, Nedd4-2, regulates several proteins, including the epithelial sodium channel. Here by examining Nedd4-2-deficient mice, the authors demonstrate that Nedd4-2 is essential for epithelial sodium channel regulation, fetal and postnatal lung function and animal survival.
doi:10.1038/ncomms1284
PMCID: PMC3104547  PMID: 21505443
12.  Acute Regulation of the Epithelial Sodium Channel in Airway Epithelia by Proteases and Trafficking 
Effective clearance of inhaled pathogens is the primary innate defense mechanism in the lung, and requires the maintenance of a proper airway surface liquid (ASL) volume to facilitate ciliary beat and optimize mucociliary clearance. Na+ absorption via the epithelial sodium channel (ENaC) is tightly regulated and, together with chloride movement, provides the optimal osmotic gradients to absorb excessive fluid in the airway lumen while preventing excessive ASL dehydration, which would compromise mucus clearance from the lung. To absorb excessive fluid from the luminal surface, a local mechanism of ENaC activation allows for an increase in Na+ absorption at times when the ASL volume is expanded. To help define these regulatory mechanisms, we examined the effects of ASL volume expansion on ENaC activity in primary human bronchial epithelial (HBE) cell cultures. We found that ENaC activity increases dramatically after rapid dilution of endogenous ASL. Approximately 35% of the increase in Na+ absorption was attributable to activation of ENaC by proteases. The remainder of the increase in Na+ current was prevented when membrane trafficking was disrupted with brefeldin A, nocodazole, or myosin light chain kinase inhibitors, demonstrating that trafficking is involved with ENaC regulation in the airway. These findings demonstrate that Na+ absorption in the airway is acutely modulated by the coordinated trafficking of channels to the luminal surface and by the proteolytic activation of ENaC in response to ASL volume expansion.
doi:10.1165/rcmb.2009-0348OC
PMCID: PMC2993091  PMID: 20097829
ENaC; HBE; airway epithelium; airway surface liquid
13.  Hypotonic Shock Modulates Na+ Current via a Cl- and Ca2+/Calmodulin Dependent Mechanism in Alveolar Epithelial Cells 
PLoS ONE  2013;8(9):e74565.
Alveolar epithelial cells are involved in Na+ absorption via the epithelial Na+ channel (ENaC), an important process for maintaining an appropriate volume of liquid lining the respiratory epithelium and for lung oedema clearance. Here, we investigated how a 20% hypotonic shock modulates the ionic current in these cells. Polarized alveolar epithelial cells isolated from rat lungs were cultured on permeant filters and their electrophysiological properties recorded. A 20% bilateral hypotonic shock induced an immediate, but transient 52% rise in total transepithelial current and a 67% increase in the amiloride-sensitive current mediated by ENaC. Amiloride pre-treatment decreased the current rise after hypotonic shock, showing that ENaC current is involved in this response. Since Cl- transport is modulated by hypotonic shock, its contribution to the basal and hypotonic-induced transepithelial current was also assessed. Apical NPPB, a broad Cl- channel inhibitor and basolateral DIOA a potassium chloride co-transporter (KCC) inhibitor reduced the total and ENaC currents, showing that transcellular Cl- transport plays a major role in that process. During hypotonic shock, a basolateral Cl- influx, partly inhibited by NPPB is essential for the hypotonic-induced current rise. Hypotonic shock promoted apical ATP secretion and increased intracellular Ca2+. While apyrase, an ATP scavenger, did not inhibit the hypotonic shock current response, W7 a calmodulin antagonist completely prevented the hypotonic current rise. These results indicate that a basolateral Cl- influx as well as Ca2+/calmodulin, but not ATP, are involved in the acute transepithelial current rise elicited by hypotonic shock.
doi:10.1371/journal.pone.0074565
PMCID: PMC3760838  PMID: 24019969
14.  Genetic Variation of SCNN1A Influences Lung Diffusing Capacity in Cystic Fibrosis 
Introduction
Epithelial Na+ Channels (ENaC) play a crucial role in ion and fluid regulation in the lung. In cystic fibrosis (CF) Na+ hyperabsorption results from ENaC over activity, leading to airway dehydration. Previous work has demonstrated functional genetic variation of SCNN1A (the gene encoding the ENaC α-subunit), manifesting as an alanine (A) to threonine (T) substitution at amino acid 663, with the αT663 variant resulting in a more active channel.
Methods
We assessed the influence of genetic variation of SCNN1A on the diffusing capacity of the lungs for carbon monoxide (DLCO) and nitric oxide (DLNO), together with alveolar capillary membrane conductance (DM), pulmonary capillary blood volume (VC), and alveolar volume (VA) at rest and during peak exercise in 18 patients with CF [10 homozygous for αA663 (AA group) and 8 with at least one T663 allele (AT/TT group)]. Due to the more active channel we hypothesized that the AT/TT group would show a greater increase in DLCO, DLNO, and DM with exercise due to exercise-mediated ENaC inhibition and subsequent attenuation of Na+ hyperabsorption.
