Regulation Of Epithelial Sodium Channel Research Articles

Mesenchymal stem cell conditioned medium alleviates acute lung injury through KGF-mediated regulation of epithelial sodium channels

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.

Critical Role of mTORC2 in Maintaining Sodium and Potassium Balance: Implications for Aldosterone-Independent Regulation of Epithelial Sodium Channel (ENaC) in the Distal Nephron

Critical Role of mTORC2 in Maintaining Sodium and Potassium Balance: Implications for Aldosterone-Independent Regulation of Epithelial Sodium Channel (ENaC) in the Distal Nephron

Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate Epithelial Sodium Channel: Role in Rapid Renal Responses to Dietary Potassium.

Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K +in vivo .

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Lessons learned about epithelial sodium channels from transgenic mouse models.

This review provides an up-to-date understanding about the regulation of epithelial sodium channel (ENaC) expression and function. In particular, we will focus on its implication in renal Na+ and K+ handling and control of blood pressure using transgenic animal models. In kidney, the highly amiloride-sensitive ENaC maintains whole body Na+ homeostasis by modulating Na+ transport via epithelia. This classical role is mostly confirmed using genetically engineered animal models. Recently identified key signaling pathways that regulate ENaC expression and function unveiled some nonclassical and unexpected channel regulatory processes. If aberrant, these dysregulated mechanisms may also result in the development of salt-dependent hypertension.The purpose of this review is to highlight the most recent findings in renal ENaC regulation and function, in considering data obtained from animal models. Increased ENaC-mediated Na+ transport is a prerequisite for salt-dependent forms of hypertension. To treat salt-sensitive hypertension it is crucial to fully understand the function and regulation of ENaC.

Tubular IL-1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages.

Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. Thirty-four-week-old db/db mice display significantly higher levels of IL-1β in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1β in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1β when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1β promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1β in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. Renal tubular epithelial cell-derived IL-1β polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1β synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

Adenosine A2B receptor activation stimulates alveolar fluid clearance through alveolar epithelial sodium channel via cAMP pathway in endotoxin-induced lung injury.

Clinical studies have established that the capacity of removing excess fluid from alveoli is impaired in most patients with acute respiratory distress syndrome. Impaired alveolar fluid clearance (AFC) correlates with poor outcomes. Adenosine A2B receptor (A2BAR) has the lowest affinity with adenosine among four adenosine receptors. It is documented that A2BAR can activate adenylyl cyclase (AC) resulting in elevated cAMP. Based on the understanding that cAMP is a key regulator of epithelial sodium channel (ENaC), which is the limited step in sodium transport, we hypothesized that A2BAR signaling may affect AFC in acute lung injury (ALI) through regulating ENaC via cAMP, thus attenuating pulmonary edema. To address this, we utilized pharmacological approaches to determine the role of A2BAR in AFC in rats with endotoxin-induced lung injury and further focused on the mechanisms in vitro. We observed elevated pulmonary A2BAR level in rats with ALI and the similar upregulation in alveolar epithelial cells exposed to LPS. A2BAR stimulation significantly attenuated pulmonary edema during ALI, an effect that was associated with enhanced AFC and increased ENaC expression. The regulatory effects of A2BAR on ENaC-α expression were further verified in cultured alveolar epithelial type II (ATII) cells. More importantly, activation of A2BAR dramatically increased amiloride-sensitive Na+ currents in ATII cells. Moreover, we observed that A2BAR activation stimulated cAMP accumulation, whereas the cAMP inhibitor abolished the regulatory effect of A2BAR on ENaC-α expression, suggesting that A2BAR activation regulates ENaC-α expression via cAMP-dependent mechanism. Together, these findings suggest that signaling through alveolar epithelial A2BAR promotes alveolar fluid balance during endotoxin-induced ALI by regulating ENaC via cAMP pathway, raising the hopes for treatment of pulmonary edema due to ALI.

