Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

In vitro and in vivo regulation of transepithelial lung alveolar sodium transport by serine proteases

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a rate-limiting step for sodium (Na+) and water absorption across lung alveolar epithelium. Recent reports suggested that ENaC is regulated by membrane-bound extracellular serine proteases, such as channel-activating proteases (CAPs). The objectives of this study were to examine the role of serine proteases in the regulation of transepithelial alveolar Na+ and water transport in vitro and in vivo and the expression of CAPs in rodent distal lung. In vitro experiments showed that inhibition of endogenous serine proteases by apical aprotinin 1) decreased ENaC-mediated currents in primary cultures of rat and mouse alveolar epithelial cells without affecting the abundance nor the electrophoretic migration pattern of biotinylated alpha- and beta-ENaC expressed at the cell surface and 2) suppressed the increase in amiloride-sensitive short-circuit current induced by the beta2-agonist terbutaline. RT-PCR experiments indicated that CAP1, CAP2, and CAP3 mRNAs were expressed in mouse alveolar epithelial cells, whereas CAP1 was also expressed in alveolar macrophages recovered by bronchoalveolar lavage. CAP1 protein was detected by Western blotting in rat and mouse alveolar epithelial cells, alveolar macrophages and bronchoalveolar lavage fluid. Finally, in vivo experiments revealed that intra-alveolar treatment with aprotinin abolished the increase in Na+-driven alveolar fluid clearance (AFC) induced by terbutaline in an in situ mouse lung model, whereas trypsin potentiated it. These results show that endogenous membrane-bound and/or secreted serine proteases such as CAPs regulate alveolar Na+ and fluid transport in vitro and in vivo in rodent lung.     

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.     

Influenza virus inhibits ENaC and lung fluid clearance

Fluid-free alveolar space is critical for normal gas exchange. Influenza virus alters fluid transport across respiratory epithelia producing rhinorrhea, middle ear effusions, and alveolar flooding. However, the mechanism of fluid retention remains unclear. We investigated influenza virus strain A/PR/8/34, which can attach and enter mammalian cells but is incapable of viral replication and productive infection in mammalian epithelia, on epithelial sodium channels (ENaC) in rat alveolar type II (ATII) cells. In parallel, we determined the effects of virus on amiloride-sensitive (i.e., ENaC-mediated) fluid clearance in rat lungs in vivo. Although influenza virus did not change the inulin permeability of ATII monolayers, it rapidly reduced the net volume transport across monolayers. Virus reduced the open probability of single ENaC channels in apical cell-attached patches. U-73122, a phospholipase C (PLC) inhibitor, and PP2, a Src inhibitor, blocked the effect of virus on ENaC. GF-109203X, a protein kinase C (PKC) inhibitor, also blocked the effect, suggesting a PKC-mediated mechanism. In parallel, intratracheal administration of influenza virus produced a rapid inhibition of amiloride-sensitive (i.e., ENaC-dependent) lung fluid transport. Together, these results show that influenza virus rapidly inhibits ENaC in ATII cells via a PLC- and Src-mediated activation of PKC but does not increase epithelial permeability in this same rapid time course. We speculate that this rapid inhibition of ENaC and formation of edema when the virus first attaches to the alveolar epithelium might facilitate subsequent influenza infection and may exacerbate influenza-mediated alveolar flooding that can lead to acute respiratory failure and death.     

Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells

Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells.     

Hypoxia and beta 2-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of beta(2)-agonists. To investigate the mechanism of beta(2)-agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O(2) for 24 h. Hypoxia reduced transepithelial sodium current and amiloride-sensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially beta and gamma) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of beta-and gamma-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxia-induced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that beta(2)-agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.     

Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective beta-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

Interleukin-1beta decreases expression of the epithelial sodium channel alpha-subunit in alveolar epithelial cells via a p38 MAPK-dependent signaling pathway

Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1beta (IL-1beta) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1beta has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1beta would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1beta on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1beta significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1beta decreased basal and dexamethasone-induced epithelial sodium channel alpha-subunit (alpha ENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1beta on alpha ENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of alpha v beta6 integrin and transforming growth factor-beta. These results indicate that IL-1beta may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in alpha ENaC expression through p38 MAPK-dependent inhibition of alpha ENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.     