Results
The AT/TT group had significantly lower pulmonary function, weight and BMI than the AA group. Both groups had similar peak workloads, relative peak oxygen consumptions, and cardiopulmonary responses to exercise. The AT/TT group demonstrated a greater increase in DLNO, DLNO/VA, and DM in response to exercise (% increases: DLNO= 18±11vs.41±38; DLNO/VA= 14±21vs.40±37; DM= 15±11vs.41±38, AAvs.AT/TT, respectively). There were no differences between groups in absolute diffusing capacity measures at peak exercise.
Conclusion
These results suggest that genetic variation of the alpha-subunit of ENaC differentially affects the diffusing capacity response to exercise in patients with CF.
doi:10.1249/MSS.0b013e318266ebc3
PMCID: PMC3501594  PMID: 22776878
exercise; cystic fibrosis; diffusing capacity; DLNO; DLCO; ENaC polymorphism
15.  Expression and Regulation of Epithelial Na+ Channels by Nucleotides in Pleural Mesothelial Cells 
Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na+ channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, α, β, γ, and two δ ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that α, β, γ, and δ ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 μM in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (Km: 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li+ > Na+ > K+. The unitary Li+ conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na+ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.
doi:10.1165/rcmb.2008-0166OC
PMCID: PMC2677435  PMID: 18927349
M9K mesothelioma cells; Ussing chamber; protein kinase A; protein kinase G; human primary mesothelial cells
16.  Regulation of the epithelial sodium channel [ENaC] in kidneys of salt-sensitive Dahl rats: Insights on alternative splicing 
The epithelial sodium channel [ENaC] is critical for the maintenance of sodium balance, extracellular fluid volume and long term blood pressure control. Monogenic disorders causing ENaC hyperactivity have led to a severe form of hereditary hypertension in humans, known as Liddle's syndrome. Similarly, in animal models, ENaC hyperactivity has been well documented in kidneys of salt-sensitive [S] Dahl rats [a genetic model of salt-sensitive hypertension] versus their normotensive control [Dahl salt-resistant [R] rats]. The purpose of the present review is to highlight the differential regulation of ENaC in kidneys of Dahl S versus R rats. A systematic overview of the putative role of alternative splicing of the main α subunit of ENaC [α ENaC] in modulating ENaC expression in kidneys of Dahl rats will be discussed. Finally, a better understanding of the meaningful contribution of ENaC in the pathogenesis of salt-sensitive hypertension will be achieved upon completion of this review.
doi:10.1186/1755-7682-2-28
PMCID: PMC2761857  PMID: 19785774
17.  The Epithelial Sodium Channel α subunit (α ENaC) alternatively spliced form "b" in Dahl rats: What's next? 
Background
The amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits α, β, & γ. While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function. To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major α ENaC transcript. While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC.
Presentation of the hypothesis
α ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner.
Testing the hypothesis
1) To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of α ENaC-b proteins to full-length α ENaC in vitro and to determine the impact of such binding on full-length α ENaC expression in vitro.
Implications of the hypothesis
Our studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.
doi:10.1186/1755-7682-3-14
PMCID: PMC2909934  PMID: 20604958
18.  Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel 
The Journal of General Physiology  2012;140(4):375-389.
Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.
doi:10.1085/jgp.201110763
PMCID: PMC3457690  PMID: 22966015
19.  Novel Role for CFTR in Fluid Absorption from the Distal Airspaces of the Lung 
The Journal of General Physiology  2002;119(2):199-208.
The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na+ absorption, the route of Cl− transport has been unclear. We applied a series of complementary approaches to define the role of Cl− transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis ΔF508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl− channel inhibitors and Cl− ion substitution, providing evidence for a transcellular route for Cl− transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in ΔF508 mice. Radioisotopic clearance studies done at 23°C (to block active fluid absorption) showed ∼20% clearance of 22Na in 30 min both without and with isoproterenol. However, the clearance of 36Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in ΔF508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl− transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in ΔF508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.
PMCID: PMC2233804  PMID: 11815669
pulmonary edema; cystic fibrosis; lung epithelium; cAMP; lung fluid balance
20.  Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel 
Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.
doi:10.1155/2011/978196
PMCID: PMC3196915  PMID: 22028593
21.  Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells 
Respiratory Research  2010;11(1):141.
Background
Hyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.
Methods
We evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.
Results
Neither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4.
