Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells.

Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane.

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, ß, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of ß- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αßγ-channels.

Laminar shear stress modulates the activity of heterologously expressed P2X(4) receptors.

P2X(4) receptors are involved in mechanotransduction processes, but it is unknown whether or not P2X(4) receptors form mechanosensitive ion channels. This study questioned, whether laminar shear stress (LSS) can modulate P2X(4) receptor activity. Mouse P2X(4) receptor was cloned and heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage-clamp experiments the application of ATP (100µM) produced a transient inward current that was decreased by about 50% upon a second ATP application, corresponding to the desensitization behavior of P2X(4) receptors. In P2X(4) expressing oocytes LSS (shear forces of ~5.1dynes/cm(2)) did not produce any effect. However, LSS modulated the response of P2X(4) to ATP. With LSS (~5.1dynes/cm(2)) the desensitization of the current due to the second ATP application was diminished. Ivermectin (IVM), a compound which stabilizes the open state of P2X(4) receptors, mimicked the effect of LSS (~5.1dynes/cm(2)), since there was no additional effect of LSS after pre-incubation with IVM detected. This indicates that LSS like IVM stabilizes the open state of the receptor, although the particular mechanism remains unknown. These data demonstrate that LSS modulates the activity of P2X(4) receptors by eliminating the desensitization of the receptors in response to ATP probably by stabilizing the open state of the channel.

Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.

Nitric oxide inhibits highly selective sodium channels and the Na+/K+-ATPase in H441 cells.

Nitric oxide (NO) is an important regulator of Na(+) reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na(+) channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na(+)/K(+)-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na(+)/K(+)-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na(+)/K(+)-ATPase α subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na(+) reabsorption by interfering with the activity of highly selective Na(+) channels and the Na(+)/K(+)-ATPase.

Expression and functional characterization of P2X receptors in mouse alveolar macrophages.

Alveolar macrophages (AM) are crucial for pulmonary host defense, and evidence emerges that ATP-gated P2X receptors are involved in inflammatory processes. This study focuses on the expression and functional characterization of P2X receptors in AM from mouse. In RT-PCR experiments, transcripts encoding the P2X1, P2X3, P2X4, P2X5, and P2X7 receptors were detected. In whole-cell patch-clamp recordings, ATP (1 mM) evoked an inward current (mouse and human AM) that was reversible upon washout, and the reversal potential was ~5 mV, indicating the activation of a non-selective conductance-a fingerprint of P2X receptors. Further characterization (mouse AM) revealed that the current was not desensitized by a second ATP application. The ATP-induced current was increased by the removal of extracellular Ca²âº (in human and mouse AM), and EC50 in mouse AM were determined with ~1 mM ATP, in the presence as well as in the absence of extracellular Ca²âº. Pharmacological characterization of mouse AM revealed that the effect was augmented by BzATP and pre-application with ivermectin, but no effect with α,ß-meATP was observed. Further, the ATP effect was reduced by PPADS (300 µM), brilliant blue G (5 µM), and about A438079 (10 µM). Although different P2X receptor transcripts were detected in mouse AM, the observed functional and pharmacological characteristics indicate primarily the participation of P2X4 and P2X7 receptors as mediators of the ATP-induced ion current in mouse AM. These suggestions were confirmed by experiments with AM from P2X7 -deficient animals, indicating a contribution of P2X4 and P2X7 receptors in pulmonary immune function.

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis.

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.

Basolateral Cl- uptake mechanisms in Xenopus laevis lung epithelium.

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl(-) uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na(+)/K(+)/2 Cl(-) cotransporter (NKCC) and HCO(3)(-)/Cl(-) anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl(-)-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl(-) gradient. Further, we were interested in the involvement of the HCO(3)(-)/Cl(-) anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (I(SC)). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO(3)(-) concentrations. Cl(-) secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO(3)(-). These experiments indicate that the AE at least partially contributes to Cl(-) secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl(-) uptake, which contrasts with the common model for Cl(-) secretion in pulmonary epithelia.

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration.

BACKGROUND: Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. METHODS: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. RESULTS: Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, ß1, and ß2, with most stable expression being noted for subunits α9, α10, ß1, and ß2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 µM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. CONCLUSIONS: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.

Carbon monoxide rapidly impairs alveolar fluid clearance by inhibiting epithelial sodium channels.

