Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis

Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells <5%. Fluid transport is regulated by ion transport, with water movement following passively. Current concepts are that TII cells are the main sites of ion transport in the lung. TI cells have been thought to provide only passive barrier, rather than active, functions. Because TI cells line most of the internal surface area of the lung, we hypothesized that TI cells could be important in the regulation of lung liquid homeostasis. We measured both Na(+) and K(+) (Rb(+)) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na(+) in an amiloride-inhibitable fashion, suggesting the presence of Na(+) channels; TI cell Na(+) uptake, per microgram of protein, is approximately 2.5 times that of TII cells. Rb(+) uptake in TI cells was approximately 3 times that in TII cells and was inhibited by 10(-4) M ouabain, the latter observation suggesting that TI cells exhibit Na(+)-, K(+)-ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (alpha, beta, and gamma) of the epithelial sodium channel ENaC and two subunits of Na(+)-, K(+)-ATPase. By Western blot analysis, TI cells contain approximately 3 times the amount of alphaENaC/microg protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.     

Deletion of the ubiquitin ligase Nedd4L in lung epithelia causes cystic fibrosis-like disease

Cystic fibrosis is caused by impaired ion transport due to mutated cystic fibrosis transmembrane conductance regulator, accompanied by elevated activity of the amiloride-sensitive epithelial Na(+) channel (ENaC). Here we show that knockout of the ubiquitin ligase Nedd4L (Nedd4-2) specifically in lung epithelia (surfactant protein C-expressing type II and Clara cells) causes cystic fibrosis-like lung disease, with airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age. These effects of Nedd4L loss are likely caused by enhanced ENaC function, as reflected by increased ENaC protein levels, increased lung dryness at birth, amiloride-sensitive dehydration of lung explants, and elevated ENaC currents in primary alveolar type II cells analyzed by patch clamp recordings. Moreover, the lung defects were rescued with administration of amiloride into the lungs of young knockout pups via nasal instillation. Our results therefore suggest that the ubiquitin ligase Nedd4L can suppress the onset of cystic fibrosis symptoms by inhibiting ENaC in lung epithelia.     

Alveolar epithelium: role in lung fluid balance and acute lung injury

The resolution of alveolar edema is regulated by active sodium and chloride transport across the pulmonary epithelium, including alveolar epithelial type I and II cells as well as distal airway epithelia. Catecholamine-dependent mechanisms can markedly upregulate alveolar fluid clearance even under pathological conditions, an effect that is mediated by both epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR). Under pathological conditions, impaired alveolar fluid clearance is associated with worse survival in patients with acute lung injury. However, there is some experimental and clinical evidence that cAMP stimulation could accelerate the resolution of pulmonary edema in the presence of acute lung injury. Clinical trials are needed to test this potential therapeutic strategy in patients with acute lung injury.     

Pharmacotherapy of the ion transport defect in cystic fibrosis: role of purinergic receptor agonists and other potential therapeutics

Cystic fibrosis (CF), is an autosomal recessive disease frequently seen in the Caucasian population. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF is characterized by enhanced airway Na(+) absorption, mediated by epithelial Na(+) channels (ENaC), and deficient Cl(-) transport. In addition, other mechanisms may contribute to the pathophysiological changes in the CF lung, such as defective regulation of HCO(3)(-) secretion. In other epithelial tissues, epithelial Na(+) conductance is either increased (intestine) or decreased (sweat duct) in CF. CFTR is a cyclic AMP-regulated epithelial Cl(-) channel, and appears to control the activity of several other transport proteins. Accordingly, defective epithelial ion transport in CF is likely to be a combination of defective Cl(-) channel function and impaired regulator function of CFTR, which in turn is linked to impaired mucociliary clearance and development of chronic lung disease. As the clinical course of CF is determined primarily by progressive lung disease, novel pharmacological strategies for the treatment of CF focus on correction of the ion transport defect in the airways. In recent years, it has been demonstrated that activation of purinergic receptors in airway epithelia by extracellular nucleotides (adenosine triphosphate/uridine triphosphate) has beneficial effects on mucus clearance in CF. Activation of the dominant class of metabotropic purinergic receptors, P2Y(2) receptors, appears to have a 2-fold benefit on ion transport in CF airways; excessive Na(+) absorption is attenuated, most likely by inhibition of the ENaC and, simultaneously, an alternative Ca(2+)-dependent Cl(-) channel is activated that may compensate for the CFTR Cl(-) channel defect. Thus activation of P2Y(2) receptors is expected to lead to improved hydration of the airway surface liquid in CF. Furthermore, purinergic activation has been shown to promote other components of mucociliary clearance such as ciliary beat frequency and mucus secretion. Clinical trials are under way to test the effect of synthetic purinergic compounds, such as the P2Y(2) receptor agonist INS37217, on the progression of lung disease in patients with CF. Administration of these compounds alone, or in combination with other drugs that inhibit accelerated Na(+) transport and help recover or increase residual activity of mutant CFTR, is most promising as successful therapy to counteract the ion transport defect in the airways of CF patients.     

