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Regulation of Na⁺ Channels in Lung Alveolar Type II Epithelial Cells

Department of Physiology, Center for Cell and Molecular Signaling; Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia; and Department of Anesthesiology, University of Alabama, Birmingham, Birmingham, Alabama

Correspondence and requests for reprints should be addressed to Douglas C. Eaton, Ph.D., Department of Physiology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322. E-mail: deaton{at}emory.edu

	ABSTRACT

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
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 -ENaC, ß-ENaC, and -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.

Key Words: ENaC • lung sodium transport • single channels • patch clamp

For gas exchange to occur optimally, the alveoli must remain open and free from fluid. In utero, the fetal lung is filled with fluid that is removed shortly after birth, mainly because active reabsorption of sodium ions (Na⁺) across the alveolar epithelium creates an osmotic force favoring reabsorption of alveolar fluid (1, 2). Insight into the nature of the Na⁺ transport pathways responsible for this reabsorption has come from electrophysiologic studies in freshly isolated and cultured alveolar type II (ATII) cells. These cells, which make up more than half of the alveolar epithelial cells but constitute much less than half of the alveolar surface area in the adult lung (3, 4), can be isolated at high purity and grown to form confluent monolayers (5, 6). We know that Na⁺ ions diffuse passively into ATII cells mostly through apically located amiloride-sensitive cation channels (7, 8) and are extruded across the basolateral membranes by the ouabain-sensitive Na⁺,K⁺-ATPase (9). The Na-permeable cation channels on the apical surface usually constitute the rate-limiting step in this process, offering more than 90% of the resistance to transcellular Na⁺ transport (10). In other sodium-transporting epithelia, apical sodium entry is mediated by epithelial sodium channels (ENaC) composed of three homologous subunits: -ENaC, ß-ENaC, and -ENaC. In the lung, in situ hybridization studies in alveoli have identified the presence of the mRNA for -ENaC and -ENaC but little ß-ENaC in vivo (8, 11, 12) and all three subunits in vitro (7, 13–15). In addition, cDNAs, which encode the subunits of amiloride-sensitive Na⁺ channels in other Na⁺ transporting epithelia, have also been cloned from airway fetal lung epithelial cells (16).

Although this review focuses on Na transport in ATII cells, alveolar type I cells have also recently been isolated from adult rat lungs and, like ATII and fetal distal lung epithelial cells, have been shown to contain aquaporins and possess very high permeability to water (17–19). Work by Borok and colleagues has shown that these cells contain proteins antigenically related to sodium transporters (such as ENaC and Na,K-ATPase) (20). Although no definitive functional studies exist at present, it seems possible that alveolar type I cells are also capable of vectorial Na⁺ transport.

	SINGLE-CHANNEL MEASUREMENTS FROM ATII CELLS: A FAMILY OF AMILORIDE-SENSITIVE CHANNELS.

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
In some epithelial tissues, Na⁺ transport appears to be a relatively simple process, beginning with tissue reabsorption of Na⁺ mediated, at a single-channel level, via Na⁺ channels that are highly selective for Na⁺ over K⁺ and that are blocked by low concentrations of the drug amiloride (21). Indeed, channels with such properties can be observed in several epithelial preparations (22–29), but examination of single channels in several other Na⁺-transporting epithelial cells, including lung cells, suggests a more complicated picture of amiloride-sensitive channels. Single-channel studies have identified at least four different amiloride-sensitive channels with either high selectivity, moderate selectivity, or no selectivity for Na⁺ over K⁺ in the apical membranes of a variety of cultured and native epithelial cells, including lung epithelial cells (Table 1). These channels differ not only in their ion selectivity but also in their unitary conductance and other biophysical characteristics; nevertheless, they all have been proposed to play some role in epithelial Na⁺ absorption.

	THE MOLECULAR BASIS FOR Na+ TRANSPORT IN LUNG EPITHELIAL CELLS

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
Expression cloning methods to isolate cDNAs from rat distal colon have yielded three separate cDNAs, which are designated as -, ß-, and -rENaC (37–39). Several investigators have suggested that the cloned ENaC subunits are also responsible for Na⁺ transport in the lung. The evidence includes immunocytochemical and Western blot studies consistent with the presence of proteins antigenically related to Na⁺ channels (40, 41) and Northern blot studies showing mRNA for these channels, although differences exist in the developmental expression and regulation of the three ENaC subunits (8, 16, 42, 43). In addition, there is functional evidence for the importance of ENaC in lung fluid absorption. Hummler and colleagues (1) showed that genetically knocking out -ENaC leads to defective lung liquid clearance and premature death in newborn mice. Kerem and colleagues (44) have recently shown that patients with ENaC-reduced function mutations who develop pseudohypoaldosteronism have excess airway liquid because of inadequate absorption of Na⁺ and water.

