Epidermal growth factor regulation in adult rat alveolar
type II cells of amiloride-sensitive cation channels
P. J. KEMP,1 Z. BOROK,2 K. J. KIM,2 R. L. LUBMAN,2 S. I. DANTO,2 AND E. D. CRANDALL2
Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care
Medicine, University of Southern California, Los Angeles, California 90033; and 1School of
Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
2Will
sodium channels; alveolar epithelium; nonselective cation
channels
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 Na1 and fluid absorption, including Na1-glucose cotransport (7, 8), Na1proline cotransport (7), Na1/H1-exchange, and Na1-Cl2
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
C1058
cotransport (11). In common with other absorptive
epithelia, the mature alveolar epithelium reabsorbs
fluid via an active Na1-linked process. In this process,
basolateral Na1-K1-ATPase activity energizes the vectorial movement of Na1 through apically positioned
Na1 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 Na1 absorption
and Cl2 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 Na1-K1-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 Na1 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
Na1-entry step is also necessary for EGF upregulation
of Na1 transport (i.e., blockade of apical Na1 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
Na1 entry with EGF treatment is via increased density
of nonselective cation channels.
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
http://www.ajpcell.org
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Kemp, P. J., Z. Borok, K. J. Kim, R. L. Lubman, S. I.
Danto, and E. D. Crandall. Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive
cation channels. Am. J. Physiol. 277 (Cell Physiol. 46):
C1058–C1065, 1999.—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 a-, b- and g-subunits
of rat epithelial Na1 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 (PNa1/PK1 < 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.
C1059
EGF AND AEC CATION CHANNELS
MATERIALS AND METHODS
Isolation and Culture of Alveolar Epithelial Type II Cells
Patch-Clamp Experiments
Solutions. See Table 1 for solution compositions and abbreviations.
Experimental. Coverslips were placed in a perfusion bath
(maximum volume 5 200 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 MV, 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
Bath 1
NaCl
Sodium isethionate
KCl
Potassium isethionate
HEPES
MgCl2
MgSO4
CaCl2
CaSO4
EGTA
D-Glucose
pH
Whole
Cell
Bath 2
Whole
Cell
Pipette
140
5
140
5
10
140
10
5
10
1.2
1.2
1
1
5
7.4
5
7.4
Single
Channel
Bath
Single
Channel
Pipette
140
140
5
5
10
1.2
10
1.2
1.2
1
1
5
7.2
5
7.4
1
5
7.2
Values are in mM (except pH).
Whole cell currents were recorded in essentially symmetrical sodium isethionate solutions during 50-ms step depolarizations (20-mV increments) from 2100 to 1120 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 (2Vp ). By convention, inward cationic current is depicted as a downward
deflection [i.e., channels open downward at negative pipette
potential (2Vp ) values and upward at positive 1Vp 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, sizefractionated by agarose gel electrophoresis under denaturing
conditions, transferred to nylon membrane (Hybond N1;
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 a-, b-, and g-subunits of rat
epithelial Na1 channel (rENaC) (Dr. B. C. Rossier, University
of Lausanne, Switzerland). Blots were washed with high
stringency (0.53 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 6 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
Cell Purity and Viability
All preparations were routinely checked for cell
purity and viability using tannic acid and trypan blue
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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 Ca21-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 3 105/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).
Table 1. Composition of solutions used in
patch-clamp studies
C1060
EGF AND AEC CATION CHANNELS
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.
Whole Cell Currents
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
Fig. 1. Rat epithelial Na1 channel (rENaC) Northern analysis. A: exemplar Northern blot that was hybridized
sequentially with [32P]cDNA probes directed against a-rENaC (3.7 kb), b-rENaC (2.2 kb), g-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
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 (2EGF) was designated
as unity and signal density obtained from cells that had been treated with EGF (1EGF) was compared with its own
control. RNA loading was normalized by reference to 18S ribosomal band (n 5 6 separate cell preparations).
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Two-day exposure of cultured alveolar epithelial type
II cells to 20 ng/ml EGF resulted in no significant
change in whole cell capacitance [2EGF, 5.95 6 0.5 pF;
1EGF, 5.7 6 0.39 pF (means 6 SE; n 5 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 EGFtreated and EGF-untreated cells (Fig. 2, C and F).
