Amiloride-sensitive Short-circuit Current Research Articles

Functional in vivo effects of an ENaC Na+ self inhibition-enhancing mutation

Background: Epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron is a critical regulator of extracellular fluid volume and K+ homeostasis. Physiologic concentrations of extracellular Na+ reduce channel activity (open probability; PO) via an allosteric mechanism termed “Na+ self-inhibition,” which involves Na+ ions binding to specific sites in the channel’s extracellular domains. Recent studies have identified human ENaC mutations that alter the Na+ self-inhibition response when assayed in vitro. Such mutations may confer sensitivity or resistance to salt-induced blood pressure changes. Using CRISPR-Cas9 technology, we generated mice carrying a mutation previously shown to enhance Na+ self-inhibition (αH282R) to assess the effects on ENaC-dependent Na+ transport in vivo. We predicted that ENaC activity would be reduced in these mice, leading to enhanced sensitivity to Na+ deprivation or K+ loading, along with accompanying compensatory changes to overcome the predicted loss of function. Methods: We measured urinary electrolyte handling in wild type (WT) and αH282R mice maintained on normal, low Na+, or high K+ diets using metabolic cages. We assessed ENaC activity ex vivo using patch clamp and Ussing chamber methods. We also measured blood chemistry and aldosterone levels in WT and αH282R mice under the dietary conditions described above. Results: When maintained on a normal chow diet (0.3% NaCl), αH2832R mice expressed ENaCs in the distal nephron with reduced PO compared to WT littermates (0.026 vs. 0.067; p<0.01), while ENaC subunit protein expression was unaltered. Amiloride-sensitive short-circuit currents were reduced in the distal colons of αH283R mice versus WT littermates (-12.6 vs. -3.9 μA/cm2; p<0.01). Plasma aldosterone levels were also higher in αH283R versus WT mice on a control diet (610 vs 288 pg/mL; p=0.02), while no other differences in baseline blood chemistry were observed. When challenged with either dietary Na+ restriction (<0.01% NaCl) or K+ loading (10% KCl), WT and αH283R mice responded similarly in terms of body weight maintenance, urinary Na+ and K+ excretion, and systemic electrolyte and metabolic parameters, with the exception of higher aldosterone levels in Na+-restricted αH282R mice compared to WT littermates (2,759 vs. 1271 pg/mL; p<0.01). Conclusions: The αH282R mutation, which enhances Na+ self-inhibition, confers reduced ENaC activity in mice. However, this loss of function may be compensated by ENaC-independent Na+/K+ handling mechanisms and/or increased aldosterone signaling during adaptation to dietary salt stressors. P30DK079307, T32DK061296. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FXYD3 facilitates Na+ and liquid absorption across human airway epithelia by increasing the transport capacity of the Na/K ATPase.

Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced FXYD3 expression using siRNA. After permeabilizing the apical membrane with nystatin, epithelia pretreated with FXYD3-targeting siRNA had lower ouabain-sensitive short-circuit currents than control epithelia. FXYD3-targeting siRNA also reduced amiloride-sensitive short-circuit currents and liquid absorption across intact epithelia. These data are consistent with FXYD3 facilitating Na+ and liquid absorption. FXYD3 may be needed to maintain the high rates of Na+ and fluid absorption observed for airway and other FXYD3-expressing epithelia.

Hypoxia Aggravates Inhibition of Alveolar Epithelial Na-Transport by Lipopolysaccharide-Stimulation of Alveolar Macrophages.

Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O2). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISCΔamil); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.

FXYD3 increases Na+ transport across human airway epithelia

The Na/K ATPase localizes within the basolateral membrane of most epithelia, where Na/K ATPase activity mediates Na+ absorption. The Na/K ATPase requires its alpha subunit for ion transport and its beta subunit for proper trafficking to the plasma membrane. The non‐essential gamma subunit of the Na/K ATPase modifies Na/K ATPase activity. Datasets within the NIH Gene Expression Omnibus indicate that FXYD3, a Na/K ATPase gamma subunit, is highly expressed by human airway epithelia. However, how FXYD3 affects airway epithelial function remains unstudied. In addition to binding the Na/K ATPase, FXYD3 can bind and modify H/K ATPases in heterologous expression systems. The H/K ATPase ATP12A acidifies human airway surface liquid. Therefore, we evaluated the role of FXYD3 in both acid secretion and Na+ absorption across human airway epithelia. Single cell RNA sequencing revealed high FXYD3 expression for all the airway cell types, and immunocytochemistry revealed FXYD3 expression within the basolateral membrane of airway epithelia. Consistent with basolateral FXYD3 localization, the airway surface liquid pH, established in part by apical H/K ATPase, was unaffected by siRNA‐mediated knockdown of FXYD3. FXYD3 knockdown decreased amiloride‐sensitive short‐circuit currents (~20% in NaCl and ~40% in NaGluconate solutions), a finding that is consistent with FXYD3 increasing Na/K ATPase activity. Consequently, fluid absorption across airway epithelia was reduced by ~20% with FXYD3 knockdown. FXYD3 may be required to efficiently return the airway surface liquid to homeostatic volumes after fluid secretion into the proximal airways.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Abstract P002: Soluble (pro)renin Receptor Contributes To Angiotensin II-Induced Hypertension In Mice