Minireview: regulation of epithelial Na+ channel trafficking

The epithelial Na(+) channel (ENaC) is a pathway for Na(+) transport across epithelia, including the kidney collecting duct, lung, and distal colon. ENaC is critical for Na(+) homeostasis and blood pressure control; defects in ENaC function and regulation are responsible for inherited forms of hypertension and hypotension and may contribute to the pathogenesis of cystic fibrosis and other lung diseases. An emerging theme is that epithelial Na(+) transport is regulated in large part through trafficking mechanisms that control ENaC expression at the cell surface. ENaC trafficking is regulated at multiple steps. Delivery of channels to the cell surface is regulated by aldosterone (and corticosteroids) and vasopressin, which increase ENaC synthesis and exocytosis, respectively. Conversely, endocytosis and degradation is controlled by a sequence located in the C terminus of alpha, beta, and gammaENaC (PPPXYXXL). This sequence functions as an endocytosis motif and as a binding site for Nedd4-2, an E3 ubiquitin protein ligase that targets ENaC for degradation. Mutations that delete or disrupt this motif cause accumulation of channels at the cell surface, resulting in Liddle's syndrome, an inherited form of hypertension. Nedd4-2 is a central convergence point for ENaC regulation by aldosterone and vasopressin; both induce phosphorylation of a common set of three Nedd4-2 residues, which blocks Nedd4-2 binding to ENaC. Thus, aldosterone and vasopressin regulate epithelial Na(+) transport in part by altering ENaC trafficking to and from the cell surface.     

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.     

cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus epithelium

BACKGROUND AND AIMS: Unlike the intestine of normal subjects, small-intestinal epithelia of cystic fibrosis patients and cystic fibrosis transmembrane conductance regulator protein-null (CFTR(-)) mice do not respond to stimulation of intracellular cyclic adenosine monophosphate with inhibition of electroneutral NaCl absorption. Because CFTR-mediated anion secretion has been associated with changes in crypt cell volume, we hypothesized that CFTR-mediated cell volume reduction in villus epithelium is required for intracellular cyclic adenosine monophosphate inhibition of Na(+)/H(+) exchanger (primarily Na(+)/H(+) exchanger 3) activity in the proximal small intestine. METHODS: Transepithelial (22)Na flux across the jejuna of CFTR(+), CFTR(-), the basolateral membrane Na(+)/K(+)/2Cl(-) co-transporter protein NKCC1(+), and NKCC1(-) mice were correlated with changes in epithelial cell volume of the midvillus region. RESULTS: Stimulation of intracellular cyclic adenosine monophosphate resulted in cessation of Na(+)/H(+) exchanger-mediated Na(+) absorption (J(ms)(NHE)) in CFTR(+) jejunum but had no effect on J(ms)(NHE) across CFTR(-) jejunum. Cell volume indices indicated an approximately 30% volume reduction of villus epithelial cells in CFTR(+) jejunum but no changes in CFTR(-) epithelium after intracellular cyclic adenosine monophosphate stimulation. In contrast, cell shrinkage induced by hypertonic medium inhibited J(ms)(NHE) in both CFTR(+) and CFTR(-) mice. Bumetanide treatment to inhibit Cl(-) secretion by blockade of the Na(+)/K(+)/2Cl(-) co-transporter, NKCC1, of stimulated CFTR(+) jejunum prevented maximal volume reduction of villus epithelium and recovered approximately 40% of J(ms)(NHE). Likewise, J(ms)(NHE) and cell volume were unaffected by intracellular cyclic adenosine monophosphate stimulation in NKCC1(-) jejuna. CONCLUSIONS: These findings show a previously unrecognized role of functional CFTR expressed in villus epithelium: regulation of Na(+)/H(+) exchanger 3-mediated Na(+) absorption by alteration of epithelial cell volume.     