	EXPRESSION OF HIGHLY SELECTIVE CATION CHANNELS IN ALVEOLAR EPITHELIA

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
The coexpression of -ENaC, ß-ENaC, and -ENaC cRNAs in Xenopus oocytes is associated with a highly sodium-selective 4- to 7-pS channel, that is, a highly selective cation channel or a highly Na⁺-selective channel (HSC) channel (37–39, 45, 46). In contrast to the numerous single channel studies from several Na⁺-transporting epithelia confirming the presence of such channels, electrophysiologic evidence for the presence of highly Na⁺-selective channels in lung epithelia is limited. However, in adult ATII cells, Jain and colleagues (13) have recently found that when the cells are grown on permeable supports in the presence of steroids and with an air interface, the predominant channel is a low conductance (6.6 ± 3.4 pS, n = 94), HSC with a P_Na/P_K of more than 80, that is inhibited by submicromolar concentrations of amiloride (K_0.5 = 37 nM) and is similar in biophysical properties to ENaCs described from other epithelia. The same investigators went on to demonstrate that it was possible to manipulate the relative surface density of the commonly described amiloride-sensitive, nonselective cation channels and the highly selective channel. The primary determinants were steroids, apical air interface (oxygen tension), and growth support: The presence of steroids, normal oxygen tension, and permeable growth supports all favor the expression of highly selective channels at the expense of nonselective channels, whereas the absence of these factors favors nonselective cation channels (Figure 1) . These findings point to the importance of the cellular environment in determining the electrophysiologic characteristics of ion channels.

View larger version (27K): [in this window] [in a new window]   Figure 1. Distribution of highly selective, 6-pS channels (highly Na+-selective channel [HSC], black bars) and nonselective (NSC), 21-pS cation channels (NSC, gray bars) in patches from alveolar type II (ATII) cells cultured under significantly different conditions (adapted from Talbot and colleagues with permission [11]). The effect of changes in culture support, presence of steroids, and liquid versus air interface on the apical surface of cells was examined. In all cases, cells were incubated in room air–5% CO2 and used for patch-clamp studies between 24 and 96 hours after plating. Effect of culture support: Cells were seeded either on glass cover slips (approximately 2 x 105 cells/cm2) or a specialized culture support that is optimized for patch clamp recording and that allows the cells to grow on a permeable support (Millipore CM) while they are submerged in medium. Effect of apical surface interface: For some cells grown on permeable supports, after they had attached to the culture surface (this usually required 2–4 hours), medium was drained from the apical surface, and cells were allowed to grow with medium on the basolateral surface and air on the apical side; alternatively, cells were cultured in an identical fashion, but without draining the medium so that cells remain submerged in medium. Effect of steroids: Aldosterone or dexamethasone was added to the culture medium. The presence or absence of each condition is marked below the bars as a plus (present) or minus (absent). When cells were grown on glass cover slips submerged in media devoid of steroids, the predominant channels seen are NSC channels with high conductance (solid bars), but when grown on permeable filters with high concentration of aldosterone (10 µM) and air interface, the predominant channels are HSC channels with low conductance (hatched bars).

	RELATIONSHIP BETWEEN LUNG CATION CHANNELS AND ENaC

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

	EFFECT OF STEROID HORMONES ON THE DIVERSITY OF APICAL CATION CHANNELS IN ATII

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

	EFFECT OF OXYGEN TENSION ON CHANNEL EXPRESSION

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
In experiments evaluating the effect of apical surface interface on expression and activity of ion channels, Jain and colleagues (13) found that the highest density of highly selective cation channels was seen when cells were cultured with their apical surface exposed to air interface. They went on to demonstrate that exposure of ATII cells to 5% oxygen was sufficient to convert virtually all apical cation channels to nonselective channels. However, when cells cultured in 5% oxygen for 24 hours were exposed to room air for 2 hours, apical membrane patches returned to a state with predominantly highly selective cation channels. This implies that the O₂ perfusion is critical for determining the type of channel in ATII cells and the character of fluid reabsorption.

	IMPLICATIONS OF Na+ CHANNEL DIVERSITY FOR LUNG FUNCTION

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 
Highly selective channels will avidly absorb Na⁺, whereas nonselective cation channels will just as likely secrete K⁺ as absorb sodium. Thus, the relative density of the two channel types could play a significant role in controlling the magnitude of lung fluid reabsorption. The role of steroids in promoting highly selective channels is not too surprising because there is considerable evidence that steroids increase ENaC expression in several tight epithelia (51, 52). Glucocorticoids are believed to have a greater effect on the lungs than mineralocorticoids and have been shown to increase -ENaC mRNA in rat and human fetal alveolar epithelial cells (43, 51) and ß-ENaC and -ENaC mRNA in A549 cells (53). One interpretation of these results is that steroids may be altering the production of subunits but that their primary function is to promote proper assembly and delivery of HSC channels to the membrane.

