INTRODUCTION

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ACTIVE Na⁺ transport across the pulmonary epithelium drives liquid from the lung lumen to the interstitium, with subsequent absorption of the fluid into the vasculature (1, 22, 23, 27). In the lung, Na⁺ reabsorption is a two-step process. The first step is passive movement of Na⁺ from the lumen across the apical membrane into the cell through Na⁺-permeable ion channels. The second step is active extrusion of Na⁺ from the cell across the basolateral membrane into the serosal space. Active amiloride-sensitive Na⁺ transport by lung epithelia has been demonstrated by several investigators (10, 19, 21, 25). Also, all three subunits, , , and , of the amiloride-sensitive Na⁺ channels that have been described in other Na⁺-transporting epithelia have also been identified in airway epithelial cells (4, 5, 24, 26, 31). Because of this observation, several investigators (1, 13, 20, 22, 23, 31) have suggested that the epithelial Na⁺ channel (ENaC) subunits known to be responsible for Na⁺ transport in other epithelial tissues must also be responsible for Na⁺ transport in the lung. Consistent with this idea is the observation by Hummler et al. (13) that genetically knocking out the -subunit of ENaC leads to defective lung liquid clearance and premature death in perinatal mice. Thus there appears to be direct evidence that, in vivo, the ENaC constitutes the limiting step for Na⁺ absorption in epithelial cells of, at least, the fetal lung. Furthermore, when observed in other epithelial tissues or if ENaC subunits are expressed in oocytes, single ENaCs are low-conductance (4-5 pS), highly sodium-selective channels. In contrast, most laboratories studying lung ENaCs report nonselective cation channels [Na⁺-to-K⁺ permeability ratio (P_Na/P_K) = 1] with a high conductance (10-45 pS) that are quite different from the highly selective, low-conductance ENaCs. In fetal distal lung epithelial cells from 20-day-gestation rat fetuses, Orser et al. (28) observed, using symmetric solutions and inside-out recordings, single channels with a conductance of 23 ± 1.1 pS and a P_Na/P_K of 0.9. These channels were blocked by amiloride applied to the apical side of the membrane. Marunaka (18) has described an Na⁺-permeable, nonselective cation channel with a linear current-voltage relationship and a single-channel conductance of 26.9 ± 0.8 pS in the fetal distal lung epithelium. Feng et al. (8) and Yue et al. (34) have also recently described nonselective cation channels in apical cell-attached and inside-out patches from adult rat alveolar type II (ATII) cells. The channels have a P_Na/P_K of 1, are voltage independent, and are inhibited by amiloride. In apical membrane patches on adult rat ATII cells in primary culture, Jain et al. (15) have also observed nonselective cation channels similar in conductance and ion selectivity to the ones described by Orser et al. (28). Thus the predominant Na⁺-permeable channels observed electrophysiologically in both adult and fetal lung cells appear to be nonselective cation channels (although some details of channel characteristics appear to differ between preparations; for a review, see Ref. 20). Although the exact role that these channels play in vivo is unclear, electrophysiological considerations and studies from epithelial cells from other organs suggest a role for this channel in Na⁺ transport in the lung. For example, Tohda et al. (30) showed that these channels may play a role in the increased reabsorption of fluid by alveolar epithelia in response to -agonist stimulation.

The apparently conflicting information obtained from electrophysiological and molecular biological experiments caused us to investigate the relationship of ENaC to the nonselective cation channels in lung epithelial cells. Antisense oligonucleotides targeting individual subunits were used to manipulate the expression of the respective subunit proteins, and the patch-clamp technique was used to characterize the resulting single channels. Our results suggest that the -subunit is responsible for the nonselective lung epithelial cation channels in rat lung epithelia.

	METHODS

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Type II pneumocyte isolation and culture. ATII cells were isolated by enzymatic digestion of lung tissue from adult Sprague-Dawley rats (100-250 g) with the use of published techniques (15). Briefly, the rats were anesthetized with pentobarbital sodium and heparinized (100 U/kg). ATII cells were digested by tracheal installation of elastase (0.4 mg/ml). The lung tissue was minced in DNase (1 mg/ml) and filtered sequentially through 100- and 20-mM nylon mesh. The cells thus obtained were collected, centrifuged, and seeded onto glass coverslips (~2 × 10⁵ cells/cm²) in Dulbecco's modified Eagle's medium-Ham's F-12 medium containing 5% fetal calf serum and antimicrobial agents and supplemented with L-glutamine and sodium bicarbonate. Cells were incubated in 90% air-10% CO₂ and used for patch-clamp studies between 24 and 96 h after being plated on glass coverslips. No significant difference in Na⁺-channel activity or characteristics was observed during this time period. Cell viability (>90%) and ATII cell purity (>95%) has been previously established in our laboratory (15).

Solutions and drugs. All solutions were made with deionized water and then passed through a 0.2-µm filter (Gelman Sciences, Bedford, MA) before use. The bath and pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl₂, 1 CaCl₂, 5 KCl, and 10 HEPES, pH 7.4 with 2 N NaOH. The contents of the bath and pipette solutions were varied as appropriate for specific protocols. All chemicals were obtained from Sigma (St. Louis, MO).

Procedure for single-channel recordings. Patch-clamp experiments were carried out at room temperature. The pipettes were pulled from filamented borosilicate glass capillaries (TW-150, World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). The pipettes were coated with Sylgard (Dow Corning) and fire polished (Narishige). The resistance of these pipettes was 5-8 M when filled with the pipette solution. We used the cell-attached configuration for most of our studies because in this configuration, the cytoplasmic constituents remain intact, thus allowing us to study the role of cytoplasmic second messengers in the regulation of ion-channel activity. Inside-out patches were also used to determine the selectivity of the channel and to determine whether the effects of agents were directly on the channel or mediated by a signaling cascade. After formation of a high-resistance seal (>50 G) between the pipette and the cell membrane, channel currents were sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) and filtered at 1 kHz with an eight-pole, low-pass Bessel filter. Data were recorded by a computer with pCLAMP 6 software (Axon Instruments). Current-amplitude histograms were made from stable continuously recorded data, and the open and closed current levels were determined from least-squares fitted Gaussian distributions. We used the product (NP_o) of the number of channels (N) times the open probability (P_o) as a measure of the activity of the channels within a patch. This product could be calculated from the single-channel recording without making any assumptions about the total N in a patch or the P_o of a single channel where T is the total recording time, i is the number of channels open, and t_i is the recording time during which i channels are open. Current-amplitude histograms provide the clearest demonstration of multiple current levels. The total N in a patch was estimated by observing the number of peaks in a current-amplitude histogram over the entire duration of the recording period. The P_o of the channels was calculated with FETCHAN in pCLAMP 6 and with locally developed software (17).

RT-PCR and Western blotting. Semiquantitative RT-PCR of total RNA obtained from rat ATII cells was performed with subunit-specific primers based on published sequences. PCR reactions were run in the linear range of amplification, with glyceraldehyde-3-phosphate dehydrogenase amplified as standard for comparison. These primers amplified a single fragment of the expected size for each RNA [579 bp for rat (r) -ENaC, 548 bp for -rENaC, and 558 bp for -rENaC]. The PCR products were not seen in control reactions without RNA. The sequences of the amplified fragments are identical to the published sequences (31). The sequences of the primers are given in Table 1.

To determine whether protein for each of the subunits was present, we used Western blot experiments to confirm the presence of protein for all three subunits in ATII cells in primary culture. Polyclonal antibodies were produced by identifying unique antigenic 20-amino acid sequences in each subunit. The peptides were produced in Emory's microchemical facility and sent to Lofstrand Laboratories where the peptides were coupled to KLH and used to immunize at least two rabbits with each peptide. This provided at least one anti-peptide antibody specific for each subunit.

	RESULTS

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Characteristics of single nonselective cation channels in the apical membrane of ATII cells. Jain et al. (15) and others (18, 28) have previously reported that the predominant Na⁺-permeable channel observed in cell-attached patches on the apical membrane of ATII cells with 140 mM NaCl in the bath and pipette is a high-conductance (20.6 ± 1.1 pS) channel (Fig. 1A). The single-channel current has a linear current-voltage relationship with no detectable rectification (Fig. 1B). The pipette potential at which current polarity reversed was estimated to be 37 mV. Because the resting membrane potential of alveolar epithelium has previously (28) been shown to be approximately 30 to 40 mV, the reversal potential appears to be close to 0 mV, which would be expected for a nonselective cation channel.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Characteristics of nonselective cation channel. A: single-channel recordings of a nonselective cation channel from a cell-attached patch in alveolar type II cell. C, closed state. Nos. on right, mV. B: current-voltage relationship showing that unit conductance of channel is 20.6 ± 1.1 pS and that current-voltage relationship is linear, with no detectable rectification. Vp, membrane potential. C: amplitude histogram of patch in A showing that open probability is ~0.2.

Ion selectivity was determined with excised inside-out patches and solutions of varying ionic compositions. The channel had a similar permeability to Na⁺ and K⁺ (P_Na/P_K = 0.97 ± 0.07; n = 7 patches). The channel P_o was decreased by amiloride (0.1-1 mM) applied to the extracellular side (i.e., in the micropipette). The P_o of untreated channels was 0.31 ± 0.01 vs. 0.03 ± 0.01 after 100 nM amiloride (P < 0.01; n = 7 patches). More than one current level was observed in 85.7% of the active patches.

Functional importance of Na⁺-permeable nonselective cation channels and their relationship to ENaC. We examined the expression of mRNA for the three subunits of the ENaC by RT-PCR experiments. Semiquantitative RT-PCR of total RNA obtained from rat type II cells was performed with subunit-specific primers. PCRs were run in the linear range of amplification, with glyceraldehyde-3-phosphate dehydrogenase amplified as a standard for comparison. These primers amplified a single fragment of the expected size for each RNA (579 bp for -rENaC, 548 bp for -rENaC, and 558 bp for -rENaC; Fig. 2A). The PCR products were not seen in the control reactions without RNA. The sequences of the amplified fragments are identical to the published sequences (31). Thus ATII cells, at least, have an RNA message for all of the ENaC subunits. To determine whether protein for each of the subunits was present, we used Western blot experiments to confirm the presence of protein for all three subunits in ATII cells in primary culture (Fig. 2B). These results show that even under conditions when the predominant Na⁺-permeable channel is the nonselective cation channel, there is an easily detectable message for all ENaC subunits and protein for the - and -subunits (we have yet to perform Western blot experiments for the -subunit). These results, of course, in no way prove that the nonselective cation channel is related to ENaC.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   RT-PCR and Western blot demonstrate presence of epithelial Na+ channel (ENaC) subunits. A: RT-PCR is capable of amplifying appropriate size cDNA for each subunit. B: Western blot demonstrates presence of - and -subunits. -Subunit has also been demonstrated in alveolar type II cells by others (21a).

Antisense oligonucleotides against -, -, and -subunits of ENaC decrease the corresponding protein.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Antisense oligonucleotide induced inhibition of ENaC subunit expression. Top: Western blot analysis (n = 1) for -subunit (A) and -subunit (B) of ENaC in rat alveolar type II cells treated with sense and antisense oligonucleotides. Bottom: densitometric estimation of protein concentration (conc) for corresponding subunit. Expression of all subunits was significantly reduced by treatment with antisense oligonucleotides.

Antisense oligonucleotide against -subunit of ENaC decreases lung epithelial cation-channel activity.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Treatment with antisense oligonucleotide directed against -subunit of ENaC produced a significant reduction in number of apical membrane cell-attached patches with cation channels. Addition of either - or -antisense oligonucleotide separately or together or sense oligonucleotide for any of the subunits (as a control) did not change cation-channel activity.

	DISCUSSION

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Whole tissue experiments on a variety of epithelial tissues including the lung imply a simple picture of Na⁺ channels. This picture is consistent with tissue reabsorption of Na⁺ and, at a single-channel level, implies that the channels should be highly selective for Na⁺ over K⁺, should open and close infrequently, and should be blocked by low concentrations of the drug amiloride (2). Indeed, channels with such properties can be observed in several epithelial preparations (9), but examination of single channels in several Na⁺-transporting epithelial cells suggests a more complicated picture of amiloride-blockable channels. Single-channel studies (3, 20) have identified at least four different amiloride-blockable channels with either high selectivity, low selectivity, or no selectivity for Na⁺ over K⁺ in the apical membranes of a variety of cultured and native epithelial cells. These channels differ in their ion selectivity, unitary conductance, and other characteristics, but they all have been proposed to play some role in Na⁺ absorption. Benos et al. (3) recently compiled and plotted selectivity versus conductance of epithelial Na⁺ channels from the published literature and showed the wide variability in these characteristics. Few of these reported channels match the characteristics of the prototypical native amiloride-sensitive Na⁺ channel that has a low conductance (4-6 pS) and high selectivity for Na⁺ (P_Na/P_K > 10). In fact, only one study (6) has reported a channel fitting this description from the lung. In this study, the investigators recorded a 4-pS amiloride-sensitive channel from outside-out patches excised from apical membrane. The channel was highly selective for Na⁺ (P_Na/P_K > 10). However, no other laboratory has subsequently reported observing this channel in lung epithelia on a consistent basis. For epithelial cells in culture, culture conditions are apparently the primary determinant of the frequency with which these different channel types are found in the apical membrane (7, 33), but ultimately, the observed variations in single-channel properties must be due to variations in subunit stoichiometry, variations in subunit types combining to form channels, or posttranslational modifications of the subunit proteins.

Molecular basis for Na⁺ transport in lung epithelial cells. The differences in the properties of Na⁺-permeable channels make a determination of the molecular basis for Na⁺ transport an important issue. Several investigators and experiments with transgenic animals (1, 13, 20, 21, 22, 23) have suggested that -, -, and -ENaC subunits known to be responsible for Na⁺ transport in other epithelial tissues are also responsible for Na⁺ transport in the lung. This would be consistent with the observation that rat adult and fetal lungs have been shown to express all three subunits of the ENaC (13). However, electrophysiological measurements suggest that if ATII cells do express ENaCs, then the functional characteristics are quite different from the highly selective, low-conductance (4-5 pS) ENaCs observed in other tissues.