Activation of (pro)renin receptor (PRR) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during angiotensin II (AngII) infusion. Soluble PRR (sPRR), the extracellular domain of PRR, is generated by multiple proteases including furin or ADAM19, and recently site-1 protease (S1P). The goal of the present study was to test the role of S1P-derived sPRR mediated AngII-induced hypertension. F1 B6129SF1/J mice were infused for 6 days with control (CTR) or AngII at 300 ng/kg/day alone or in combination with S1P inhibitor PF-429242 (PF) and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated AngII-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histadine-tagged sPRR termed as sPRR-His. Mutagenesis of overlapping recognition site for S1P and furin in PRR for was generated in mice by CRISPR strategy (termed as mutant mice). Mutant mice were fertile and developed normally with a 50% reduction plasma sPRR. These mice exhibited blunted hypertensive response to AngII infusion accompanied with suppressed intrarenal renin levels. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mpkCCD cells exposed for 24 h to AngII, AngII + PF, or AngII + PF + sPRR-His. AngII-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that sPRR derived from S1P or in combination with furin mediates AngII-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Sorting Nexins 1, 2, 3 and 17 Regulate Epithelial Sodium Channel Trafficking

The epithelial sodium channel (ENaC) facilitates sodium absorption of polarized epithelia and therefore, is required for regulation of salt and water homeostasis. ENaC’s apical membrane population is strictly controlled, with loss of this control leading to hyper‐ or hypotensive disorders such as Liddle’s Syndrome, or Pseudohypoaldosteronism type 1, respectively. Retromer and retriever are conserved endosome‐localized protein trafficking complexes that mediate recycling of membrane proteins back to the cell surface either directly, via recycling endosomes or via the trans‐Golgi network. Both retromer and retriever associate with accessory proteins including sorting nexins (SNX) proteins which play a role in the establishment of a tubulation complex leading to the formation of endosomal tubule‐shaped extensions which contain the cargo selected for recycling. We hypothesized that retromer and/or retriever are required for ENaC recycling. Using three epithelial cell lines (Fischer rat thyroid (FRT), mouse cortical collecting duct (mCCD) or human embryonic kidney cells (HEK293), we found that siRNA knockdown of retromer and retriever associated SNX proteins reduced ENaC amiloride‐sensitive short circuit current (Isc‐Amil). SNX1 and SNX2 are reported to be essential for cargo recycling through retromer. Individually, knockdown of SNX1 (n=6) or SNX2 (n=8) reduced Isc‐Amil by 38±9% and 39±8% compared with control FRT epithelia, respectively. Double‐knockdown of those SNXs reduced Isc‐Amil by 47±1% (n=6) compared with control FRT epithelia. SNX3 is known to bind directly with the retromer complex. Knockdown of SNX3 decreased Isc‐Amil by 47±5% FRT (n=9) and 35±4% in mCCD (n=9) epithelia compared with control epithelia. Additionally, it is known that SNX17 mediates cargo retrieval via the retriever complex. Knockdown of SNX17 lowered Isc‐Amil by 56±1% (n=9) in FRT and 35±6% (n=9) in mCCD epithelia compared with control epithelia. With cell surface biotinylation experiments and using FRT cells, knockdown of SNX3 and SNX17 reduced the ENaC cell surface population by 68±25% (n=3) and 77±17% (n=3), respectively, thus, attributing to the reduced Isc‐Amil. Furthermore, co‐immunoprecipitation experiments of αβHAγENaC overexpressed in HEK293 cells, demonstrated a protein‐protein interaction between retriever associated SNX17 and ENaC (n=3) suggesting SNX17 may act as a cargo binding protein between ENaC and the retriever complex. However, there was no evidence for an interaction between SNX3 and ENaC (n=3). Together, our findings suggest that SNX protein complexes play a role in ENaC recycling to the plasma membrane in polarized epithelia. Additionally, these data suggest that both the retromer and retriever recycling complexes may aid in the trafficking of ENaC to the plasma membrane.Support or Funding InformationThis work was supported by a grant from the New Zealand Lottery Health Board. Further support was provided by the School of Biomedical Sciences and the Department of Physiology of the University of Otago.

