INTRODUCTION

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THE EXISTENCE of active Na⁺ reabsorption across the adult alveolar epithelium in vivo in a number of species, including human, has been well documented (for a review, see Ref. 17). In brief, plasma or normal saline containing 5% bovine serum albumin instilled in the alveolar spaces of anesthetized animals or isolated perfused lungs is reabsorbed within a few hours. Coinstillation of amiloride or 5-(N-ethyl-N-isopropyl)-amiloride into the alveolar space or injection of ouabain into the circulation significantly decreases the rate of fluid reabsorption (16, 20, 29).

Additional insight into the nature of the transporters was derived from electrophysiological measurements performed on alveolar type II (ATII) cells. Patch-clamp studies (7, 30) demonstrated the presence of nonselective and moderately selective Na⁺ channels in the apical membranes of ATII cells maintained in primary culture. Furthermore, when grown to confluence and mounted in Ussing chambers, ATII cells generate a spontaneous potential difference and short-circuit current (I_sc) that are inhibited to a large extent by amiloride and ouabain (4). Based on the results of these studies, we now believe that Na⁺ diffuses passively across the ATII apical membrane, mainly through cation channels (23), down a favorable electrochemical gradient maintained by Na⁺-K⁺-ATPase and then are actively transported across the basolateral membrane by the ouabain-sensitive Na⁺-K⁺-ATPase. K⁺ leaves ATII cells, driven by their favorable electrochemical gradient, through K⁺ channels located in the basolateral membrane, whereas Cl crosses through the paracellular junctions in response to the transepithelial potential difference.

The importance of active Na⁺ transport in fluid clearance across the injured alveolar epithelium was demonstrated by two different studies. First, instillation of phenamil, an irreversible blocker of epithelial Na⁺ channels, into the lungs of rats exposed to hyperoxia resulted in higher levels of lung water compared with rats receiving vehicle alone (29). Second, a positive correlation has been established between active Na⁺ transport across the alveolar space of patients with acute lung injury and the rate of resolution of noncardiogenic pulmonary edema (18). The demonstration that Na⁺ transport across the alveolar epithelium in vivo and ex vivo, as well as across ATII cells, can be upregulated by -agonists (2, 4, 5) has led to speculation that these agents may 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-chlorophenylthio)-cAMP (8-CPT-cAMP) increase Na⁺ transport across alveolar epithelial cells. Activation of ₂-receptors, shown to be present on ATII cell surfaces (3), generally stimulates adenylate cyclase that, in turn, increases intracellular cAMP levels and activates Na⁺ channels in a number of epithelial cells and tissues (8, 12). 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 (11, 19). Based on the results of their experiments in Ussing chambers, Jiang et al. (11) 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.

Herein, we present evidence indicating that increased levels of cAMP in the ATII cell cytoplasm activate amiloride-sensitive Na⁺ channels. Results of patch-clamp studies convincingly show that -agonists increase the open probability (P_o) and mean open time (₁)of amiloride-sensitive single Na⁺ channels located in the apical membranes of rat ATII cells maintained in primary culture without altering their single-channel unitary conductance. Furthermore, protein kinase A (PKA) in the presence of ATP phosphorylated a putative Na⁺-channel protein isolated from rabbit ATII cells and activated an amiloride-sensitive single channel when this protein was reconstituted in planar lipid bilayers. Finally, we believe that the results of I_sc measurements across cultured rat and rabbit ATII cells obtained from a number of laboratories (4, 19) indicate that cAMP-stimulated vectorial Na⁺ transport cannot be explained solely by activation of Cl channels.