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.     

Interdependency of beta-adrenergic receptors and CFTR in regulation of alveolar active Na+ transport

Beta-adrenergic receptors (betaAR) regulate active Na+ transport in the alveolar epithelium and accelerate clearance of excess airspace fluid. Accumulating data indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) is important for upregulation of the active ion transport that is needed to maintain alveolar fluid homeostasis during pulmonary edema. We hypothesized that betaAR regulation of alveolar active transport may be mediated via a CFTR dependent pathway. To test this hypothesis we used a recombinant adenovirus that expresses a human CFTR cDNA (adCFTR) to increase CFTR function in the alveolar epithelium of normal rats and mice. Alveolar fluid clearance (AFC), an index of alveolar active Na+ transport, was 92% greater in CFTR overexpressing lungs than controls. Addition of the Cl- channel blockers NPPB, glibenclamide, or bumetanide and experiments using Cl- free alveolar instillate solutions indicate that the accelerated AFC in this model is due to increased Cl- channel function. Conversely, CFTR overexpression in mice with no beta1- or beta2-adrenergic receptors had no effect on AFC. Overexpression of a human beta2AR in the alveolar epithelium significantly increased AFC in normal mice but had no effect in mice with a non-functional human CFTR gene (Deltaphi508 mutation). These studies indicate that upregulation of alveolar CFTR function speeds clearance of excess fluid from the airspace and that CFTRs effect on active Na+ transport requires the betaAR. These studies reveal a previously undetected interdependency between CFTR and betaAR that is essential for upregulation of active Na+ transport and fluid clearance in the alveolus.     

Difference in the effect of phloridzin on alveolar fluid absorption in anesthetized rats and in ex vivo rat lungs.

We reexamined the effect of phloridizin on alveolar fluid absorption by utilizing ex vivo rat lungs, which are considered to be a useful tool to investigate electrolyte and fluid transport across alveolar epithelium. Alveolar fluid absorption was almost completely reduced by 10(-3) M phloridzin with 10(-4) M amiloride as reported previously. However, we found that phloridzin alone was also able to significantly reduce alveolar fluid absorption. We then examined the effect of phloridzin on lung metabolism and compared the data with those determined in the presence of iodoacetic acid (IAA) and NaCN. Phloridzin reduced alveolar glucose uptake with no decrease in lung ATP content. Both IAA and NaCN decreased lung ATP content significantly. Our data indicate that the effect of phloridzin on alveolar fluid absorption in ex vivo rat lungs is not the secondary effect to the alteration of lung energy metabolism. Therefore our data support the current concept that Na(+)-glucose cotransport is involved with transalveolar active Na+ transport, which is a separated pathway from amiloride-sensitive Na+ channels.     

Effect of inflammatory stimuli on airway ion transport

The airway epithelium controls the chemical and physical properties of airway surface fluid and consequently mucociliary clearance. The treatment for 24-48 hours of human bronchial epithelial cells with interferon-gamma or interleukin-4 leads to marked changes in transepithelial ion transport properties. Both cytokines downregulate the activity of the epithelial Na+ channel and, at the same time, upregulate Ca2+-dependent Cl- secretion. Interleukin-4 also increases the expression and function of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results suggest that some inflammatory stimuli may change the balance between fluid absorption and secretion to favor hydration of the airway surface and consequently mucus clearance.     