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Low expression of the beta-ENaC subunit impairs lung fluid clearance in the mouse

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the beta-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the beta-ENaC gene locus resulting in low levels of beta-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that beta-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. beta-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but alpha- and gamma-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The beta2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in alpha- and gamma-ENaC protein expression observed in mutant mouse lung, low expression of beta-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate beta-ENaC expression appears to be required for AFC stimulation by beta2-agonists.     

Apical trypsin increases ion transport and resistance by a phospholipase C-dependent rise of Ca2+

We investigated the mechanisms by which serine proteases alter lung fluid clearance in rat lungs and vectorial ion transport in airway and alveolar epithelial cells. Inhibition of endogenous protease activity by intratracheal instillation of soybean trypsin inhibitor (SBTI) or alpha(1)-antitrypsin decreased amiloride-sensitive lung fluid clearance across rat fluid-filled lungs; instillation of trypsin partially restored this effect. Gelatin zymography demonstrated SBTI-inhibitable trypsin-like activity in rat lung lavage fluid. Apical trypsin and human neutrophil elastase, but not agonists of protease activated receptors, increased Na(+) and Cl(-) short-circuit currents (I(sc)) and transepithelial resistance (R(TE)) across human bronchial and nasal epithelial cells and rat alveolar type II cells, mounted in Ussing chambers, for at least 2 h. The increase in I(sc) was fully reversed by amiloride and glibenclamide. The increase in R(TE) was not prevented by ouabain, suggesting that trypsin decreased paracellular conductance. Apical trypsin also induced a transient increase in intracellular Ca(2+) in human airway cells; treatment of these cells with BAPTA-AM mitigated the trypsin-induced increases of intracellular Ca(2+) and of I(sc) and R(TE). Increasing intracellular Ca(2+) in airway cells with either ionomycin or thapsigargin reproduced the increase in I(sc), whereas inhibitors of phospholipase C (PLC) prevented the increases in both Ca(2+) and I(sc). These data indicate trypsin-like proteases and elastase, either present in lung cells or released by inflammatory cells into the alveolar space, play an important role in the clearance of alveolar fluid by increasing ion transport and paracellular resistance via a PLC-initiated rise of intracellular Ca(2+).     

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O(2) on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 muM) for 3 days before exposure to hypoxia (1.5% O(2)). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (-30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na(+)-K(+)-ATPase and epithelial Na(+) channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of alpha- and gamma-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of alpha(1)-Na(+)-K(+)-ATPase and alpha-ENaC increased in normoxia and hypoxia, and gamma-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of alpha(1)-Na(+)-K(+)-ATPase and alpha-, beta-, and gamma-ENaC of A549 cells in normoxia and hypoxia (1.5% O(2)) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.     

A novel function of ionotropic gamma-aminobutyric acid receptors involving alveolar fluid homeostasis

Polarized distribution of chloride channels on the plasma membrane of epithelial cells is required for fluid transport across the epithelium of fluid-transporting organs. Ionotropic gamma-aminobutyric acid receptors are primary ligand-gated chloride channels that mediate inhibitory neurotransmission. Traditionally, these receptors are not considered to be contributors to fluid transport. Here, we report a novel function of gamma-aminobutyric acid receptors involving alveolar fluid homeostasis in adult lungs. We demonstrated the expression of functional ionotropic gamma-aminobutyric acid receptors on the apical plasma membrane of alveolar epithelial type II cells. gamma-Aminobutyric acid significantly increased chloride efflux in the isolated type II cells and inhibited apical to basolateral chloride transport on type II cell monolayers. Reduction of the gamma-aminobutyric acid receptor pi subunit using RNA interference abolished the gamma-aminobutyric acid-mediated chloride transport. In intact rat lungs, gamma-aminobutyric acid inhibited both basal and beta agonist-stimulated alveolar fluid clearance. Thus, we provide molecular and pharmacological evidence that ionotropic gamma-aminobutyric acid receptors contribute to fluid transport in the lung via luminal secretion of chloride. This finding may have the potential to develop clinical approaches for pulmonary diseases involving abnormal fluid dynamics.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Redox regulation of epithelial sodium channels examined in alveolar type 1 and 2 cells patch-clamped in lung slice tissue

The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 +/- 3.2 and 22.5 +/- 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 microm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 +/- 0.26 to 0.82 +/- 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (O2.) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that O2. and NO signaling plays an important role in maintaining lung fluid balance.