However, whole cell conductance density of EGFtreated cells (169.6 6 3.5 pS/pF; n 5 8) was significantly (P , 0.05) higher than that of untreated cells
(99 6 13.1 pS/pF; n 5 9).
With the use of a concentration of amiloride (10 µM)
known to inhibit maximally all classes of amiloridesensitive Na1 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 6 13.1
to 78.6 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 6 3.5 to
116.8 6 22.4 pS/pF (see exemplar currents in Fig. 2, D
and E). Mean current density vs. voltage relationships
are shown in Fig. 2C (2EGF) and 2F (1EGF). 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 amilorideresistant 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 6 4.8 to
52.8 6 15.0 pS/pF (P , 0.01).
The apparently inconsistent observations that EGF
treatment caused more than a doubling of the Na1
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 (Na1 containing) for whole cell bath
solution 2 (K1 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 cationselective permeability pathway in these cells, we employed excised, inside-out membrane patches from both
control and EGF-treated cells.
EGF AND AEC CATION CHANNELS
C1061
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 Na1-containing solutions, the
channel had linear current-voltage relationships (Fig.
3B), with a unitary conductance of 24.7 6 0.8 pS (n 5 7)
in treated cells and 24.1 6 0.7 pS in untreated cells (n 5
10). No channel activity could be recorded when the
pipette solution contained 10 µM amiloride (n 5 8, data
not shown). In untreated cells, exchanging the intracellular (single channel bath) solution for solutions where
Na1 had been substituted isosmotically by different
monovalent cations (Cl2 salts; Fig. 4A, n 5 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 Na1 . Cs1 . Rb1 $ K1 . Li1 (with Na1
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 PNa1/PK1 of 1.0:0.72 (Fig. 4C,
n 5 3). Channels from both cohorts (6EGF) 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 260 mV (2Vp ), EGF-treated cells had a mean Po of
0.42 6 0.07 (Fig. 5, C and D), whereas in untreated cells
Po was 0.38 6 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.
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,
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Fig. 2. Whole cell Na1 current recordings. A, B, D, E: typical whole cell Na1 currents recorded in alveolar epithelial
type II cells that had been cultured for 48 h in the absence (2EGF, A and B) or presence (1EGF, D and E) of EGF. A
and D show control recordings while B and E show Na1 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 2100 to 1120 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 5 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.
C1062
EGF AND AEC CATION CHANNELS
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 Na1
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.
DISCUSSION
We have previously shown that EGF increases shortcircuit current (Isc ) across alveolar epithelial cell monolayers over a relatively delayed time course, beginning
Fig. 4. Permeability sequence for single
channels. A: typical single channel recording from an untreated cell. The patch was
held at a potential of 160 mV (5 2Vp ), and
the intracellular Na1-containing solution
changed sequentially for those containing
the cations as indicated on left (all Cl2
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 5 4) and treated (C; n 5 3) cells. Lines
were calculated using the constant field
equation for current in an iterative fitting
protocol.
at 10–12 h and becoming maximal at 24–36 h (1).
Although EGF-induced signal transduction events occur within minutes (28), changes in Na1 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
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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 Na1-containing solution at the pipette
potentials (2Vp ) indicated at left. By convention, inward cationic current is depicted as a downward deflection (i.e., channels are opening downward at negative
2Vp values and upward at positive 2Vp
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 5
7) or untreated (open symbols, n 5 10) with
EGF for 48 h. Regression lines were fitted
by the method of least squares.
EGF AND AEC CATION CHANNELS
C1063
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 160 mV in symmetrical Na1containing solutions. Channel closed state is
indicated by the arrows at right.
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 Na1 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 a-, b-, and g-rENaC
are all present in primary cultured cells at 48 h.
However, it seems unlikely that EGF increases whole
cell Na1 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 a/b ENaC
dimers carrying this component of the current, 2) the
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 260 mV in symmetrical Na1-containing
solutions.
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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 Cl2
conductances found in the alveolar epithelium (10)
demonstrates significant permeability to glutamate
(14), we chose to employ isethionate as the substitute
for Cl2 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.