Tetramethylpyrazine stimulates cystic fibrosis transmembrane conductance regulator-mediated anion secretion in distal colon of rodents

AIM: To investigate the effect of tetramethylpyrazine (IMP), an active compound from Ligustium Wollichii Franchat, on electrolyte transport across the distal colon of rodents and the mechanism involved. METHODS: The short-circuit current (Isc) technique in conjunction with pharmacological agents and specific inhibitors were used in analyzing the electrolyte transport across the distal colon of rodents. The underlying cellular signaling mechanism was investigated by radioimmunoassay analysis (RIA) and a special mouse model of cystic fibrosis. RESULTS: TMP stimulated a concentration-dependent rise in Isc, which was dependent on both Cl- and HCO3-, and inhibited by apical application of diphenylamine-2,2'-dicarboxylic acid (DPC) and glibenclamide, but resistant to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). Removal of Na+ from basolateral solution almost completely abolished the Isc response to TMP, but it was insensitive to apical Na+ replacement or apical Na+ channel blocker, amiloride. Pretreatment of colonic mucosa with BAPTA-AM, a membrane-permeable selective Ca2+ chelator, did not significantly alter the TMP-induced Isc. No additive effect of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was observed on the TMP-induced Isc, but it was significantly reduced by a protein kinase A inhibitor, H89. RIA results showed that TMP (1 mmol/L) elicited a significant increase in cellular cAMP production, which was similar to that elicited by the adenylate cyclase activator, forskolin (10 μmol/L). The TMP-elicited Isc as well as forskolin- or IBMX-induced Isc were abolished in mice with homozygous mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) presenting defective CFTR functions and secretions. CONCLUSION: TMP may stimulate cAMP-dependent and CFTR-mediated Cl- and HCO3- secretion. This may have implications in the future development of alternative treatment for constipation.     

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na(+) currents generated by epithelial Na(+) channels (ENaC). Half-maximal stimulation occurred at 25 degrees C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a "Deg" mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.     

Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis

For more than 20 years, the abnormally thick mucus (mucoviscidosis) in cystic fibrosis has been widely shown to be linked to a genetic defect in the cystic fibrosis transmembrane conductance regulator Cl(-) channel. The defect is widely thought to cause mucus to become dehydrated as a result of basic defects in Cl(-) dependent fluid transport. However, this widely held explanation is inconsistent with the known physiological properties and functions of organs affected by cystic fibrosis. During the process of releasing highly condensed mucins from intracellular granules, Ca(2+) and H(+) cations must be removed to enable the mucins to expand by as much as 1000 times, forming extracellular mucus-gel networks. Over the past few years, that HCO(3)(-) transport is also defective in patients with cystic fibrosis has become apparent. I propose that HCO(3)(-) is crucial to normal mucin expansion because it forms complexes with these cations. Thus, because HCO(3)(-) secretion is defective in cystic fibrosis, mucins in organs affected by cystic fibrosis tend to remain aggregated, poorly solubilised, and less transportable. If the hypothesis is valid, pathogenesis in cystic fibrosis could be due as much to defective transport of HCO(3)(-) as to defective Cl(-) transport.     

Hepatocyte growth factor inhibits amiloride-sensitive Na(+) channel function in cystic fibrosis airway epithelium in vitro.

Cystic fibrosis (CF) airway epithelial cells have a reduced cAMP-dependent Cl(-)conductance channel (CFTR) function but an increased level of amiloride-sensitive Na(+)channel (ENaC) activity. Recently, expression of the alpha -subunit of the ENaC protein complex was shown to be down-regulated by activation of the extracellular signal-regulated protein kinase (ERK) pathway. In the present study we have examined the actions of a potent regulator of the ERK pathway, recombinant human hepatocyte growth factor (rhHGF), on the function of ENaC in confluent, polarized monolayers of both primary cultures of CF airway cells and an SV40-transformed CF nasal epithelial cell line (JME CF/15). Treatment of JME/CF 15 cells with rhHGF at concentrations of 100 ng/ml and above was found to dramatically decrease the activity of amiloride-sensitive Na(+)transport. This effect required basolateral exposure of the cytokine. Addition of 100 ng/ml rhHGF to JME/CF 15 cells decreased I(eq)with a t(1/2)of;18 h, with a maximal inhibition of;90% by 36 h. By 48 h, stimulation with rhHGF induced a down-regulation of its receptor, c-met, expressed in these cells. The decrease in I(eq)of JME/CF 15 monolayers was not immediately reversed upon removal of rhHGF. Treatment with rhHGF did not appear to affect monolayer resistances nor Cl(-)currents induced by mediators such as isoproterenol, histamine or bradykinin. Studies with primary cultures of CF airway cell sheets demonstrated comparable sensitivity and time-course properties for the inhibition of amiloride-sensitive currents following rhHGF addition. These observations are consistent with the possible application of an extracellular signalling molecule, such as the cytokine HGF, to reduce the abnormally high activity of amiloride-sensitive Na(+)ion channels observed in CF airway cells.     

