INTRODUCTION

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

SODIUM TRANSPORT IS IMPORTANT in the lung. 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 (29, 64). Studies showing reabsorption of intratracheally instilled isotonic fluid or plasma from the alveolar spaces of adult anesthetized animals and resected human lungs, and the partial inhibition of this process by amiloride and ouabain, indicate that adult alveolar epithelial cells are also capable of actively transporting Na⁺ (reviewed in Ref. 57). Whether active Na⁺ transport plays an important role in maintaining the normal alveoli free of fluid remains to be established. On the other hand, a variety of studies have clearly established that active Na⁺ transport limits the degree of alveolar edema in pathological conditions in which the alveolar epithelium has been damaged. For example, intratracheal instillation of a Na⁺ channel blocker in rats exposed to hyperoxia increased the amount of extravascular lung water (89). Conversely, intratracheal instillation of adenoviral vectors containing copies of the Na⁺-K⁺- ATPase genes increased survival of rats exposed to hyperoxia (19). Moreover, patients with acute lung injury who are still able to concentrate alveolar protein (as a result of active Na⁺ reabsorption) have a better prognosis than those who cannot (58, 88).

	Na+ CHANNELS IN LUNG EPITHELIAL CELLS

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

Insight into the nature and regulation of transport pathways has come from electrophysiological studies in freshly isolated and cultured alveolar type II cells (ATII) cells. These cells, which make up 67% of the alveolar epithelial cells but constitute only 3% of the alveolar surface area in the adult lung, can be isolated at high purity and grown to form confluent monolayers (16, 53). We know that Na⁺ diffuse passively into ATII cells through apically located amiloride-sensitive, amiloride-insensitive, and cGMP-gated cation channels (32, 90) and are extruded across the basolateral membranes by the ouabain-sensitive Na⁺-K⁺-ATPase (20). The 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 (56). In situ hybridization studies have also identified the presence of at least two of the three subunits of the cloned epithelial Na⁺ channel [- amiloride-sensitive epithelial Na⁺ channels (ENaC) and -ENaC] in both adult and fetal alveolar epithelial cells in vivo (21, 81, 90) and all three subunits in vitro (32, 33, 71, 77). In addition, cDNAs that encode the subunits of amiloride-sensitive Na⁺ channels in other Na⁺ transporting epithelia have also been cloned from airway fetal lung epithelial cells (87).

Recently, alveolar type I (ATI) cells have also been isolated from adult rat lungs and, like ATII and fetal distal lung epithelial (FDLE) cells, have been shown to contain aquaporins and possess very high permeability to water (23, 73, 85). Preliminary information (presently unpublished) also indicate that these cells contain proteins antigenically related to Na⁺ transporters (such as ENaC and Na⁺-K⁺-ATPase). Although no definitive functional studies exist at present, it seems possible that ATI cells are also capable of vectorial Na⁺ transport.

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

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

In some epithelial tissues, Na⁺ transport appears to be a relatively simple process beginning with tissue reabsorption of Na⁺ via Na⁺ channels that, at a single-channel level, are highly selective for Na⁺ over K⁺ and that are blocked by low concentrations of the drug amiloride (2). Indeed, channels with such properties can be observed in several epithelial preparations (17, 36, 42, 44, 49, 55, 66, 69), but examination of single channels in several other Na⁺-transporting epithelial cells, including lung cells, suggests a more complicated picture of amiloride-blockable channels. Single-channel studies have identified at least four different amiloride-blockable 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 characteristics; nevertheless, they all have been proposed to play some role in epithelial Na⁺ absorption.