Conclusions
These results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.
doi:10.1186/1465-9921-11-141
PMCID: PMC2959028  PMID: 20932306
22.  Ketamine Inhibits Lung Fluid Clearance through Reducing Alveolar Sodium Transport 
Ketamine is a broadly used anaesthetic for analgosedation. Accumulating clinical evidence shows that ketamine causes pulmonary edema with unknown mechanisms. We measured the effects of ketamine on alveolar fluid clearance in human lung lobes ex vivo. Our results showed that intratracheal instillation of ketamine markedly decreased the reabsorption of 5% bovine serum albumin instillate. In the presence of amiloride (a specific ENaC blocker), fluid resolution was not further decreased, suggesting that ketamine could decrease amiloride-sensitive fraction of AFC associated with ENaC. Moreover, we measured the regulation of amiloride-sensitive currents by ketamine in A549 cells using whole-cell patch clamp mode. Our results suggested that ketamine decreased amiloride-sensitive Na+ currents (ENaC activity) in a dose-dependent fashion. These data demonstrate that reduction in lung ENaC activity and lung fluid clearance following administration of ketamine may be the crucial step of the pathogenesis of resultant pulmonary edema.
doi:10.1155/2011/460596
PMCID: PMC3185271  PMID: 21976965
23.  The Alternatively Spliced Form “b” of the Epithelial Sodium Channel α subunit (α ENaC): Any Prior Evidence of its Existence? 
The epithelial sodium channel (ENaC) is critical in maintaining sodium balance across aldosterone-responsive epithelia. ENaC is a combined channel formed of three subunits (αβγ) with α ENaC subunit being the most critical for channel functionality. In a previous report, we have demonstrated the existence and mRNA expression levels of four alternatively spliced forms of the α ENaC subunit denoted by -a, -b, -c and -d in kidney cortex of Dahl S and R rats. Of the four alternatively spliced forms presently identified, α ENaC-b is considered the most interesting for the following reasons: Aside from being a salt-sensitive transcript, α ENaC-b mRNA expression is ∼32 fold higher than α ENaC wildtype in kidney cortex of Dahl rats. Additionally, the splice site used to generate α ENaC-b is conserved across species. Finally, α ENaC-b mRNA expression is significantly higher in salt-resistant Dahl R rats versus salt-sensitive Dahl S rats. As such, this commentary aims to highlight some of the previously published research articles that described the existence of an additional protein band on α ENaC western blots that could account for α ENaC-b in other rat species.
PMCID: PMC2937307  PMID: 20842211
α ENaC-b; Dahl rats; salt-sensitive hypertension; Western analysis
24.  Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells 
Physiological Reports  2014;2(3):e00269.
Abstract
Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19‐day gestational age rat fetuses. Equivalent short‐circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose‐dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5‐(N‐Ethyl‐N‐isopropyl)amiloride inhibition of ISC was not affected, however, benzamil‐sensitive ISC was increased in insulin‐stimulated monolayers. The application of LY‐294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF‐1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF‐1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.
Insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC, which is dependent on the activity of PI3K and AKT, but not on the activity of SGK1 in alveolar cells.
doi:10.1002/phy2.269
PMCID: PMC4002249  PMID: 24760523
AKT; alveolar cells; epithelial Na+ channel (ENaC); insulin; SGK1
25.  Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis 
Airway surface liquid (ASL) absorption is initiated by Na+ entry via epithelial Na+ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surface. We and others have recently reported that a protease/anti-protease balance regulates ENaC in human airway epithelial cells (HAEC) and provides a mechanism for autoregulation of ASL volume. In cystic fibrosis (CF), this balance is disturbed, leading to constitutive proteolytic activation of ENaC and the pathological Na+ hyperabsorption characteristic of this airway disease. Prostasin is a glycosylphosphatidylinositol-anchored serine protease that activates ENaC and is expressed on the surface epithelium lining the airway. In this report we present evidence that prostasin expression is regulated by the ASL volume, allowing for increased proteolytic activation of ENaC when the ASL volume is high. Prostasin activity is further regulated by the cognate serpin protease nexin-1 (PN-1), which is expressed in HAEC and inhibits Na+ absorption by forming an inactive complex with prostasin and preventing the proteolytic processing of prostasin. Whereas these mechanisms regulate prostasin expression in response to ASL volume in non-CF epithelia, HAEC cultured from CF patients express >50% more prostasin on the epithelial surface. These findings suggest that a proteolytic cascade involving prostasin, an upstream prostasin-activating protease, and PN-1 regulate Na+ absorption in the airway and that abnormal prostasin expression contributes to excessive proteolytic activation of ENaC in CF patients.
doi:10.1152/ajplung.00437.2007
PMCID: PMC2387194  PMID: 18310226
epithelial sodium channels; protease nexin 1; bronchial epithelial cultures; matriptase

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