	SINGLE-CHANNEL RECORDINGS IN ATII CELLS: EFFECTS OF CAMP/PKA

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Single channels with diverse biophysical properties have been detected in ATII cells isolated from the lungs of adult rabbits, rats, and guinea pigs (for a review, see Ref. 15). Yue et al. (30) isolated ATII cells from the lungs of rats by elastase digestion and plated them on fibronectin-treated coverslips for 12-24 h. In cells patched in the cell-attached mode, single-channel currents were observed for holding potentials between 80 and +30 mV, with a single-channel conductance of 27 ± 3 pS in 10-15% of successful patches (Figs. 1 and 2). The addition of 10 µM terbutaline (a potent ₂-agonist known to increase intracellular cAMP levels) to the bath increased the ₁ and P_o of these channels without affecting their unitary conductance (Table 1). Pretreatment of ATII cells with propranolol (10 µM), a ₂-antagonist, obviated the terbutaline-induced increases in P_o and ₁. Because ATII cells in primary culture orient themselves so that their basal membranes are attached to the substratum and the apical membranes are pointing upward (14), channels in excised or cell-attached patches are likely to be located in their apical membranes.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Sample recordings of single-channel currents from a cell-attached patch obtained from an alveolar type II (ATII) cell cultured for 24 h. Ionic composition of pipette and bath solutions was (in mM) 150 sodium glutamate, 5 HEPES, 2 CaCl2, and 1 EGTA, pH 7.2. Applied potential difference across patch membrane was 20 mV (extracellular side as reference). A: single-channel current obtained under control conditions (downward deflection indicates inward positive currents). B: single-channel current across the same cell 5 min after terbutaline (10 µM) was added to bath solution. [Reprinted from Yue et al. (30) with permission.]

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Current-voltage (I-V) relationships for single-channel currents obtained from ATII cells patched in cell-attached mode before [control (cont)] and 5 min after addition of 10 µM terbutaline (Terb) to bath solution. Ionic composition of pipette and bath solutions was (in mM) 150 sodium glutamate, 5 HEPES, 2 CaCl2, and 1 EGTA, pH 7.2. Lines were fitted through data points with linear regression and extrapolated to 0 current. Notice absence of currents for applied potential differences exceeding +30 mV (extracellular side as reference). Values are means ± SE; n = 7. In most cases, SE was smaller than width of symbols. [Reprinted from Yue et al. (30) with permission.]

View this table: [in this window] [in a new window]   Table 1.   Po and 1 of single channels in cell-attached and inside-out patches of rat ATII cells maintained in primary culture

Single-channel currents with a conductance of 25 ± 2 pS and Na⁺-to-K⁺ permeability ratio (P_Na/P_K) of 7:1 (Figs. 3-5) were also recorded across 50% of ATII cells patched in the inside-out mode (30). The addition of 1 µM amiloride or 5-(N-ethyl-N-isopropyl)-amiloride to the pipette solution (150 mM sodium glutamate) blocked single-channel activity almost completely. The addition of 250 U/ml of PKA, 1 mM ATP, and 5 mM MgCl₂ to the bath solution (150 mM sodium glutamate) increased the single-channel ₁ and P_o without altering the unitary conductance (Figs. 3-5, Table 1). The observed increase in P_o with PKA may have been brought about by direct phosphorylation of Na⁺-channel proteins or phosphorylation of cytoskeletal proteins such as actin, ankyrin, spectrin, or fondrin interacting with Na⁺-channel proteins (22, 26).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Sample recordings of single channels recorded across an ATII cell patched in inside-out mode. Ionic composition of pipette solution was (in mM) 150 sodium glutamate, 5 HEPES, 2 CaCl2, and 1 EGTA, pH 7.2, and that of bath solution was (in mM) 150 sodium glutamate, 5 HEPES, and 1 EGTA, pH 7.2. Applied potential difference across patch membrane was 60 mV (extracellular side as reference). A: single-channel current recorded under control conditions. B: single-channel current obtained across the same cell 5 min after addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 to bath (cytoplasmic) side of patch. [Reprinted from Yue et al. (30) with permission.]

View larger version (16K): [in this window] [in a new window]   Fig. 4.   I-V relationships for inside-out patches in ATII cells recorded with symmetrical and nonsymmetrical conditions. Ionic compositions for symmetrical solutions were (in mM) 150 sodium glutamate, 5 HEPES, 2 CaCl2, and 1 EGTA, pH 7.2, for pipette and 150 sodium glutamate, 5 HEPES, and 1 EGTA, pH 7.2, for bath. For nonsymmetrical solutions, sodium glutamate in bath was replaced by 150 mM potassium glutamate. PNa+/PK+, Na+-to-K+ permeability ratio. Values are means ± SE; n = 6 cells. In most cases, SE was smaller than width of symbols. Lines were fitted through points by linear regression. [Reprinted from Yue et al. (30) with permission.]