	ELECTROPHYSIOLOGY OF Na+ CHANNELS IN LUNG ALVEOLAR EPITHELIAL CELLS

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

Several investigators have confirmed the presence of cation channels in both FDLE cells (46, 48, 67) and adult type II pneumocytes (22, 31, 90, 91) [as reviewed recently by Matalon and O'Brodovich (55)]. Using FDLE cells from 20-day-gestation rat fetuses cultured on collagen-coated coverslips, Orser et al. (67) reported single channels with a conductance of 23 ± 1.1 pS and Na⁺-to-K⁺ permeability of 0.9. These channels were blocked by amiloride applied to the apical side of the membrane. Marunaka and colleagues (48, 51, 70) have also described an amiloride-sensitive, Na⁺-permeable, nonselective cation (NSC) channel with a linear current-voltage relationship and single-channel conductance of 26.9 ± 0.8 pS in the FDLE. The exact role these channels play in vivo is unclear, especially because their activation requires a very high (intracellular) concentration of Ca²⁺ (>100 µM). It is unlikely that cells are able to achieve such high concentrations of Ca²⁺ under normal circumstances, except possibly when stimulated by -adrenergic agents (84). Marunaka et al. (51) also described another amiloride-sensitive NSC channel from FDLE cells with lower conductance (12 pS) that is activated by insulin (but not by terbutaline) and requires a lower Ca²⁺ concentration for its activity. Yet another NSC channel that may be less dependent on Ca²⁺ was reported from guinea pig FDLE cells by MacGregor et al. (46).

Similarly, several investigators have reported cation channels with variable single-channel characteristics from adult ATII pneumocytes. Feng et al. (22) described a NSC channel in apical cell-attached and inside-out patches from rat ATII cells. The channels have a Na⁺-to-K⁺ permeability ratio of close to 1, are voltage independent, are inhibited by amiloride, but remain in the closed state unless the intracellular Ca²⁺ concentration is higher than 10 µM. Yue et al. (91) reported the existence of amiloride-sensitive 25- to 27-pS channels in both cell-attached and inside-out patches of rat type II cells that are activated by cAMP. Ion substitution studies showed that these channels have a Na⁺-to-K⁺ selectivity of 6:1. Jain and his co-workers (12, 31, 32) also reported a NSC channel in the apical membrane patches of ATII cells. This amiloride-sensitive channel has a unitary conductance of 20 pS, a Na⁺-to-K⁺ permeability ratio of 1, and is not strongly dependent on Ca²⁺. It is activated by cAMP (12) but suppressed by cGMP (31). A 27-pS amiloride-sensitive channel was also identified in cell-attached patches of ATII cells isolated from the lungs of rats exposed to sublethal hyperoxia (90). However, if these NSC channels were the only pathways for Na⁺ absorption across the alveolar epithelium in vivo, one would expect to find higher than normal K⁺ concentrations in the adult epithelial lining fluid. Indeed, Nielson and Lewis (61) used alveolar micropuncture techniques to measure the ionic composition of the alveolar epithelial lining fluid of anesthetized rabbits and did find elevated K⁺ levels. The measured K⁺ concentration (7.4 meq/l) was much higher than expected and was reduced significantly after application of amiloride. These data are consistent with the presence of low-selectivity Na⁺ channels in the alveolar epithelium. Thus it is interesting that in other studies investigators have identified in ATII cells the presence of both highly selective Na⁺ (33) and K⁺ channels (14). Thus it is very likely that a variety of Na⁺ channels exist in the alveolar epithelium in vivo.

	MOLECULAR BASIS FOR Na+ TRANSPORT IN LUNG EPITHELIAL CELLS

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

Expression cloning methods to isolate cDNAs from rat (r) distal colon have yielded three separate cDNAs that are designated as -, -, and -rENaC (6, 7, 45). 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 (52, 54) and Northern blot studies showing mRNA for these transporters, although differences exist in the developmental expression and regulation of the three ENaC subunits (63, 82, 87, 90). Voilley et al. (87) have also cloned a Na⁺ channel from fetal lung tissue using homology to cDNA from rat colon ENaC. In addition, there is functional evidence for the importance of ENaC in lung fluid absorption. Hummler et al. (29) showed that genetically knocking out -ENaC leads to defective lung liquid clearance and premature death in newborn mice. Kerem et al. (37) recently showed that patients with ENaC loss of function mutations who develop pseudohypoaldosteronism have excess airway liquid due to inadequate absorption of Na⁺ and water. Studies utilizing nasal potential differences in human neonates have also suggested that immaturity of Na⁺ transport mechanisms contribute to the development of transient tachypnea of the newborn and respiratory distress syndrome (1, 26). However, it is not clear whether ENaC channels are the only functional Na⁺-permeant channels in the lung because recent studies point to the presence of amiloride-insensitive channels in alveolar epithelia that are cyclic nucleotide gated (15, 75). Furthermore, the higher than expected K⁺ concentrations in the alveolar lining fluid of anesthetized rabbits and the reduction of these values after application of amiloride (61) are consistent with K⁺ efflux through NSC or poorly selective cation channels in alveolar epithelial cells.