The effect of oxygen tension on ATII cells also has implication for normal and pathologic lung function. Under conditions of acute lung injury when fluid begins to accumulate in alveoli, oxygen delivery to the ATII cells will be reduced. Because this leads to a transition from highly selective to nonselective apical channels, it is likely that the ability to reabsorb fluid will be reduced. This will lead to increased edema and a positive feedback loop that will rapidly lead to alveolar flooding. This positive feedback may explain the rapid degradation in the condition of some patients with acute lung injury and the development of acute respiratory distress syndrome. Agents that can promote increased activity of ENaC (particularly if they favor the re-establishment of apical highly selective channels) may be beneficial in treatment of adult respiratory distress syndrome.

Taken together, the results of these studies indicate that the apical membranes of ATII cells contain (1) Ca⁺²-activated NSC (composed of ENaC subunits alone) and Na⁺-selective channels (composed of ENaC and ßENaC or ENaC subunits) and (2) a Ca⁺²-insensitive, 4-pS Na⁺-selective channel, with biophysical properties similar to those of -ENaC, ß-ENaC, and -ENaC reconstituted in Xenopus oocytes (37, 38). It is likely that all these types of channels are expressed in vivo and environmental and hormonal stimuli are capable of altering the biophysical properties of channels in the lung (Figure 2) .

View larger version (28K): [in this window] [in a new window]   Figure 2. The central hypothesis of this article. The diversity in amiloride-sensitive, epithelial sodium channels arises because of different combinations of epithelial sodium channel (ENaC) subunits assembled into channels. Which subunits are assembled depends on the environmental conditions.

	REGULATION OF Na+ CHANNELS BY cAMP

TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

Presently, there is controversy concerning the mechanisms by which ß₂ agonists, such as terbutaline, or lipid-soluble analogues of cAMP, such as CPT-cAMP, increase Na⁺ transport across alveolar epithelial cells. Activation of ß₂ receptors on ATII cells (57) generally stimulates adenylate cyclase, which in turn increases intracellular cAMP levels and activates Na⁺ channels in a number of epithelial cells and tissues (27, 58). However, I_sc measurements across rabbit and rat ATII cells indicate that cAMP-induced responses are considerably more complex and involve both Na⁺ and Cl^- conductive pathways (59, 60). Based on the results of their experiments in Ussing chambers, Jiang and colleagues (59) proposed that agents that increase cAMP activate apical cystic fibrosis transmembrane conductance regulator (CFTR)-type Cl^- channels. Because the resting membrane potential of ATII cells is approximately -40 mV, stimulation of Cl^- channels will result in influx of Cl^- ions, hyperpolarization of the apical membrane, and creation of a favorable driving force for increased Na⁺ transport. These investigators did not find any evidence of activation of Na⁺ channels by cAMP in their experimental model. This is in marked contrast to Ussing chamber measurements on another epithelial cell line, A6, that also has both CFTR and ENaCs in its apical membrane. In this work, Chalfant and colleagues (61) found that an increase in intracellular cAMP produced an initial and rapid increase in chloride current followed by a slower increase of amiloride-sensitive sodium current. Under open circuit conditions, the final sodium and chloride current was equal, representing a net salt flux from the lumen to serosa across the epithelial cell layer.

In addition, the results of a number of patch clamp studies also argue against the Jiang and colleagues hypothesis. In several studies, the addition of terbutaline to the basolateral side of ATII cells patched in the cell-attached mode increased ENaC single-channel activity (32, 34). The exact effect of cAMP depended on the type of ENaC in the patch. For NSC ENaCs, cAMP approximately doubled the open probability (P_O) by increasing the mean open time of the NSC channels without affecting single channel conductance (32, 34). For HSC ENaCs, cAMP increased the activity by increasing the number of channels per unit area of apical membrane (N) presumably by promoting channel insertion with little or no change in P_O (34). The effects of increased cAMP were totally blocked by the ß-receptor antagonist propranolol and by the protein kinase A (PKA) blocker H89. Other agents that increase intracellular cAMP also activate both HSC and NSC channels. For example, the adenylyl cyclase activator, forskolin, has essentially the same effect as ß₂-agonists to activate NSC and HSC channels in ATII cells activity (32, 34) and NSC channels in the continuous alveolar cell line, A549, which are a good model for ATII cells. This result was similar to that of Marunaka and Eaton who reported that exposure of A6 cells to agents that increase cAMP levels also resulted in an increase in the density of single channels but not in their P_O, consistent with (although not proof of) the insertion of new channels in the apical cell membranes (27). However, recently Butterworth and colleagues (62) have shown that increases in cAMP lead to increased exocytosis and endocytosis in A6 cells and increased sodium channels in the apical membrane. These observations are also consistent with the findings of Kleyman and colleagues who reported that exposure of A6 cells to increased intracellular cAMP doubled the amount of ENaC protein in the apical membrane of A6 cells (63).