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption.

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (−55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction—both of which contribute to Na+ malabsorption and diarrhea.

Dexmedetomidine enhances human lung fluid clearance through improving alveolar sodium transport.

Accumulating evidence shows that dexmedetomidine can attenuate lung edema with acute lung injury in experimental mouse and rat models, but the mechanisms of dexmedetomidine on human alveolar fluid transport are still unknown. We measured the effects of dexmedetomidine on alveolar fluid clearance in human lung lobes ex vivo. Moreover, we measured the regulation of transepithelial Na+ transport by dexmedetomidine in H441 cells by electrophysiological technique and Western blot method. Our results showed that intratracheal instillation of dexmedetomidine markedly increased the reabsorption of 5% bovine serum albumin instillate (19.8 ± 1.4%, P < 0.01 vs. Control, n = 5). Further studies suggested that dexmedetomidine increased amiloride-sensitive short-circuit currents in permeabilized H441 monolayers and whole cell amiloride-sensitive Na+ currents in a dose-dependent fashion. Real-time PCR and Western blot results showed that dexmedetomidine could enhance the mRNA and protein expression of α-ENaC subunit, while inhibiting the phosphorylation of ERK1/2 . These data demonstrate that dexmedetomidine could improve human lung fluid clearance and lung epithelial Na+ channel activity, and these effects may be mediated through the enhancement of α-ENaC expression and inhibition of ERK1/2 pathway.

1,25-Dihydroxyvitamin D Enhances Alveolar Fluid Clearance by Upregulating the Expression of Epithelial Sodium Channels

Vitamin D is implicated in the pathogenesis of asthma, acute lung injury, and other respiratory diseases. 1,25-Dihydroxyvitamin D (1,25(OH)2D3), the hormonal form of vitamin D, has been shown to reduce vascular permeability and ameliorate lung edema. Therefore, we speculate that 1,25(OH)2D3 may regulate alveolar Na+ transport via targeting epithelial Na+ channels (ENaC), a crucial pathway for alveolar fluid clearance. In vivo total alveolar fluid clearance was 39.4 ± 3.8% in 1,25(OH)2D3-treated mice, significantly greater than vehicle-treated controls (24.7 ± 1.9 %, n = 10, p < 0.05). 1,25(OH)2D3 increased amiloride-sensitive short-circuit currents in H441 monolayers, and whole-cell patch-clamp data confirmed that ENaC currents in single H441 cell were enhanced in 1,25(OH)2D3-treated cells. Western blot showed that the expression of α-ENaC was significantly elevated in 1,25(OH)2D3-treated mouse lungs and 1,25(OH)2D3-treated H441 cells. These observations suggest that vitamin D augments transalveolar fluid clearance, and vitamin D therapy may potentially be used to ameliorate pulmonary edema.

Harvest and primary culture of the murine aldosterone-sensitive distal nephron.

The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm(2)) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.

Apical volume expansion results in an immediate ENaC activation but in delayed onset of water resorption (718.1)

Apical volume expansion results in an activation of the epithelial sodium channel ENaC. Its activation is proposed to facilitate water resorption and thus to counteract increases of apical surface liquid (ASL) volume e.g. in lung edema. In this study we investigated the adaptation of epithelial water resorption to apical volume expansion (AVE) in NCI‐H441 epithelia. Cells were cultivated at air‐liquid interface. AVE was introduced by adding isotonic NaCl solution onto the apical surface. Water resorption was measured by the recently established deuterium oxide dilution method (Neubauer et al. 2013, Anal Chem). ENaC channel activation was quantified in Ussing chamber experiments as amiloride sensitive short circuit currents. As expected, ENaC activity increases to its maximum within 15 min after AVE. Water resorption does not follow ENaC activation but starts 4 to 6 h after AVE. When ASL volume is reduced to 3.7 ±0.2 μl / 0.33 cm^2 water resorption stops and ENaC activity is reduced to basal levels. Our data demonstrate that AVE results in a rapid ENaC upregulation but ENaC activation is not the only trigger of onset of water resorption. However, off set of water resorption is associated with ENaC down regulation.Supported by BIU D.5007Grant Funding Source: Supported by BIU.5007