	EXPRESSION OF HIGHLY SELECTIVE CATION CHANNELS IN ALVEOLAR EPITHELIA

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

The coexpression of -, -, and -ENaC cRNAs in Xenopus laevis oocytes is associated with a highly Na⁺-selective, 4- to 5-pS channel, that is, a highly selective cation channel or HSC channel (6, 7, 28, 45, 68). In contrast to the numerous single-channel studies from several Na⁺-transporting epithelia confirming the presence of such channels, electrophysiological evidence for the presence of highly Na⁺-selective channels in lung epithelia is limited. In adult ATII cells, Jain et al. (33) 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), highly Na⁺-selective channel (HSC) with a Na⁺-to-K⁺ permeability of >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. These findings point to the importance of the cellular environment in determining the electrophysiological characteristics of ion channels. Although environmental variables can be most easily determined and controlled under in vitro conditions, the cellular environment in vivo may also alter the ion channel expression in ATII cells. For example, as mentioned above, measurement of K⁺ concentrations in the alveolar epithelial lining fluid by micropuncture indicates high K⁺ values, suggesting K⁺ efflux through NSC or poorly selective cation channels in alveolar epithelial cells. However, it is possible that local trauma from the insertion of the micropipette may have resulted in a local hypoxic environment favoring the switch from highly selective to nonselective sodium channels.

	RELATIONSHIP BETWEEN LUNG CATION CHANNELS AND ENaC

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

The available molecular biological and biochemical data suggest the presence of ENaC subunits in ATII cells. It has been proposed that different combinations of the various subunits comprising the channel (, , and ) could produce channels with varying unitary conductances and regulatory properties (79). Channels with alternative subunits could explain at least some of the functional diversity observed in electrophysiological recordings. In fact, Jain and co-workers (32, 33) have shown that, as expected, HSC channels are composed of , , and  subunits, whereas NSC channels appear to be composed of -subunits alone, and moderately selective channels are some combination of  with  or .

Together, the results of these studies indicate that the apical membranes of ATII and FDLE cells contain the following: 1) Ca²⁺-activated NSC (composed of -ENaC subunits) and Na⁺-selective channels (composed of - and - or -ENaC subunits) and 2) a Ca²⁺-insensitive, 4-pS Na⁺-selective channel, with biophysical properties similar to those of -, -, and -ENaC reconstituted in Xenopus oocytes (6, 7). 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.

As mentioned above, all three ENaC subunits can be detected by Western blotting in both fetal and adult ATII cells (82), but these experiments provide little information about the relative amounts of the different subunits. However, in situ hybridization studies examining message levels have provided some evidence for the relative abundance of the subunits. In two studies, - and - but not -ENaC were identified in the alveolar epithelium (63, 90). The failure to detect -ENaC in these studies was probably due to its very low abundance because in two other studies (21, 80) -ENaC was detected but at very low levels compared with  and . Thus -ENaC may be the rate-limiting subunit in formation of channels and trafficking to the surface membrane. This idea is supported by work demonstrating that -ENaC expression can be increased by various hormones such as dexamethasone (40) with an increase in channel activity and changes in the biophysical properties of the channel as if, in response to increased production of  protein, the channels in the apical membrane were changing from  only or - channels to the highly selective -- channels.

	REGULATION OF Na+ CHANNELS BY CAMP

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

The demonstration that Na⁺ transport across the alveolar epithelium in vivo and ex vivo, as well as across ATII cells, can be upregulated by -adrenergic agonists (3, 11-13) has led to speculation that these agents might be useful in limiting alveolar edema and decreasing morbidity and mortality in patients with acute lung injury.

Presently, there is controversy concerning the mechanisms by which -agonists, such as terbutaline, or lipid-soluble analogs of cAMP, such as 8-(4-chlorophenylthiol)-cAMP, increase Na⁺ transport across alveolar epithelial cells. Activation of ₂ receptors on ATII cells (9) generally stimulates adenylate cyclase that, in turn, increases intracellular cAMP levels and activates Na⁺ channels in a number of epithelial cells and tissues (25, 49). However, short-circuit current measurements across rabbit and rat ATII cells indicate that cAMP-induced responses are considerably more complex and involve both Na⁺ and Cl conductive pathways (34, 60). On the basis of the results of their experiments in Ussing chambers, Jiang et al. (34) 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 around 40 mV, stimulation of Cl channels will result in influx of Cl, 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 et al. (10) found that an increase in intracellular cAMP produced an initial and rapid increase in Cl current followed by a slower increase of amiloride-sensitive Na⁺ current. Under open-circuit conditions, the final Na⁺ and Cl current was equal, representing a net salt flux across the epithelial cell layer.