Aldosterone Mediated Regulation of Epithelial Sodium Channel (ENaC) Subunits in the Rat Hypothalamus

Current evidence suggests that the epithelial Na+ channel (ENaC) in the brain plays a significant role in the development of hypertension. ENaC is present in vasopressin (VP) neurons in the hypothalamus, suggesting that ENaC in VP neurons is involved in the regulation of blood pressure. Our recent study demonstrated that high dietary salt intake caused an increase in the expression and activity of ENaC that were responsible for the more depolarized basal membrane potential in VP neurons. A known regulator of ENaC expression, the mineralocorticoid receptor (MR), is present in VP neurons, suggesting that ENaC expression in VP neurons is regulated by aldosterone. In this study, the effects of aldosterone and corticosterone on ENaC were examined in acute hypothalamic slices. Real-time PCR and Western blot analysis showed that aldosterone and corticosterone treatment resulted in a significant increase in the expression of γENaC, but not α- or βENaC, and that this expression was attenuated by MR and glucocorticoid receptor (GR) antagonists. Moreover, chromatin immunoprecipitation demonstrated that the aldosterone-MR complex directly interacts with the promoter region of the γENaC gene. However, the treatment with aldosterone did not cause subcellular translocation of ENaC toward the plasma membrane nor an increase in ENaC Na+-leak current. These results indicate that expression of γENaC in VP neurons is induced by aldosterone and corticosterone through their MR and GR, respectively; however, aldosterone or corticosterone alone is not sufficient enough to increase ENaC current when they are applied to hypothalamic slices in vitro.

Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC)

Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear. In this study, we propose a new component for the protein machinery involved in the regulation of ENaC, the missing-in-metastasis (MIM) protein. The MIM protein contains an IMD domain (for interaction with PIP2 -rich plasma membrane regions and Rac GTPases; this domain also possesses F-actin bundling activity), a PRD domain (for interaction with cortactin), and a WH2 domain (interaction with G-actin). The patch-clamp electrophysiological technique in whole-cell configuration was used to test the involvement of MIM in the actin-dependent regulation of ENaC. Co-transfection of ENaC subunits with the wild-type MIM protein (or its mutant forms) caused a significant reduction in ENaC-mediated integral ion currents. The analysis of the F-actin structure after the transfection of MIM plasmids showed the important role played by the domains PRD and WH2 of the MIM protein in cytoskeletal rearrangements. These results suggest that the MIM protein may be a part of the complex of actin-binding proteins which is responsible for the actin-dependent regulation of ENaC in the CCD.

The endothelial mineralocorticoid receptor: mediator of the switch from vascular health to disease.

Endothelial dysfunction is an early feature of vascular disease induced by cardiovascular risk factors (CRFs). In growing populations with obesity, diabetes, hypertension, and heart failure, mineralocorticoid receptor antagonism improves endothelial function. This review summarizes recent advances in our understanding of the specific role of endothelial cell mineralocorticoid receptor in vascular function in health and disease. Using transgenic mice with mineralocorticoid receptor expression specifically modulated in endothelial cells, recent studies support the emerging concept that while endothelial cell mineralocorticoid receptor may be protective in health, in the presence of CRFs, endothelial cell mineralocorticoid receptor activity contributes to endothelial dysfunction and progression of vascular disease. Proposed mechanisms include a role for endothelial cell mineralocorticoid receptor in decreased nitric oxide production and bioavailability, increased vascular oxidative stress, regulation of epithelial sodium channels that enhance vascular stiffness, and increased endothelial cell adhesion molecules promoting inflammation. The role of endothelial cell mineralocorticoid receptor may also depend on the sex, race, or vascular bed involved. Recent advances support the idea that endothelial cell mineralocorticoid receptor is a mediator of the switch from vascular health to disease in response to CRFs. Further investigation of the molecular mechanism is underway to identify therapeutic interventions that will limit the detrimental effects of endothelial cell mineralocorticoid receptor in patients at cardiovascular risk.

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity.

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.

Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator: CUTTING EDGE AND CLEAVAGE SITES

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.

Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency.

Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation, etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane-permeabilized cells. A decrease in the Na(+)-K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid reabsorption by MTEs. ERK1/2, but not Akt, phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs. those of wild-type controls. Expression of caspase 8, however, did not differ between wild-type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)-K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.

Regulation of ion channels in cystic fibrosis (869.8)

Cystic Fibrosis (CF) is an autosomal recessive disease, characterized by mutations in the CFTR gene, incorrect protein folding, and impaired chloride secretion. In addition to the maintenance of chloride secretion, CFTR is also suggested to be an important regulator of epithelial sodium channel (ENaC) activity. We previously identified the chloride channel ClCN2 as coexpressed with the mutant CFTR and hypothesized that in addition to restoring chloride secretion, ClCN2 can act as a negative regulator of ENaC. Coimmunoprecipitation analysis shows a novel physical interaction between ClCN2 and ENaC in pulmonary epithelium, which is accompanied by changes in ClCN2 and ENaC protein localization, as well as transepithelial resistance. Furthermore, ClCN2 has been shown to play an important role in the regulation of epithelial cell junctions and to this end, we show that changes in ClCN2 localization and activity may also affect cell junctions in CF cells. In conclusion, our data is the first to identify a novel interaction between ClCN2 and ENaC subunits. We also show potential changes in junction formation which may be regulated by changes in ClCN2 activity and localization. Therefore, when CFTR is nonfunctional, as in Cystic Fibrosis, ClCN2 may regulate ENaC activity and tight junction formation, which suggests that modulation of ClCN2 activity might be an advantageous target for treatment of the disease.Grant Funding Source: Supported by 5T32HL72748‐11: Ruth L Kirschstein National Research Service Award