The fact that the cAMP effect is mediated by PKA is not surprising, but it does raise the question of the mechanism by which PKA increases channel activity. PKA is known to promote microfilament- and microtubule-mediated exocytosis (64–69), and disrupting microfilaments blocks the cAMP-mediated increase in sodium transport in A6 cells (70). In addition, PKA is known to phosphorylate other cytoskeletal proteins (such as ankyrin, spectrin, or fodrin) (71, 72) that could be involved in exocytosis or stabilization of ENaCs in the membrane. Therefore, the increase in HSC channel density would seem to be a predictable response to activation of PKA.

Obviously, the PKA-mediated increase in P_O for NSC channels must involve a different mechanism. One possibility is that PKA may be directly phosphorylating the channel proteins. This possibility is supported by studies of ATII cells patched in the inside–out mode where addition of exogenous PKA with 1-mM ATP and 5-mM MgCl₂ to the bath solution more than doubled the mean open time and almost doubled the P_O of single channels (32). The addition of PKA plus ATP to the presumed cytoplasmic side of planar bilayers containing the putative immunopurified ATII Na⁺ channel protein also resulted in a doubling of single-channel P_O (73). These data support the hypothesis that phosphorylation of the Na⁺ channel complex is involved in cAMP-mediated increase in P_O of NSC channels.

Although phosphorylation of channel subunits appears to play a role in PKA-mediated activation of NSC channels, other factors may also be important. Besides increasing intracellular concentrations of cAMP, the ß₂-agonist terbutaline also substantially increases intracellular calcium in adult ATII cells (34). The large terbutaline-induced increase in intracellular calcium appears sufficient to activate NSC channels in adult ATII cells (34) because application of comparable calcium concentrations to the cytosolic surface of excised, cell-free patches activated channels to almost the same extent as ß₂ agonists. One interesting possibility is that some of the diversity in NSC channel properties could reflect the combination of the phosphorylation state of the channel and the local calcium concentration. In a dephosphorylated state, only very high levels of intracellular calcium could activate NSC channels, whereas phosphorylated channels could be spontaneously active with normal levels of intracellular calcium, but small increases in intracellular calcium could produce an additional increase in channel activity.

There may also be a contribution of channel turnover in the activation of NSC channels. In fetal distal lung epithelial cells, brefeldin A blocked the ability of terbutaline to stimulate the P_O of NSC channels (74). Brefeldin is usually associated with inhibition of protein trafficking through the Golgi apparatus; thus, its effect on the action of terbutaline suggests a requirement for membrane trafficking. However, membrane trafficking is usually associated with increases in intracellular calcium: The effect of brefeldin may, therefore, only reflect a reduction in the agonist-induced calcium increase. Clearly, terbutaline increases Na⁺ transport across ATII and fetal distal lung epithelial cells via a number of different mechanisms.

In conclusion, it seems clear that ion channels that are members of the ENaC family of proteins play an important role in lung fluid balance. In ATII 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. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of -ENaC, ß-ENaC, and -ENaC subunits to form channels with different biophysical properties and different mechanisms for regulation. In particular, when only -ENaC subunits are assembled together to form channels, the result is a 21- to 28-pS nonselective cation channel that is sensitive to intracellular calcium and whose P_O is increased by cAMP. In marked contrast, when -ENaC, ß-ENaC, and -ENaC subunits are assembled together, the result is a 4- to 6-pS channel that is highly selective for Na⁺ over K⁺ and is insensitive to intracellular calcium, and cAMP increases channel number with no effect on P_O. The exact mechanism by which cAMP and ß-adrenergic agonists increase salt and water transport in the lung remains unclear, although single-channel measurements appear to argue for a concurrent effect to increase activity of both Na⁺ and Cl^- channels. Nonetheless, the diversity of channel expression and functional properties leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport.

	FOOTNOTES

 
Supported by HL63306 to L.J. and HL071621, DK56305, and DK-50268 to D.C.E.

(Received in original form June 19, 2003; accepted in final form October 14, 2003)

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TOP ABSTRACT SINGLE-CHANNEL MEASUREMENTS FROM... THE MOLECULAR BASIS FOR... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG CATION... EFFECT OF STEROID HORMONES... EFFECT OF OXYGEN TENSION... IMPLICATIONS OF Na+ CHANNEL... REGULATION OF Na+ CHANNELS... REFERENCES

 

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