C1064
EGF AND AEC CATION CHANNELS
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 Na1 and K1 (PK1/PNa1 5 1.15) and were highly
selective for cations (PCl2/PNa1 , 0.05), as we similarly
found. Channel activity was Ca21 dependent, requiring
at least 10 µM Ca21 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 Ca21-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 Ca21 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 < 0.6) by b2-agonists at
1 µM Ca21 when intracellular Cl2 concentration ([Cl2]i )
is reduced to between 40 and 20 mM (17). This suggests
that the activity that we record at the artificially
elevated [Cl2]i and supramaximal [Ca21]i is of physiological significance. We did not specifically test whether
lowering bath Ca21 would inactivate the channels,
which would definitively show these channels to be
identical to those described above. However, their
relative nonselectivity between Na1 and K1 indicates
that they are in some respects different from those
described by Yue and Matalon (27), which were seven
times more permeable to Na1 than to K1. Importantly,
a similar nonspecific cation channel in fetal alveolar
epithelial type II cells appears to be the major b2-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 amiloridesensitive component of the whole cell current is ,30%
of the total current and is carried by ionic channels that
select poorly among cation species.
We thank Drs. Cecilia Canessa and Bernard Rossier for Na1
channel subunit cDNAs. We note with appreciation the expert
technical support of Li Ma, Martha Jean Foster, and Susie Parra.
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.
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: [email protected]).
Received 25 February 1999; accepted in final form 30 August 1999.
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.
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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 a/b-ENaC has been
reported to have a single channel conductance of 5.1 pS
and a higher permeability to Li1 than Na1 (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
Na1 flux (and, therefore, fluid absorption), an amiloridesensitive, nonselective cation channel becomes the likely
EGF-responsive Na1 entry pathway. Kizer et al. (12)
showed that expression of a-rENaC alone results in
formation of a 24-pS channel that cannot distinguish
between Na1 and K1, whereas Jain et al. (9) have
recently suggested that a-rENaC alone (or in combination with proteins other than b- or g-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 a-rENaC is more efficiently
translated than b- or g-rENaC in both EGF-treated and
untreated cells. Thus, while we recognize the possibility that expression of a-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 Ca21 concentration ([Ca21]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 [Ca21]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
EGF AND AEC CATION CHANNELS
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
mal growth-factor receptor is increased following bleomycininduced lung injury in rats. Am. J. Respir. Cell Mol. Biol. 11:
540–551, 1994.
Marunaka, Y. Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium. Jpn. J. Physiol. 47:
499–511, 1997.
Marunaka, Y., N. Niisato, H. O’Brodovich, and D. C. Eaton.
Regulation of an amiloride-sensitive Na1-permeable channel by
a b2-adrenergic agonist, cytosolic Ca21 and Cl2 in fetal rat
alveolar epithelium. J. Physiol. (Lond.) 515: 669–683, 1999.
Matalon, S., K. L. Kirk, J. K. Bubien, Y. S. Oh, P. Hu, G. Yue,
R. Shoemaker, E. J. Cragoe, Jr., and D. J. Benos. Immunocytochemical and functional characterization of Na1 conductance
in adult alveolar pneumocytes. Am. J. Physiol. 262 (Cell Physiol.
31): C1228–C1238, 1992.
McNicholas, C. M., and C. M. Canessa. Diversity of channels
generated by different combinations of epithelial sodium channel
subunits. J. Gen. Physiol. 109: 681–692, 1997.
Nielson, V. G., M. D. DuVall, M. S. Baird, and S. Matalon.
cAMP activation of chloride and fluid secretion across the rabbit
alveolar epithelium. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol.
19): L1127–L1133, 1998.
Orser, B. A., M. Birtlik, L. Fedorka, and H. B. O’Brodovich.
Cation selective channel in fetal alveolar type II epithelium.
Biochim. Biophys. Acta 1094: 19–24, 1991.
Raaberg, L., E. Nexo, S. Buckley, W. Luo, M. L. Snead, and
D. Warburton. Epidermal growth-factor transcription, translation, and signal transduction by rat type-ii pneumocytes in
culture. Am. J. Respir. Cell Mol. Biol. 6: 44–49, 1992.
Raaberg, L., E. Nexo, P. E. Jorgensen, S. S. Poulsen, and M.
Jakab. Fetal effects of epidermal growth-factor deficiency induced in rats by autoantibodies against epidermal growth-factor.
Pediatr. Res. 37: 175–181, 1995.
Strandjord, T. P., J. G. Clark, D. E. Guralnick, and D. K.
Madtes. Immunolocalization of transforming growth-factoralpha, epidermal growth-factor (EGF), and EGF-receptor in
normal and injured developing human lung. Pediatr. Res. 38:
851–856, 1995.
Sznajder, J. I., K. M. Ridge, D. B. Yeates, J. Ilekis, and W.
Olivera. Epidermal growth factor increases lung liquid clearance in rat lungs. J. Appl. Physiol. 85: 1004–1010, 1998.
Warburton, D., R. Seth, L. Shum, P. G. Horcher, F. L. Hall, Z.
Werb, and H. C. Slavkin. Epigenetic role of epidermal growth
factor expression and signalling in embyonic mouse lung morphogenesis. Dev. Biol. 149: 123–133, 1992.
Yue, G., and S. Matalon. Single-channel Na1 currents in
alveolar type-II (ATII) cells from normal and oxygen adapted rats
(Abstract). FASEB J. 8: A665, 1994.
Zhang, X. L., S. Veeraraghavan, R. L. Lubman, S. F. HammAlvarez, Z. Borok, S. I. Danto, and E. D. Crandall. Epidermal growth factor-induced signalling mechanisms in alveolar
epithelial cells (Abstract). Am. J. Respir. Crit. Care Med. 157:
A425, 1998.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 18, 2017
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.
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.
4. Danto, S. I., Z. Borok, X. L. Zhang, M. Z. Lopez, P. Patel,
E. D. Crandall, and R. L. Lubman. Mechanisms of EGFinduced stimulation of sodium reabsorption by alveolar epithelial cells. Am. J. Physiol. 275 (Cell Physiol. 44): C82–C92, 1998.
5. Feng, Z. P., R. B. Clark, and Y. Berthiaume. Identification of
nonselective cation channels in cultured adult rat alveolar
type-II cells. Am. J. Respir. Cell Mol. Biol. 9: 248–254, 1993.
6. Fyfe, G. K., P. J. Kemp, E. J. Cragoe, Jr., and R. E. Olver.
Conductive cation transport in apical membrane vesicles prepared from fetal lung. Biochim. Biophys. Acta 1224: 355–364,
1994.
7. Hardin, J. A., J. K. Wong, C. I. Cheeseman, and D. G. Gall.
Effect of luminal epidermal growth factor on enterocyte glucose
and proline transport. Am. J. Physiol. 271 (Gastrointest. Liver
Physiol. 34): G509–G515, 1996.
8. Horvath, K., I. D. Hill, P. Devarajan, D. Mehta, S. C.
Thomas, R. B. Lu, and E. Lebenthal. Short-term effect of
epidermal growth factor (EGF) on sodium and glucose cotransport of isolated jejunal epithelial cells. Biochim. Biophys. Acta
1222: 215–222, 1994.
9. Jain, L., X.-J. Chen, B. Malik, O. Al-Khalili, and D. C. Eaton.
Antisense oligonucleotides against the a-subunit of ENaC decrease lung epithelial cation-channel activity. Am. J. Physiol. 276
(Lung Cell. Mol. Physiol. 20): L1046–L1051, 1999.
10. Kemp, P. J., G. G. MacGregor, and R. E. Olver. G proteinregulated large-conductance chloride channels in feshly isolated
fetal type II alveolar epithelial cells. Am. J. Physiol. 265 (Lung
Cell. Mol. Physiol. 9): L323–L329, 1993.
11. Khurana, S., S. K. Nath, S. A. Levine, J. M. Bowser, C. M.
Tse, M. E. Cohen, and M. Donowitz. Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na1/H1 exchange. J. Biol.
Chem. 271: 9919–9927, 1996.
12. Kizer, N., X. L. Guo, and K. Hruska. Reconstitution of
stretch-activated cation channels by expression of a-subunit of
the epithelial sodium channel cloned from osteoblasts. Proc.
Natl. Acad. Sci. USA 94: 1013–1018, 1997.
13. Lesur, O., K. Arsalane, and D. Lane. Lung alveolar epithelial
cell migration in vitro: modulators and regulation processes.
Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L311–L319,
1996.
14. Litchfield, S. J., R. E. Olver, G. G. MacGregor, and P. J.
Kemp. Amiloride-sensitive whole-cell currents in freshly isolated fetal guinea-pig type-ii pneumocytes. J. Physiol. (Lond.)
475P: P108–P109, 1994.
15. Madtes, D. K., H. K. Busby, T. P. Strandjord, and J. G.
Clark. Expression of transforming growth-factor-a and epider-
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