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Current amplitudes for inside-out patches shown in Fig. 3. Small shift in conductance after addition of PKA (250 U/ml), ATP (1 mM), and MgCl2 (5 mM) was not significant (see Table 1). [Reprinted from Yue et al. (30) with permission.]

Nonselective (P_Na/P_K = 1), voltage-independent, Ca⁺²-activated [intracellular Ca²⁺ concentration > 0.1 mM] cation channels with a conductance of 20.4 pS in symmetric NaCl (150 mM) solutions have also been identified in rat ATII cells patched in the inside-out mode after culture on collagen-coated coverslips for 24 and 72 h (7). The effects of cAMP/PKA on these channels have not been reported. However, the biophysical properties of these Ca⁺²-activated channels are almost identical to those recorded in fetal distal lung epithelial cells (21). Marunaka et al. (13) and Tohda et al. (28) reported that the addition of 10 µM terbutaline to the bath solution of fetal distal lung epithelial cells, patched in the cell-attached mode, increased the P_o of the Ca⁺²-activated, nonselective cation channels. Furthermore, in the presence of brefeldin A, terbutaline did not alter the density of the Ca⁺²-activated cation channel, indicating that terbutaline may promote the trafficking of this cation channel to the apical cell surface (9).

	RECONSTITUTION OF IMMUNOPURIFIED ATII CELL NA+-CHANNEL PROTEIN INTO PLANAR LIPID BILAYERS: EFFECTS OF PKA

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A putative Na⁺-channel protein was isolated from freshly isolated rabbit ATII cells with ion-exchange chromatography followed by immunoaffinity purification in a column coated with a polyclonal antibody raised against purified bovine renal Na⁺-channel protein. The ATII Na⁺-channel protein consists of two peptides with molecular masses of ~130 and 70 kDa (25). When this protein was reconstituted in lipid bilayers, single-channel currents with linear current-voltage relationships and a unitary conductance of 25 pS were seen (25), in agreement with what was observed in rat ATII cells patched in the cell-attached or inside-out modes (10, 30). The addition of PKA and ATP to the presumed cytoplasmic side of the bilayer increased the P_o from 0.40 ± 0.14 to 0.8 ± 0.12 without altering the channel unitary conductance. In additional studies, Berdiev et al. (1) showed that PKA phosphorylated both the 135- and 70-kDa polypeptides of the immunopurified ATII Na⁺-channel protein. Studies in A6 cells have shown that PKA phosphorylates a subunit of the Na⁺-channel protein and that the level of phosphorylation correlates with vectorial Na⁺ transport (24).

	CHANGES IN ISC ACROSS ATII CELL MONOLAYERS BY CAMP

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Patch-clamp measurements provide definitive evidence for the existence of ion channels on cell membranes and considerable insight as to the factors responsible for their regulation. There is considerable interest in correlating these findings with macroscopic measurements of Na⁺ transport across confluent ATII monolayers. Jiang et al. (11) isolated and purified ATII cells from the lungs of adult rats and cultured them on large Transwell membrane filters using serum-free medium until they formed confluent monolayers. Six days later, ATII cells developed large I_sc values, 60% of which were inhibited by 10 µM amiloride. The basolateral addition of terbutaline (2 µM) resulted in a rapid decrease in I_sc followed by a gradual recovery to its baseline value (Fig. 6). When amiloride was added before terbutaline, I_sc decreased but failed to return to its baseline value. Finally, when ATII monolayers were bathed in a Cl-free solution, the response to terbutaline was almost completely abolished. Based on these findings, they proposed that terbutaline stimulated Cl absorption through an apical Cl conductance. The Cl influx hyperpolarized the cell membrane, increasing the driving force for Na⁺ entry. Thus their measurements indicate that terbutaline stimulates Na⁺ transport without directly activating apical Na⁺ channels.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Representative short-circuit current (Isc) tracings showing effects of Terb (2 µM) added into basolateral compartment of an Ussing chamber containing confluent monolayers of rat ATII cells in presence and absence of apical amiloride (Amil). [Redrawn from Jiang et al. (11) with permission.]