Effect of apical hyperosmotic sodium challenge and amiloride on sodium transport in human bronchial epithelial cells from cystic fibrosis donors

Hypertonic saline (HS) inhalation therapy benefits cystic fibrosis (CF) patients [Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006; Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT; the National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. N Engl J Med 354: 229-240, 2006]. Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na(+) channel blocker amiloride (Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: 1) Ussing chambers to measure amiloride-sensitive short-circuit currents (INa); 2) continuous perfusion Ussing chambers; and 3) near "thin-film" conditions in which the airway surface of the inserts was exposed to a small volume (30 μl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near thin-film experiments; Tarran R, Boucher RC. Methods Mol Med 70: 479-492, 2002). HS solutions (660 mosmol/kgH2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT), and capacitance (CT) were measured by transepithelial impedance analysis (Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, Bridges RJ. Am J Physiol Lung Cell Mol Physiol 290: L558-L569, 2006; Singh AK, Singh S, Devor DC, Frizzell RA, van Driessche W, Bridges RJ. Methods Mol Med 70: 129-142, 2002). Exposure to apical HS inhibited INa, GT, and CT. The INa inhibition required 60 min of reexposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was <2.5 min. Under near thin-film conditions, apical exposure to HS inhibited INa, but as osmotically driven water moved to the apical surface, the aqueous apical volume increased, leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Our conclusions are that exposure to HS inhibits hBE INa and that amiloride diminishes this effect.

Regulation of the delta and alpha epithelial sodium channel (ENaC) by ubiquitination and Nedd8

The δ epithelial sodium channel (δENaC) is a proton-activated, sodium-selective, amiloride-sensitive ion channel in the ENaC/degenerin family of ion channels involved in blood pressure regulation and mechanosensation. Other ENaC family members are subject to ubiquitin modification leading to internalization from the cell surface, and degradation of the channel. Here, we show that δENaC is also modified by ubiquitin on three intracellular lysine residues. Absence of these lysines abolished ubiquitin modification of δENaC and increased cell surface levels of δENaC. Although the HECT-domain ubiquitin ligase Nedd4-2 reduced amiloride-sensitive current generated by δβγENaC-containing channels, δENaC does not contain a binding site for Nedd4-2; therefore, this effect is probably mediated by the βγENaC subunits. Nedd8, a ubiquitin-like protein that regulates RING-domain E3 ubiquitin ligases, promoted δENaC ubiquitination, decreased both the intracellular and cell surface δENaC populations, and decreased δβγENaC amiloride-sensitive short circuit current (Isc -amiloride) in a mammalian epithelium. Nedd8 also promoted α- and γENaC ubiquitination, decreased the cell surface pools, and decreased αβγENaC Isc -amiloride. Conversely, XIAP, a single subunit RING E3 ligase, decreased ubiquitinated δENaC, increased the δENaC cell surface pool and increased δβγENaC Isc -amiloride. Therefore δ- and α - βγENaC channel function may be influenced by RING-domain E3 ubiquitin ligases.

Severe Defects in Absorptive Ion Transport in Distal Colons of Mice That Lack ClC-2 Channels

The fluid secretion model predicts that intestinal obstruction disorders can be alleviated by promoting epithelial Cl(-) secretion. The adenosine 3',5'-cyclic monophosphate (cAMP)-activated anion channel CFTR mediates Cl(-)-dependent fluid secretion in the intestine. Although the role of the ClC-2 channel has not been determined in the intestine, this voltage-gated Cl(-) channel might compensate for the secretory defects observed in patients with cystic fibrosis and other chronic constipation disorders. We investigated whether mice that lack ClC-2 channels (Clcn2(-/-)) have defects in intestinal ion transport. Immunolocalization and immunoblot analyses were used to determine the cellular localization and the amount of ClC-2 expressed in mouse early distal colon (EDC) and late distal colon (LDC). Colon sheets from wild-type and Clcn2(-/-) littermates were mounted in Ussing chambers to determine transepithelial bioelectrical parameters and Na(+), K(+), and Cl(-) fluxes. Expression of ClC-2 was higher in the basolateral membrane of surface cells in the EDC compared with the LDC, with little expression in crypts. Neither cAMP nor Ca(2+)-induced secretion of Cl(-) was affected in the EDC or LDC of Clcn2(-/-) mice, whereas the amiloride-sensitive short-circuit current was increased approximately 3-fold in Clcn2(-/-) EDC compared with control littermates. Conversely, electroneutral Na(+), K(+), and Cl(-) absorption was dramatically reduced in colons of Clcn2(-/-) mice. Basolateral ClC-2 channels are required for colonic electroneutral absorption of NaCl and KCl. The increase in the amiloride-sensitive short-circuit current in Clcn2(-/-) mice revealed a compensatory mechanism that is activated in the colons of mice that lack the ClC-2 channel.