In addition, the results of a number of patch-clamp studies also argue against the Jiang hypothesis. In several studies, addition of terbutaline to the basolateral side of ATII cells patched in the cell-attached mode increased ENaC single-channel activity (12, 91). 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 (12, 91). For HSC ENaCs, cAMP increased the activity by increasing the number of channels per unit area of apical membrane, presumably by promoting channel insertion with little or no change in P_o (12). The effects of increased cAMP were totally blocked by the -antagonist propranolol and by the protein kinase A (PKA) blocker H-89. 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 -agonists to activate NSC and HSC channels in ATII cells activity (12, 91) and NSC channels in continuous alveolar cell line, A549, which are a good model for ATII cells. This result was similar to those of Marunaka and Eaton (49), 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. However, recently Butterworth et al. (5) showed that increases in cAMP lead to increased exo- and endocytosis in A6 cells and increased Na⁺ channels in the apical membrane. These observations are also consistent with the findings of Kleyman et al. (38), who reported that exposure of A6 cells to increasing intracellular cAMP doubled the amount of ENaC protein in the apical membrane of A6 cells.

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 (4, 18, 24, 35, 39, 47), and the disruption of microfilaments blocks the cAMP-mediated increase in Na⁺ transport in A6 cells (86). In addition, PKA is known to phosphorylate other cytoskeletal proteins [such as ankyrin, spectrin, or fodrin (72, 78)] 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 (91). 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 (76). 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 -agonist terbutaline also substantially increases intracellular Ca²⁺ in both FDLE cells (50, 59, 62) and adult ATII cells (12). The large terbutaline-induced increase in intracellular Ca²⁺ appears sufficient to activate NSC channels in fetal distal lung cells (51, 84) and adult ATII cells (12) because application of comparable Ca²⁺ concentrations to the cytosolic surface of excised, cell-free patches activated channels to almost the same extent as -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 Ca²⁺ concentration. In a dephosphorylated state, only very high levels of intracellular Ca²⁺ could activate NSC channels, whereas phosphorylated channels could be spontaneously active with normal levels of intracellular Ca²⁺ and small increases in intracellular Ca²⁺ could produce an additional increase in channel activity.

There may also be a contribution of channel turnover in the activation of NSC channels. In FDLE cells, brefeldin A blocked the ability of terbutaline to stimulate the P_o of NSC channels (30). Brefeldin is usually associated with inhibition of protein trafficking through the Golgi so its effect on the action of terbutaline suggests a requirement for membrane trafficking. However, membrane trafficking is usually associated with increases in intracellular Ca²⁺: the effect of brefeldin may, therefore, only reflect a reduction in the -agonist-induced Ca²⁺ increase. Clearly, terbutaline increases Na⁺ transport across ATII and FDLE cells via a number of different mechanisms.

	CONCLUSION

TOP ABSTRACT INTRODUCTION Na+ CHANNELS IN LUNG... SINGLE-CHANNEL MEASUREMENTS... ELECTROPHYSIOLOGY OF Na+... MOLECULAR BASIS FOR Na+... EXPRESSION OF HIGHLY SELECTIVE... RELATIONSHIP BETWEEN LUNG... REGULATION OF Na+ CHANNELS... CONCLUSION REFERENCES

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, Na⁺-permeable channels. This significant diversity appears to play a role in both normal lung physiology and in pathological states. At least part of the diversity of Na⁺-permeable channels in lung arises from assembling different combinations of -, -, 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 NSC channel that is sensitive to intracellular Ca²⁺ and whose P_o is increased by cAMP. In marked contrast, when -, -, and -ENaC subunits are assembled together, the result is a 4- to 6-pS channel that is highly selective for Na⁺ over K⁺ and insensitive to intracellular Ca²⁺; in addition, cAMP increases the channel number with no effect on P_o. The exact mechanism by which cAMP and -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.