Carbon monoxide (CO) is currently being evaluated as a therapeutic modality in the treatment of patients with acute lung injury and acute respiratory distress syndrome. No study has assessed the effects of CO on transepithelial ion transport and alveolar fluid reabsorption, two key aspects of alveolocapillary barrier function that are perturbed in acute lung injury/acute respiratory distress syndrome. Both CO gas (250 ppm) and CO donated by the CO donor, CO-releasing molecule (CORM)-3 (100 microM in epithelial lining fluid), applied to healthy, isolated, ventilated, and perfused rabbit lungs, significantly blocked (22)Na(+) clearance from the alveolar compartment, and blocked alveolar fluid reabsorption after fluid challenge. Apical application of two CO donors, CORM-3 or CORM-A1 (100 microM), irreversibly inhibited amiloride-sensitive short-circuit currents in H441 human bronchiolar epithelial cells and primary rat alveolar type II cells by up to 40%. Using a nystatin permabilization approach, the CO effect was localized to amiloride-sensitive channels on the apical surface. This effect was abolished by hemoglobin, a scavenger of CO, and was not observed when inactive forms of CO donors were employed. The effects of CO were not blocked by 8-bromoguanosine-3',5'-cyclic guanosine monophosphate, soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), or inhibitors of trafficking events (phalloidin oleate, MG-132, and brefeldin A), but the amiloride affinity of H441 cells was reduced after CO exposure. These data indicate that CO rapidly inhibits sodium absorption across the airway epithelium by cyclic guanosine monophosphate- and trafficking-independent mechanisms, which may rely on critical histidine residues in amiloride-sensitive channels or associated regulatory proteins on the apical surface of lung epithelial cells.

Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability.

Epithelial cells are exposed to a variety of mechanical forces, but little is known about the impact of these forces on epithelial ion channels. Here we show that mechanical activation of epithelial sodium channels (ENaCs), which are essential for electrolyte and water balance, occurs via an increased ion channel open probability. ENaC activity of heterologously expressed rat (rENaC) and Xenopus (xENaC) orthologs was measured by whole-cell as well as single-channel recordings. Laminar shear stress (LSS), producing shear forces in physiologically relevant ranges, was used to mechanically stimulate ENaCs and was able to activate ENaC currents in whole-cell recordings. Preceding pharmacological activation of rENaC with Zn2+ and xENaC with gadolinium and glibenclamide largely prevented LSS-activated currents. In contrast, proteolytic cleavage with trypsin potentiated the LSS effect on rENaC whereas the LSS effect on xENaC was reversed (inhibition of xENaC current). Further, we found that exposure of excised outside-out patches to LSS led to an increased ion channel open probability without affecting the number of active channels. We suggest that mechano-sensitivity of ENaC may represent a ubiquitous feature for the physiology of epithelia, providing a putative mechanism for coupling transepithelial Na+ reabsorption to luminal transport.

CFTR-dependent Cl- secretion in Xenopus laevis lung epithelium.

In our present study we used preparations from Xenopus laevis lungs to perform electrophysiological Ussing chamber measurements, unidirectional flux measurements, and employed molecular approaches to elucidate the presence and function of a cystic fibrosis transmembrane conductance regulator (CFTR) homolog in this tissue. Application of different CFTR blockers (NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid (NFA), glibenclamide, lonidamine, CFTR(inh)-172) to the apical side of the tissues was able to significantly decrease the measured short circuit current (I(SC)) indicating a Cl(-) secretion due to luminal located CFTR channels. This was further supported by a net (36)Cl(-) secretion determined by radioactive tracer flux experiments. Further, Xenopus pulmonary epithelia responded to apical chlorzoxazone exposure - a CFTR activator - and this activated current was inhibited by CFTR(inh)-172. We performed reverse transcription-PCR (RT-PCR) and Western blot analysis and with both approaches we found characteristic signals indicating the presence of a CFTR homolog in Xenopus lung. In addition, we were able to detect CFTR in apical membranes of Xenopus lung slices with immunohistological techniques. We conclude that Xenopus lung epithelium exhibits functional CFTR channels and that this tissue represents a valuable model for the investigation of ion transport properties in pulmonary epithelia.

Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 µM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 µM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 µM) and ACh responses were not affected by nicotine or mecamylamine (25 µM). Luminal application of IBMX (I, 100 µM) and forskolin (F, 10 µM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 µM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

Evidence for expression and function of angiotensin II receptor type 1 in pulmonary epithelial cells.

There is a growing evidence that the peptide hormone angiotensin II (ANGII) can act as an auto-/paracrine mediator to regulate epithelial ion transport processes. The present study focused on the impact of ANGII on transepithelial ion transport in pulmonary epithelia. Transcripts for the ANGII receptor type 1 (ATR1) were detected in lungs of Xenopus laevis and H441 cells (human pulmonary epithelial cell line). Native Xenopus lung preparations were used for Ussing chamber recordings and apically applied ANGII (10µM) induced a significant increase of short-circuit current (ISC: 8±2%, n=13). Pre-incubation with losartan (LOS), an antagonist of ATR1 prevented the effect of ANGII on ISC. Transcripts for ATR1 in Xenopus lungs and H441 cells were detected and an increase of ISC was observed by ANGII in native Xenopus lung epithelia. This indicates that ANGII is a potential auto-/paracrine mediator for ion transport regulation in pulmonary epithelia.