Regulation of Alveolar Epithelial Na+ Channels by ERK1/2 in Chlorine-Breathing Mice

The mechanisms by which the exposure of mice to Cl(2) 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 Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) 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 Cl(2), 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 Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.

Impaired aldosterone responsiveness in corticosteroid binding globulin deficient mice

Corticosteroid binding globulin (CBG) is the high affinity plasma carrier protein for cortisol. It keeps the steroids inactive, prevents them from degradation and defines the amount of free hormone acting on target tissues. Previous findings have shown insufficient responsiveness of corticosterone in peripheral tissues in CBG⁻(/)⁻ mice despite elevated free plasma corticosterone. In the large intestine, glucocorticoids synergistically enhance the pro-absorptive effects of aldosterone. We therefore hypothesized that CBG⁻(/)⁻ mice have reduced responsiveness to aldosterone. We used CBG⁻(/)⁻ and CBG(+/+) mice to investigate distal colonic electrogenic Na(+) absorption. An Ussing chamber was used to quantify amiloride-sensitive Na(+) transport in distal colonic mucosa (ΔI(sc) (amil)) as a measure of the physiological effect of aldosterone. No differences were observed in ΔI(sc) (amil) or aldosterone levels in animals on control diet. When Na(+) restricted, CBG(+/+) mice responded with a marked up-regulation of ΔI(sc) (amil) (25-fold). In CBG⁻(/)⁻ mice this up-regulation was greatly attenuated as seen in a markedly reduced amiloride-sensitive short circuit current (reduced by ∼50%), a reduced ability to lower faecal Na(+) excretion and a significantly attenuated up-regulation of the ENaC channel γ-subunit. Diet-induced increases of total plasma aldosterone were similar in both genotypes, but CBG⁻(/)⁻ mice had an increased free plasma aldosterone fraction. This study defines the functional hyporesponsiveness and aldosterone resistance in distal colon of CBG⁻(/)⁻ mice. This resistance occurs despite sufficient free corticosterone plasma level. Thus, steroid actions require an intrinsic but unknown function of CBG, which allows the sufficient supply of the hormone/s to the target tissue.

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl(-) channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na(+) conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl(-) current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl(-) current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn(2+) sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn(2+) (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl(-) channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na(+) transport in this surface epithelium under basal physiological conditions.

Cholera Toxin Enhances ENaC‐Mediated Sodium Absorption Across Cultured Human Mammary Epithelial Cells

Mechanisms that control ion transport across human mammary gland and contribute to the unique low sodium concentration in human milk have not been fully described. When grown on permeable supports, MCF10A cells, an epithelial cell line derived from human mammary gland, exhibited amiloride‐sensitive ion transport, suggesting activity of the epithelial Na+ channel, ENaC. When cultured in the presence of cholera toxin, MCF10A cells exhibited significantly greater amiloride sensitive short circuit current (Isc) at all time points tested (2 h to 7 d). Maximal amiloride‐sensitive Isc was observed with 1 d exposure to cholera toxin (4.76 ± 0.73 μA cm−2) when compared to vehicle‐treated monolayers (1.19 ± 0.27 μA cm−2). Effects of cholera toxin on Isc were neither mimicked by forskolin nor inhibited by H‐89 (10 μM), a protein kinase A (PKA) inhibitor. RT‐PCR analyses showed that cholera toxin increased the copy number for mRNA coding for each of the three ENaC subunits by 2 to 4 fold. Taken together, these results suggest that human mammary epithelial cells express ENaC, a mechanism that can remove Na+ from milk. Furthermore, the results suggest a novel mechanism to regulate ENaC expression that involves cholera toxin, but that is independent of cAMP and PKA. [NIH (P20‐RR017686) and KS Ag Exp Stn support]

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.