Genetic variation in NEDD4L, an epithelial sodium channel regulator, is associated with cardiovascular disease and cardiovascular death

We have previously shown that genetic variance in NEDD4L, a regulating protein of a sodium channel in the distal nephron, has been associated with marginally higher blood pressure and enhanced salt sensitivity. Here, we tested if the genetic NEDD4L variation previously associated with salt sensitivity is related to population blood pressure, incidence of cardiovascular disease (CVD) and mortality. We genotyped the rs4149601 A→G and rs2288774 T→C NEDD4L variants in 27,564 participants of the Malmö Diet and Cancer Study. The genotype combination previously shown to be associated with salt sensitivity (rs4149601 GG+rs2288774 CC), which was present in 9.6% of participants, was related to cross sectional blood pressure as well as to CVD incidence and mortality during a median follow-up time of 14 years using Cox regression models. Carriers of the NEDD4L salt sensitivity-associated genotype had (mean ± SEM) higher systolic (142 ± 0.4 vs. 141 ± 0.1 mmHg, P = 0.002) and diastolic (86.0 ± 0.5 vs. 85.6 ± 0.2 mmHg, P = 0.025) blood pressure and multivariate adjusted hazards ratio (95% confidence interval) of CVD 1.13 (1.02-1.25, P = 0.018), coronary events 1.20 (1.06-1.37; P = 0.005) and cardiovascular mortality 1.17 (0.99-1.37; P = 0.055) than noncarriers but there was no significant difference in the incidence of stroke and total mortality. The NEDD4L salt sensitivity-associated genotype was associated with higher blood pressure, which may translate into increased risk for CVD morbidity and mortality. Interestingly, there was no association with stroke suggesting that the association is partially blood pressure independent.

Glutathione regulation of epithelial sodium channels (ENaC) in alveolar type1 and type 2 cells.

We used single channel patch clamp recordings to examine the effect of oxidative stress, via H2O2 accumulation, and alteration of the glutathione redox potential on the activity of ENaC. We found that addition of 20 μM H2O2 significantly increased ENaC open probability (Po) from 0.34+0.16 to 0.470+0.16 in alveolar type 1 (AT1) and 0.17+0.05 to 0.44+0.07 in type 2 (AT2) cells; P<0.05. We also found a linear decrease in ENaC activity as the GSH/GSSG redox potential became less negative (n=19; P<0.05). Sequentially treating cells with H2O2 and then GSH resulted in significant decreases in ENaC activity. Likewise, singular treatment of GSSG significnatly decreased ENaC Po from 0.39 + 0.06 to 0.13 + 0.05 and decreased the number of active channels in the membrane, thus implicating GSSG as an important pro‐injury signaling molecule in edematous lung disorders. Fluorescent maleimide labeling and co‐immunoprecipitation studies show that α‐ENaC subunit is glutathionylated. Real time X‐ray imaging studies show that GSH and GSSG are important determinants of alveolar fluid clearance in vivo. From these results we conclude that ENaC is regulated by changes in the glutathione redox potential. Research was supported by R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH

Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells

We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

Thiol-Mediated Regulation of Epithelial Sodium Channels

Thiol-Mediated Regulation of Epithelial Sodium Channels

Physiological regulation of epithelial sodium channel by proteolysis

Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric disease. A variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved α and γ-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1), which is glycophosphatidylinositol-anchored to the apical cell surface, mediates important extracellular cleavage. Soluble protease activity is very low in urine under physiological conditions but rises in proteinuria. In nephrotic syndrome, the dominant soluble protease activity is plasmin, which is formed from filtered plasminogen via urokinase-type plasminogen activator. Plasmin activates ENaC directly at high concentrations and through prostasin at lower concentrations. The discovery of serine protease-mediated activation of renal ENaC in physiological and pathophysiological conditions opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets.