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Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels

P. J. Kemp1, Z. Borok2, K. J. Kim2, R. L. Lubman2, S. I. Danto2, and E. D. Crandall2

2 Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033; and 1 School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 µM) produced ~20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha -, beta - and gamma -subunits of rat epithelial Na+ channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (PNa+/PK+ approx  1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

sodium channels; alveolar epithelium; nonselective cation channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPIDERMAL GROWTH FACTOR (EGF) is important to normal lung development (2, 23, 24, 26) and appears to play a pivotal role, in conjunction with a number of other peptide growth factors, in recovery from lung injury (15). Although EGF provides a potent cell proliferative signal, there is an accumulating body of evidence to suggest that, in addition, EGF can regulate cellular function independently of cell division. In the small intestine (an example of an absorptive epithelium), EGF stimulates a number of processes known to be important in transepithelial Na+ and fluid absorption, including Na+-glucose cotransport (7, 8), Na+-proline cotransport (7), Na+/H+-exchange, and Na+-Cl- cotransport (11). In common with other absorptive epithelia, the mature alveolar epithelium reabsorbs fluid via an active Na+-linked process. In this process, basolateral Na+-K+-ATPase activity energizes the vectorial movement of Na+ through apically positioned Na+ and cation channels.

Regulation of the composition and volume of the alveolar subphase is crucial for effective gas exchange in the adult lung. The efficiency of transepithelial water transport is dependent on balance of Na+ absorption and Cl- secretion (20) and epithelial barrier integrity. In injury states, absorptive driving force is exceeded by fluid backflux across a damaged epithelium, and alveolar flooding ensues. EGF and its receptor are both expressed in alveolar epithelial cells of the postnatal lung (22), and their expression rises dramatically in lung injury (24). It has been shown that EGF stimulates alveolar epithelial cell migration (13) and differentiation as well as alveolar fluid absorption (25). Therefore, it seems likely that EGF signaling represents an important mechanism that helps coordinate the process of recovery from lung injury by stimulating epithelial repopulation, restoration of barrier integrity, and clearance of alveolar fluid overload.

Recent observations that EGF treatment of cultured monolayers of alveolar epithelial cells augments both short-circuit current (Isc) (1) and Na+-K+-ATPase expression/activity (4) strongly support the notion that EGF may be capable of increasing fluid reabsorption in the mature lung, a suggestion that has now also been demonstrated in vivo (25). Although the mechanism of this upregulation of Na+ transport by EGF (in vivo and in culture) appears to be intimately linked to increased pump transcription, translation (4), and activity (1, 4) (thereby increasing the driving force), increase in the Na+-entry step is also necessary for EGF upregulation of Na+ transport (i.e., blockade of apical Na+ entry with benzamil during EGF exposure partially suppresses the EGF-evoked rise in Isc). However, to date, there have been no direct electrophysiological observations of modulation of the conductive entry step by this growth factor. The studies described herein address this directly by use of whole cell and single channel configurations of the patch-clamp technique and describe the effects of 48-h treatment of freshly isolated alveolar epithelial type II cells with EGF on amiloride-sensitive and amiloride-insensitive whole cell currents. Furthermore, it provides evidence, at the single channel level, which suggests that the mechanism for upregulation of Na+ entry with EGF treatment is via increased density of nonselective cation channels.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Culture of Alveolar Epithelial Type II Cells

Solutions. Unless otherwise stated, all chemicals were of the highest grade available and were purchased from Sigma Chemical (St. Louis, MO). Solution I contained (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 1.0 CaCl2, and 10 D-glucose, pH 7.4 with NaOH. Solution II contained (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 1.0 EGTA, and 10 D-glucose, pH 7.4 with NaOH. Neutralization solution contained (in mM) 136 NaCl, 2.2 NaPO4, 5.3 KCl, 10 HEPES, 5.6 D-glucose, and 2 EDTA supplemented with 1% BSA and 0.1% soybean trypsin inhibitor. Minimum completely defined serum-free (MDSF) medium contained 1:1 DMEM/Ham's F-12 nutrient mix (Sigma) supplemented with 1.25 mg/ml BSA, 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 100 U/ml sodium-penicillin G, and 100 µg/ml streptomycin.

Experimental. Adult male Sprague-Dawley rats were anesthetized with pentobarbital sodium, and alveolar type II cells were isolated as previously described with the use of elastase digestion and differential adhesion on IgG-coated plates (1). Briefly, alveolar macrophages were removed by 10 times lavage with the Ca2+-free solution II, and the pulmonary vascular bed was cleared of blood by transcardial perfusion with ice-cold PBS. Lungs were removed and instilled to slightly more than physiological volume via a tracheal catheter, with solution I containing 2.0-2.5 U/ml elastase (Worthington Biochemical, Freehold, NJ) and incubated for 20 min at 37°C. The lungs were then chopped finely in neutralization solution and filtered sequentially through filters of mesh sizes 100, 40, and 15 µm before being plated onto IgG-coated bacteriological plates. Contaminating cells were allowed to adhere to the plates for 1 h at 37°C before the type II cell-enriched supernatant was collected and centrifuged at 150 g. The cell pellet was then resuspended in MDSF and cells seeded, at a density of 2 × 105/cm2, onto glass coverslips and incubated in the presence or absence of added EGF (20 ng/ml) in a humidified air-CO2 mixture (19:1) at 37°C for 36-48 h. The concentration of EGF used in the present study is identical to that previously determined to maximally increase Isc across alveolar epithelial monolayers (1). Although it is above normal circulating in vivo blood levels of EGF, it is likely within the range locally attainable for alveolar epithelial cells in the setting of lung injury or by pharmacological intervention (25).

Patch-Clamp Experiments

Solutions. See Table 1 for solution compositions and abbreviations.

                              
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Table 1.   Composition of solutions used in patch-clamp studies

Experimental. Coverslips were placed in a perfusion bath (maximum volume = 200 µl; flow rate ~5 ml/min) mounted on the stage of a Nikon TM-D inverted microscope and were viewed using phase-contrast optics. Only those cells containing the granular inclusions typical of type II cell morphology were chosen for study. Pipettes were manufactured from thin-walled, filamented borosilicate glass (World Precision Instruments, Sarasota, FL) using a two-stage puller (Narishige PB-7). Pipettes used for whole cell and single channel recording had resistances of 4-5 and 10-12 MOmega , respectively. Voltage clamp was achieved using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) in capacitative (single channel recording) or resistive (whole cell recording) feedback modes. Voltage protocols were generated and current recording/analysis was achieved, using the pCLAMP 6.03 suite of software (Axon Instruments).

Whole cell currents were recorded in essentially symmetrical sodium isethionate solutions during 50-ms step depolarizations (20-mV increments) from -100 to +120 mV at 0.5 Hz. Holding potential was 0 mV. Single channel activity was recorded in the inside-out configuration and, therefore, all figures quote minus the pipette potential (-Vp). By convention, inward cationic current is depicted as a downward deflection [i.e., channels open downward at negative pipette potential (-Vp) values and upward at positive +Vp potentials].

RNA Isolation and Northern Blotting

Solutions. Hybridization buffer contained 1 M NaPO4 (pH 7.0), 7% SDS and 1% BSA.

Experimental. Total RNA was isolated from the cells by the phenol-guanidinium-chloroform method of Chomczynski and Sacchi (3). RNA was denatured with formaldehyde, size-fractionated by agarose gel electrophoresis under denaturing conditions, transferred to nylon membrane (Hybond N+; Amersham Life Science, Cleveland, OH) and immobilized by ultraviolet cross-linking. Blots were prehybridized for 2 h and then hybridized for 16 h at 65°C in hybridization buffer with the 32P-labeled cDNA probes for alpha -, beta -, and gamma -subunits of rat epithelial Na+ channel (rENaC) (Dr. B. C. Rossier, University of Lausanne, Switzerland). Blots were washed with high stringency (0.5× standard sodium citrate), visualized by autoradiography, and quanititated by densitometry. Blots were reprobed for 18S rRNA to normalize differences in RNA loading.

Statistical Analysis

Where appropriate, data are presented as means ± SE. Comparisons between EGF-treated and EGF-untreated cells employed unpaired Student's t-test. Comparisons between current and conductance densities in the absence or presence of amiloride employed paired Student's t-test. In the current density vs. voltage plots, analysis of covariance was employed to test statistically the difference before and after addition of amiloride. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Purity and Viability

All preparations were routinely checked for cell purity and viability using tannic acid and trypan blue dye exclusion, respectively. Over 90% of cells stained positive for lamellar bodies, and viability always exceeded 90%.

rENaC Northern Blotting

The Northern blot shown in Fig. 1 demonstrates clearly that exposure of cells for 2 days to EGF resulted in a significant (P < 0.01) decrease in steady-state mRNA levels of all three rENaC subunits.


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Fig. 1.   Rat epithelial Na+ channel (rENaC) Northern analysis. A: exemplar Northern blot that was hybridized sequentially with [32P]cDNA probes directed against alpha -rENaC (3.7 kb), beta -rENaC (2.2 kb), gamma -rENaC (3.2 kb), and 18S mRNAs. Total RNA was extracted from alveolar epithelial type II cells that had been cultured in serum-free medium for 48 h in the absence (left lane) or presence (right lane) of epidermal growth factor (EGF). RNA loading = 5 µg/lane. B: mean densitometric quantification of 6 Northern blots prepared from 4 separate cell isolation/purifications. Signal density obtained for each rENaC subunit mRNA from untreated cells (-EGF) was designated as unity and signal density obtained from cells that had been treated with EGF (+EGF) was compared with its own control. RNA loading was normalized by reference to 18S ribosomal band (n = 6 separate cell preparations).

Whole Cell Currents

Two-day exposure of cultured alveolar epithelial type II cells to 20 ng/ml EGF resulted in no significant change in whole cell capacitance [-EGF, 5.95 ± 0.5 pF; +EGF, 5.7 ± 0.39 pF (means ± SE; n = 21 and 23, respectively)]. With the use of whole cell bath solution 1 and whole cell pipette solution (see Table 1), the families of currents (Fig. 2, A, B, D, and E) elicited by the standard voltage-stepping protocol evoked essentially linear current-voltage relationships in both EGF-treated and EGF-untreated cells (Fig. 2, C and F). However, whole cell conductance density of EGF-treated cells (169.6 ± 3.5 pS/pF; n = 8) was significantly (P < 0.05) higher than that of untreated cells (99 ± 13.1 pS/pF; n = 9).


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Fig. 2.   Whole cell Na+ current recordings. A, B, D, E: typical whole cell Na+ currents recorded in alveolar epithelial type II cells that had been cultured for 48 h in the absence (-EGF, A and B) or presence (+EGF, D and E) of EGF. A and D show control recordings while B and E show Na+ currents after a 10-min treatment with 10 µM amiloride. Currents were recorded in essentially symmetrical sodium isethionate (NaIse) solutions during 50-ms step depolarizations (20-mV increments) from -100 to +120 mV at 0.5 Hz. Holding potential was 0 mV with sodium isethionate bath and whole cell bath solution 1 pipette solutions (see Table 1). C and F: mean current-density vs. voltage relationships for untreated (C) and EGF-treated (F) cells in the absence and presence of amiloride (Amil; n = 8 treated and 9 untreated cells). G: comparison of the separate components of the currents. Open bars, untreated cells; solid bars, EGF-treated cells. EGF more than doubled the amiloride-sensitive component of the whole cell currents.

With the use of a concentration of amiloride (10 µM) known to inhibit maximally all classes of amiloride-sensitive Na+ channels in tissues, isolated cells, cell monolayers, and subcellular cell fractions, current densities in untreated and treated cells were reduced at all test potentials (Fig. 2, A, B, D, and E). Conductance density of cells not treated with EGF was significantly (P < 0.003) reduced by amiloride ~20% from 99 ± 13.1 to 78.6 ± 10.5 pS/pF (see exemplar currents in Fig. 2, A and B), whereas that of EGF-treated cells was significantly (P < 0.01) reduced ~30% from 169.6 ± 3.5 to 116.8 ± 22.4 pS/pF (see exemplar currents in Fig. 2, D and E). Mean current density vs. voltage relationships are shown in Fig. 2C (-EGF) and 2F (+EGF). Analysis of covariance showed that amiloride caused a significant (P < 0.05) decrease in whole cell current densities in both EGF-treated and untreated cells. The amiloride-resistant components of current (treated vs. untreated) were not significantly different from each other (P > 0.1), suggesting that EGF selectively increased amiloride-sensitive channels in these cells. Calculation of the absolute magnitude of this amiloride-sensitive component of the whole cell currents (Fig. 2G) showed that EGF exposure resulted in a significant (P < 0.05) increase in the conductance density from 20.6 ± 4.8 to 52.8 ± 15.0 pS/pF (P < 0.01).

The apparently inconsistent observations that EGF treatment caused more than a doubling of the Na+ current density (Fig. 2) but about a halving of rENaC subunit mRNA expression prompted us to investigate further the nature of the currents. Substituting whole cell bath solution 1 (Na+ containing) for whole cell bath solution 2 (K+ containing) resulted in only a mild reduction of the inward currents (data not shown), which suggested that the majority of the channels underlying the whole cell currents discriminated poorly between cations. To examine the possibility that a nonspecific cation conductance is a major cation-selective permeability pathway in these cells, we employed excised, inside-out membrane patches from both control and EGF-treated cells.

Single Channel Currents

Nonselective cation channels have been described previously in fetal and postnatal alveolar epithelial type II cells (5, 21). However, long-term regulation of these channels has not been observed to date. We investigated the possibility that EGF increases the magnitude of amiloride-sensitive whole cell currents via changes in the biophysical properties of these single channels.

With the use of the single channel bath solution and single channel pipette solution (see Table 1), the most common channel type observed in inside-out, excised membrane patches from both treated and untreated cells (Fig. 3A) was similar to that described previously (5). In these symmetrical Na+-containing solutions, the channel had linear current-voltage relationships (Fig. 3B), with a unitary conductance of 24.7 ± 0.8 pS (n = 7) in treated cells and 24.1 ± 0.7 pS in untreated cells (n = 10). No channel activity could be recorded when the pipette solution contained 10 µM amiloride (n = 8, data not shown). In untreated cells, exchanging the intracellular (single channel bath) solution for solutions where Na+ had been substituted isosmotically by different monovalent cations (Cl- salts; Fig. 4A, n = 4) resulted in small positive perturbations in reversal potentials and decreases in the magnitude of the outward currents (Fig. 4B). These changes were consistent with the channel being selective for cations. This maneuver allowed calculation of a permeability sequence for the channel as Na+ > Cs+ > Rb+ >=  K+ > Li+ (with Na+ permeability being unity, the ratio was 1.0:0.72:0.56:0.48:0.40). Similarly, in EGF-treated cells, substitution with CsCl resulted in a positive shift in reversal potential and a calculated PNa+/PK+ of 1.0:0.72 (Fig. 4C, n = 3). Channels from both cohorts (±EGF) were insensitive to the effects of the classical anion channel blocker DIDS, even at 100 µM (data not shown). In addition to the almost identical conductance and selectivity of the channels observed in EGF-treated and untreated cells, there was also a strong similarity between open state probability (Po) at potentials similar to the predicted membrane potential in intact cells. At -60 mV (-Vp), EGF-treated cells had a mean Po of 0.42 ± 0.07 (Fig. 5, C and D), whereas in untreated cells Po was 0.38 ± 0.12 (Fig. 5, A and B). Because of the low probability of recording only one channel per patch in the EGF-treated group (see below), further rigorous kinetic comparison was not attempted.


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Fig. 3.   Single channel recordings in excised inside-out patches. A: typical family of currents recorded from an EGF-exposed alveolar epithelial type II cell in symmetrical Na+-containing solution at the pipette potentials (-Vp) indicated at left. By convention, inward cationic current is depicted as a downward deflection (i.e., channels are opening downward at negative -Vp values and upward at positive -Vp potentials). Channel closed state is indicated by the arrows at right. Note that there is a minimum of 3 channel levels in this example. B: mean current-voltage relationships for channels recorded from cells that had been treated (closed symbols, n = 7) or untreated (open symbols, n = 10) with EGF for 48 h. Regression lines were fitted by the method of least squares.



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Fig. 4.   Permeability sequence for single channels. A: typical single channel recording from an untreated cell. The patch was held at a potential of +60 mV (= -Vp), and the intracellular Na+-containing solution changed sequentially for those containing the cations as indicated on left (all Cl- salts). B and C: typical current-voltage relationships of channels recorded with different cation-containing solutions as described in A in patches from untreated (B; n = 4) and treated (C; n = 3) cells. Lines were calculated using the constant field equation for current in an iterative fitting protocol.



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Fig. 5.   Open-state probability of the single channels. Exemplar single channel recordings (B, D) and equivalent amplitude histograms (A, C) from cells either untreated (A, B) or treated (C, D) with EGF for 48 h. Patches were held at +60 mV in symmetrical Na+-containing solutions. Channel closed state is indicated by the arrows at right.

The major difference between single channels recorded in the two groups of cells was the observation that EGF-treated cells had a higher channel density than untreated cells (Fig. 6). More than one-third of the patches obtained from untreated cells contained no channel activity. In contrast, only 15% of patches from EGF-treated cells were quiescent. Furthermore, the modal number of channels per patch increased from one to two with EGF exposure. In EGF-treated cells, 70% of the patches contained two or more channels, whereas this was reduced to <20% in untreated cells. The observation that nonspecific cation single channel density is increased by EGF treatment is consistent with the whole cell data in Fig. 2. Furthermore, taken together with the Northern blot data in Fig. 1, these data suggest that the EGF-evoked increase in Na+ current is not likely due to the increased functional expression of classical trimeric rENaC but rather to the expression of 25-pS, nonselective cation channels.


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Fig. 6.   Channel density. Frequency histograms plotting number of active nonselective cation channels per patch in excised membrane patches obtained from cells untreated (A) or treated (B) with EGF for 48 h. Patches were held at -60 mV in symmetrical Na+-containing solutions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that EGF increases short-circuit current (Isc) across alveolar epithelial cell monolayers over a relatively delayed time course, beginning at 10-12 h and becoming maximal at 24-36 h (1). Although EGF-induced signal transduction events occur within minutes (28), changes in Na+ pump and channel expression occur over hours and immediately precede Isc changes (4). The present study was designed to investigate the mechanisms by which this chronic effect, which appears to require changes in gene and protein expression, occurs in response to EGF.

This study demonstrates that EGF causes an increase in total alveolar epithelial type II cell whole cell cation current and that ~20-30% of this current is amiloride sensitive. The largest component of the whole cell conductance in both treated and untreated cells was insensitive to blockade by amiloride. This is not the first observation in alveolar epithelial type II cells of significant amiloride insensitivity in whole cell currents (18). It has been suggested that the residual current may be carried by the anion species employed. Indeed, in many studies, glutamate has been used as the major charge-carrying anion (e.g., see Ref. 18). Based on the observation that at least one of the Cl- conductances found in the alveolar epithelium (10) demonstrates significant permeability to glutamate (14), we chose to employ isethionate as the substitute for Cl- in this study. Isethionate is also the least permeable anion tested in the fetal alveolar apical membrane vesicle experimental system (6). With the use of isethionate, the whole cell currents were essentially nonrectifying [in contrast to the previous report (18)] but still contained a sizable amiloride-sensitive component. This component was not investigated further in this study.

Figure 2G shows that EGF more than doubled the amiloride-sensitive whole cell current in alveolar epithelial type II cells. Although it is generally accepted that rENaC underlies Na+ currents in fetal alveolar epithelial type II cells, its contribution to adult cell currents is less well established. The Northern analysis in Fig. 1 shows clearly that mRNA encoding alpha -, beta -, and gamma -rENaC are all present in primary cultured cells at 48 h. However, it seems unlikely that EGF increases whole cell Na+ currents by inducing rENaC transcription and translation, since steady-state mRNA for all three subunits is markedly decreased by EGF. Taken together, the electrophysiological recordings and rENaC Northern analysis suggests three possibilities: 1) the EGF-dependent increase in amiloride-sensitive current results from an increase in the probability of alpha /beta ENaC dimers carrying this component of the current, 2) the available rENaC trimer function is posttranslationally upregulated by EGF, or 3) another cation channel is being upregulated by EGF. The first two possibilities seem unlikely, since recombinant alpha /beta -ENaC has been reported to have a single channel conductance of 5.1 pS and a higher permeability to Li+ than Na+ (19), whereas the vast majority of single channel recordings that we obtained (see below) did not demonstrate channels with conductances small enough to represent classical rENaC subunits (4-6 pS). Therefore, it appears that the classical rENaC pathway may not be the major mechanism contributing to the EGF-induced increase of the conductance in these cells. Because the previous Isc data show that EGF is an important modulator of electrogenic Na+ flux (and, therefore, fluid absorption), an amiloride-sensitive, nonselective cation channel becomes the likely EGF-responsive Na+ entry pathway. Kizer et al. (12) showed that expression of alpha -rENaC alone results in formation of a 24-pS channel that cannot distinguish between Na+ and K+, whereas Jain et al. (9) have recently suggested that alpha -rENaC alone (or in combination with proteins other than beta - or gamma -rENaC) forms the major cation-selective channel in adult alveolar epithelium. These observations are compatible with our electrophysiological findings, although the Northern analysis data would require that alpha -rENaC is more efficiently translated than beta - or gamma -rENaC in both EGF-treated and untreated cells. Thus, while we recognize the possibility that expression of alpha -rENaC alone could result in formation of nonselective 24-pS cation channels (12), a decrease in all three rENaC subunit mRNA levels has not been associated with increased rENaC channels in any setting reported to date. Until further data are available concerning rENaC subunit protein abundance in these cells and/or the molecular identity of the nonselective cation channel is available, however, the issue will remain unresolved.

With the use of a cytosolic (bath) solution that contained the same low intracellular Ca2+ concentration ([Ca2+]i) as that used in the whole cell recording experiments, only 2/37 patches demonstrated single channel events. These events had a calculated conductance of <10 pS and may have been rENaC. However, event frequency was too low for systematic study, and activity rapidly ran down. EGF treatment did not appear to increase the likelihood of observing these infrequent and small events (data not shown). When a solution with 1 mM [Ca2+]i was employed, single channels were seen in >60% and 85% of patches from EGF-untreated and EGF-treated cells, respectively.

Figures 3, 4 and 5 show that the major cation channel in the plasma membrane of adult alveolar epithelial type II cells has a unitary conductance of ~25 pS, selects poorly among monovalent cations, and has a Po of ~0.4 at quasiphysiological membrane potential. EGF exposure does not result in any significant alteration in conductance, selectivity, or Po but causes increased expression/membrane insertion of these channels, as evidenced by the increased density shown in Fig. 6. The biophysical properties of the nonselective cation channels observed in this study closely resemble those found by Feng et al. (5) in adult type II cells, which had a linear slope conductance of 20.4 pS in a symmetrical NaCl solution similar to that used in the current study. These channels were approximately equally permeable to Na+ and K+ (PK+/PNa+ = 1.15) and were highly selective for cations (PCl-/PNa+ < 0.05), as we similarly found. Channel activity was Ca2+ dependent, requiring at least 10 µM Ca2+ on the cytosolic side of an inside-out patch to activate the channel. Similar channels have also been reported in fetal alveolar type II epithelial cells (21), which were reversibly inactivated when the bath was exchanged with a Ca2+-free solution. As noted in Table 1, the bath solution used for single channel patch experiments reported herein (single channel bath solution) contained 1 mM CaCl2. This solution bathes the cytosolic face of the inside-out membrane patch and contains sufficient Ca2+ to fully activate the channels. Although this is out of the range normally seen in living cells, similar channels become almost maximally activated (to Po approx  0.6) by beta 2-agonists at 1 µM Ca2+ when intracellular Cl- concentration ([Cl-]i) is reduced to between 40 and 20 mM (17). This suggests that the activity that we record at the artificially elevated [Cl-]i and supramaximal [Ca2+]i is of physiological significance. We did not specifically test whether lowering bath Ca2+ would inactivate the channels, which would definitively show these channels to be identical to those described above. However, their relative nonselectivity between Na+ and K+ indicates that they are in some respects different from those described by Yue and Matalon (27), which were seven times more permeable to Na+ than to K+. Importantly, a similar nonspecific cation channel in fetal alveolar epithelial type II cells appears to be the major beta 2-agonist regulatable conductance (16).

In summary, we have shown that exposure of cultured, subconfluent adult alveolar epithelial type II cells to EGF causes a marked increase in whole cell cation current and nonselective cation channel density. Under these conditions, it appears that the amiloride-sensitive component of the whole cell current is <30% of the total current and is carried by ionic channels that select poorly among cation species.


    ACKNOWLEDGEMENTS

We thank Drs. Cecilia Canessa and Bernard Rossier for Na+ channel subunit cDNAs. We note with appreciation the expert technical support of Li Ma, Martha Jean Foster, and Susie Parra.


    FOOTNOTES

This work was supported, in part, by a Human Frontiers Science Program Fellowship to P. J. Kemp, by the American Lung Association, the American Heart Association, by National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-38658, HL-51928, HL-38658, and HL-46943, and by the Hastings Foundation.

E. D. Crandall is Hastings Professor of Medicine and Kenneth T. Norris, Jr., Chair of Medicine.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. J. Kemp, School of Biomedical Sciences, Worsely Medical and Dental Bldg., Univ. of Leeds, Leeds LS2 9JT, UK (E-mail: p.z.kemp{at}leeds.ac.uk).

Received 25 February 1999; accepted in final form 30 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Borok, Z., A. Hami, S. I. Danto, R. L. Lubman, K.-J. Kim, and E. D. Crandall. Effects of EGF on alveolar epithelial junctional permeability and active sodium transport. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L559-L565, 1996[Abstract/Free Full Text].

2.   Catterton, W. Z., M. B. Escibedi, W. R. Sexson, M. E. Gray, and H. W. Sundell. Effect of epidermal growth factor on lung maturation in fetal rabbits. Pediatr. Res. 13: 104-108, 1979[Abstract].

3.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

4.   Danto, S. I., Z. Borok, X. L. Zhang, M. Z. Lopez, P. Patel, E. D. Crandall, and R. L. Lubman. Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells. Am. J. Physiol. 275 (Cell Physiol. 44): C82-C92, 1998[Abstract/Free Full Text].

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