Contribution of active Na+ and Cl- fluxes to net ion transport by alveolar epithelium

Changes in bioelectric properties of alveolar epithelial cell monolayers due to pharmacological agents such as beta-agonists, amiloride and ouabain have recently been reported. In order to determine specifically which ionic species contribute to these changes, fluxes of Na+ and Cl- across primary cultured monolayers of rat type II pneumocytes were directly measured. Monolayers were mounted in modified flux chambers and short-circuited. Unidirectional fluxes of 22Na (or 36Cl) and [14C]-mannitol were measured simultaneously. Experimental maneuvers included apical (A) exposure to 10 microM amiloride, basolateral (B) exposure to 1 mM ouabain, or basolateral exposure to 20 microM terbutaline. Results show that baseline monolayers actively reabsorb Na+ (about 0.14 micro Eq.cm-2.h-1) from the apical fluid, while mannitol and Cl- appear to traverse the alveolar epithelium passively. Active Na+ reabsorption was abolished by amiloride or ouabain, while Cl- and mannitol fluxes were unaffected. Terbutaline, on the other hand, markedly increased net absorption of Na+ and caused active transport of Cl- in the A to B direction. Passive mannitol flow was somewhat increased with terbutaline. These data indicate that active Na+ reabsorption across alveolar epithelial monolayers is dependent on intact Na+,K(+)-ATPase activity and cell Na+ entry (probably via Na+ channels), and can be stimulated by beta-agonists. Beta-agonists also cause active reabsorption of Cl- (passive under other conditions).     

Prolactin increases Na+ transport across adult bullfrog skin via stimulation of both ENaC and Na+/K+-pump

PRL is involved in osmoregulation in lower vertebrates. Its serum concentration starts to increase during the metamorphosis of bullfrog tadpoles. Adult bullfrog skin transports Na(+) from the apical to the basolateral side across the skin. PRL is involved in the regulation of this transport. We investigated the effect of ovine PRL on the epithelial Na(+) channel (ENaC), Na(+)/K(+)-pump, and basolateral K(+) channels, which regulate Na(+) transport across adult bullfrog skin, by measuring the short-circuit current (SCC). At 0.1 microg/ml, PRL had no effect on the SCC. PRL (1 microg/ml) was sufficient to stimulate the SCC since 1 and 10 microg/ml of PRL each increased SCC 1.8-fold. Current-fluctuation analysis revealed that PRL (10 microg/ml) increased the density of active ENaC almost 1.8-fold. The effect of PRL on the Na(+)/K(+)-pump was investigated using apically nystatin-permeabilized skin with Ca-free Na-Ringers' solution on each side. PRL (10 microg/ml) increased SCC in this condition around 1.1-fold, suggesting that PRL stimulates the Na(+)/K(+)-pump [although PRL (1 microg/ml) had no effect on this SCC]. The effect of PRL on basolateral K(+) channels was investigated using apically nystatin-permeabilized skin with high-K Ringer's solution on the apical side. PRL (10 microg/ml) had no effect on the SCC, suggesting that PRL does not affect basolateral K(+) channels. Thus, although PRL stimulates the Na(+)/K(+)-pump, this effect probably contributes less than that on ENaC to the regulation of Na(+) transport across adult bullfrog skin.     

Regulation of Na+ channels in lung alveolar type II epithelial cells

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and pathologic states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins designated alpha-ENaC, beta-ENaC, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this article, we discuss the regulation of ENaCs composed of varying subunits and some of the implications of the regulation for normal pulmonary function.     

WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo

Homeostasis of intravascular volume, Na(+), Cl(-), and K(+) is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin-angiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K(+) channels, and paracellular Cl(-) flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na(+) channel (ENaC), the major mediator of aldosterone-sensitive Na(+) (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC beta- and gamma-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na(+) flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na(+) absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.