Why Do We have to Move Fluid to be Able to Breathe?

The ability to breathe air represents a fundamental step in vertebrate evolution that was accompanied by several anatomical and physiological adaptations. The morphology of the air-blood barrier is highly conserved within air-breathing vertebrates. It is formed by three different plies, which are represented by the alveolar epithelium, the basal lamina, and the endothelial layer. Besides these conserved morphological elements, another common feature of vertebrate lungs is that they contain a certain amount of fluid that covers the alveolar epithelium. The volume and composition of the alveolar fluid is regulated by transepithelial ion transport mechanisms expressed in alveolar epithelial cells. These transport mechanisms have been reviewed extensively. Therefore, the present review focuses on the properties and functional significance of the alveolar fluid. How does the fluid enter the alveoli? What is the fate of the fluid in the alveoli? What is the function of the alveolar fluid in the lungs? The review highlights the importance of the alveolar fluid, its volume and its composition. Maintenance of the fluid volume and composition within certain limits is critical to facilitate gas exchange. We propose that the alveolar fluid is an essential element of the air-blood barrier. Therefore, it is appropriate to refer to this barrier as being formed by four plies, namely (1) the thin fluid layer covering the apical membrane of the epithelial cells, (2) the epithelial cell layer, (3) the basal membrane, and (4) the endothelial cells.

Amiloride-sensitive sodium channels and pulmonary edema.

The development of pulmonary edema can be considered as a combination of alveolar flooding via increased fluid filtration, impaired alveolar-capillary barrier integrity, and disturbed resolution due to decreased alveolar fluid clearance. An important mechanism regulating alveolar fluid clearance is sodium transport across the alveolar epithelium. Transepithelial sodium transport is largely dependent on the activity of sodium channels in alveolar epithelial cells. This paper describes how sodium channels contribute to alveolar fluid clearance under physiological conditions and how deregulation of sodium channel activity might contribute to the pathogenesis of lung diseases associated with pulmonary edema. Furthermore, sodium channels as putative molecular targets for the treatment of pulmonary edema are discussed.

Epithelial Na+ channels derived from human lung are activated by shear force.

During breathing the pulmonary epithelial cells are permanently exposed to physical forces and shear force (SF) in particular. In our present study we questioned whether the lung epithelial Na(+) channel (hENaC) responds to shear force. For this purpose ENaC was cloned from human lung tissue, expressed in Xenopus oocytes and functionally characterized by electrophysiological techniques. Shear force in physiological relevant ranges was applied via a fluid stream. By the application of SF we obtained an increased inward current indicating an activation of hENaC. The SF-induced effect was reversible and interestingly, the response to SF was augmented by trypsin due to proteolytic cleavage. The direct activation of hENaC by SF was confirmed in outside-out single channel experiments. In five out of nine recordings an increased NP(O) was observed. From our observations we conclude that lung-derived hENaCs are directly activated by SF and this may represent an important feature for the regulation of pulmonary Na(+) reabsorption and pulmonary fluid homeostasis.

Mechano-sensitivity of ENaC: may the (shear) force be with you.

The epithelial Na+ channel (ENaC) is the rate-limiting step for Na+ absorption in various vertebrate epithelia and deeply enmeshed in the control of salt and water homeostasis. The phylogenetic relationship of ENaC molecules to mechano-sensitive Degenerins from Caenorhabditis elegans indicates that ENaC might be mechano-sensitive as well. Primarily, it was suggested that ENaC might be activated by membrane stretch. However, this issue still remains to be clarified because controversial results were published. Recent publications indicate that shear stress represents an adequate stimulus, activating ENaC via increasing the single-channel open probability. Basing on the experimental evidence published within the past years and integrating this knowledge into a model related to the mechano-sensitive receptor complex known from C. elegans, we introduce a putative mechanism concerning the mechano-sensitivity of ENaC. We suggest that mechano-sensitive ENaC activation represents a nonhormonal regulatory mechanism. This feature could be of considerable physiological significance because many Na+-absorbing epithelia are exposed to shear forces. Furthermore, it may explain the wide distribution of ENaC proteins in nonepithelial tissues. Nevertheless, it remains a challenge for future studies to explore the mechanism how ENaC is controlled by mechanical forces and shear stress in particular.