However, results from other laboratories cannot be explained by this model. For example, Cheek et al. (4) showed that the addition of terbutaline to the basolateral bath of an Ussing chamber containing confluent monolayers of rat ATII cells resulted in a transient decrease in I_sc followed by a gradual increase to steady-state levels, surpassing the baseline (Fig. 7). About 80% of I_sc after terbutaline stimulation was abolished by the addition of 10 µM amiloride to the apical compartment. The initial decrease in I_sc is consistent with the findings of Jiang et al. (11) and is most likely due to activation of a Cl conductance. However, Jiang et al.'s model cannot account for the increase in I_sc above its initial value after terbutaline stimulation. Instead, Cheek et al.'s (4) data suggest that terbutaline stimulates Na⁺-conductive pathways secondary to an increase in cAMP. Indeed, this group reported that terbutaline stimulated the net apical-to-basolateral flux of ²²NaCl, and the magnitude of stimulation was similar to the steady-state increase in I_sc. An important experiment that needs to be done is to repeat these measurements in monolayers pretreated with amiloride and show that, under those conditions, the response of I_sc to terbutaline is blunted.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Isc responses of rat ATII cell monolayers to Terb (100 µM) added to basolateral compartment of an Ussing chamber. At indicated time, Amil (10 µM) was added into apical compartment. [Modified from Cheek et al. (4) with permission.]

In another study, Nielsen et al. (19) assessed the effects of forskolin and 8-CPT-cAMP on the I_sc of rabbit ATII cells grown to form tight confluent monolayers. Either forskolin (10 µM) or 8-CPT-cAMP (100 µM) produced an early biphasic change in the I_sc followed by a slow steady-state increase to a value that was 3.4 ± 0.2 µA/cm² higher than baseline, in agreement with the data of Cheek et al. (4) (Fig. 8). In addition, Nielsen et al. (19) showed that the addition of forskolin to monolayers pretreated with amiloride resulted in a sustained increase in I_sc. There are several explanations for these findings: first, forskolin may have reversed the effect of amiloride; second, it may have stimulated Na⁺ absorption across nonamiloride-sensitive pathways; and third, it may have induced Cl secretion across cAMP-activated Cl channels. In any event, their data cannot be explained by the model of Jiang et al. (11). Current experiments in a number of laboratories are attempting to identify the mechanisms involved and the factors that may reconcile these apparently contradictory results.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Isc recordings from ATII cell monolayers mounted in Ussing chambers. Addition of forskolin (Forsk; 10 µM) increases Isc either before (A) or after (B) Amil (10 µM) administration. Further addition of cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide (Glib; 200 µM) and/or loop diuretic bumetanide (Bumet; 100 µM) decreased Forsk-mediated Isc. Results represent a typical experiment. [Modified from Nielsen et al. (19) with permission.]

	CONCLUSIONS AND UNANSWERED QUESTIONS

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Measurement of single-channel activity in cell-attached and inside-out patches of dispersed ATII cells maintained in primary culture provides strong support for the hypothesis that agents that increase cAMP increase the activity of moderately selective, amiloride-sensitive Na⁺ channels located in their apical membranes. Whether this increase is brought up by insertion of new channels in the membrane from a cytoplasmic pool or by activation of previously inactive channels remains to be demonstrated.

However, there is a clear discrepancy among the aforementioned patch-clamp studies of Yue et al. (30) and results obtained across confluent monolayers of cultured ATII cells. There is clear evidence that these monolayers contain both amiloride-sensitive and CFTR-type Cl channels in their apical membranes. Furthermore, all published studies agree that terbutaline, forskolin, or 8-CPT-cAMP activate Cl currents across cultured ATII cells. However, the proposed hypothesis that increased Na⁺ transport is due solely to an increased driving force for Na⁺ secondary to activation of Cl channels cannot explain the sustained increase in I_sc reported in at least two studies and contradicts the direct measurement of single-channel activation by terbutaline and PKA.

In trying to explain these apparently contradictory findings, one has to consider that cultured ATII cells undergo a number of important phenotypic changes and may be transformed to ATI cells as shown by their increased immunoreactivity to monoclonal antibodies raised against ATI cells (6). For example, it is possible that cultured but not freshly isolated rat ATII cells express functional CFTR. As shown by Stutts et al. (27), agents that increase cAMP activate amiloride-sensitive currents across cells transfected with rat epithelial Na⁺ channel but not rat epithelial Na⁺ channel and CFTR. One way to resolve this controversy will be to seed ATII cells on clear semipermeable filters and attempt to perform both patch-clamp and I_sc measurements in the same filter.