Expression of highly selective sodium channels in alveolar
type II cells is determined by culture conditions
Lucky
Jain1,3,
Xi-Juan
Chen1,
Semra
Ramosevac1,3,
Lou Ann
Brown1, and
Douglas C.
Eaton1,2,3
Departments of 1 Pediatrics and 2 Physiology and
3 Center for Cell and Molecular Signaling, Emory University
School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
Alveolar fluid
clearance in the developing and mature lungs is believed to be mediated
by some form of epithelial Na channels (ENaC). However, single-channel
studies using isolated alveolar type II (ATII) cells have failed to
demonstrate consistently the presence of highly selective
Na+ channels that would be expected from ENaC expression.
We postulated that in vitro culture conditions might be responsible for
alterations in the biophysical properties of Na+
conductances observed in cultured ATII cells. When ATII cells were
grown on glass plates submerged in media that lacked steroids, the
predominant channel was a 21-pS nonselective cation channel (NSC) with
a Na+-to-K+ selectivity of 1; however, when
grown on permeable supports in the presence of steroids and air
interface, the predominant channel was a low-conductance (6.6 ± 3.4 pS, n = 94), highly Na+-selective
channel (HSC) with a
PNa/PK >80 that is
inhibited by submicromolar concentrations of amiloride
(K0.5 = 37 nM) and is similar in
biophysical properties to ENaC channels described in other epithelia.
To establish the relationship of this HSC channel to the cloned ENaC,
we employed antisense oligonucleotide methods to inhibit the individual
subunit proteins of ENaC (
,
, and
) and used patch-clamp
techniques to determine the density of this channel in apical membrane
patches of ATII cells. Overnight treatment of cells with antisense
oligonucleotides to any of the three subunits of ENaC resulted in a
significant decrease in the density of HSC channels in the apical
membrane cell-attached patches. Taken together, these results show that
when grown on permeable supports in the presence of steroids and air
interface, the predominant channels expressed in ATII cells have
single-channel characteristics resembling channels that are associated
with the coexpression of the three cloned ENaC subunits
-,
-, and
-ENaC.
single-channel recording; air interface; antisense
oligonucleotides; epithelial sodium channels
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INTRODUCTION |
FETAL TRANSITION TO AIR
BREATHING after birth is characterized by rapid clearance of
fetal lung fluid (6). Although factors that promote the
sudden increase in lung fluid reabsorption at birth are largely
unknown, an increase in amiloride-sensitive Na+ transport
mediated by cation channels located on the apical surface of alveolar
epithelial cells is considered a key step in this process. Several
lines of investigation point to the presence of and a functional role
for epithelial Na+ channels (ENaC) in lung epithelia, but
it is not clear whether these channels are related to the ENaC that has
been cloned and characterized in other epithelial tissues. In other
epithelial tissues, ENaC is a heteromultimeric protein consisting of
some combination of three subunits, designated
-,
-, and
-ENaC. The exact stoichiometry of the subunits forming a channel is
controversial. In rat lung, the expression of rat
-ENaC (
-rENaC)
mRNA in adult and fetal alveolar type II (ATII) cells has been
demonstrated by a variety of techniques (20, 42, 61).
Similarly,
- and
-subunit mRNAs have also been detected in ATII
cells but are less abundant (58, 61). Evidence for a
functional role for ENaC in fetal lung fluid clearance at birth comes
from experiments by Hummler et al. (28), who have shown
that newborn
-subunit knockout mice failed to clear their lung fluid
and died within 48 h of birth.
The coexpression of
-,
-, and
-ENaC cRNAs in Xenopus
oocytes is associated with a highly Na+-selective, 4- to 5-pS channel, that is, a highly selective cation (HSC)
channel (8). Indeed, single-channel studies from several sodium-transporting epithelia have confirmed the presence of such channels (3, 19). In contrast to these studies,
electrophysiological evidence for the presence of HSC channels in lung
epithelia is generally lacking. With the exception of one group
(58) that reported a 4-pS sodium channel in fetal distal
lung epithelial cells with single-channel characteristics similar to
those expected for coexpression of
-,
-, and
-ENaC,
patch-clamp studies from several laboratories, including ours
(22, 31, 37, 45, 60), have shown that the predominant
Na+-permeant channel in ATII cells is a nonselective cation
(NSC) channel. The electrophysiological characteristics of these NSCs (high conductance, low Na+ selectivity) are quite different
from the low-conductance HSCs associated with ENaC expression reported
from other tissues (41, 46).
Thus there is conflicting information about the presence and role of
ENaC in distal alveolar epithelia and its role in lung fluid clearance.
However, because the biochemical and molecular biological information
is based on isolated tissue, whereas electrophysiological data are from
cells in culture, it is possible that the process of isolation of ATII
cells and in vitro culture conditions alter the electrophysiological
characteristics of ion channels. It is well known that the phenotype of
ATII cells in tissue culture is unstable, and a recent study has shown
that airway epithelia can be maintained in a differentiated state for
longer periods in vitro by use of air-liquid interface cultures
(13). We therefore decided to test whether altering in
vitro culture conditions would affect Na+ channels in the
apical membranes of ATII cells. In particular, we used a
steroid-containing enriched medium, grew the cells on permeable
supports, and, for some of the cells, exposed the apical surface of the
cells to humidified air to mimic the conditions in the lung. Depending
on culture conditions, we observed two channel types. The first one was
a 21-pS NSC channel similar to the one reported by us (31,
32) and others (22, 37, 45, 60), which is seen most
consistently when cells are cultured in the absence of steroids and the
apical surface of cultured cells is exposed to a liquid interface. The
second type is a 6-pS HSC that is similar in properties to channels
associated with expression of ENaC subunits in other epithelia and is
best seen when the apical surface of cells cultured on permeable
supports in the presence of steroids is exposed to air interface.
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METHODS |
Type II pneumocyte isolation and cell culture.
ATII cells were isolated by enzymatic digestion of lung tissue from
adult Sprague-Dawley rats (200-250 g) by use of published techniques (12). Briefly, the rats were anesthetized with
pentobarbital sodium and were heparinized (100 units/kg). ATII cells
were digested by tracheal instillation of elastase (0.4 mg/ml). Lung
tissue was minced in DNase type IV from bovine pancreas (1 mg/ml;
Sigma, St. Louis, MO) and filtered sequentially through 100- and
20-µm nylon mesh. The purification was based on differential
adherence of cells to dishes coated with rat IgG. Nonadherent ATII
cells were collected, centrifuged, and seeded onto permeable supports in a highly enriched medium: 3 parts Coon's modification of Ham's F-12 and 7 parts Liebovitz's L-15 with 10 µM aldosterone or 1 µM
dexamethasone. The high concentration of steroid was used to ensure
that, even after 5 days in culture and significant cellular metabolism,
there would be sufficient steroid to activate both mineralocorticoid
and glucocorticoid receptors. (For experiments in which the specific
effects of steroids were investigated, lower concentrations of steroids
with appropriate antagonists were used.) After isolation, some cells
were allowed to grow on glass coverslips, and others were allowed to
attach to a specialized culture support (38) that was
optimized for patch-clamp recording and allowed the cells to grow on a
permeable support (Millipore) while submerged in medium. After the
cells had attached to the culture surface (this usually required
2-4 h), medium was drained from the apical surface, and cells were
allowed to grow with medium on the basolateral surface and air on the
apical side. Alternatively, cells were cultured in an identical fashion
but without the medium being drained so that cells would remain
submerged. Cells were incubated in 95% air-5% CO2 and
used for patch-clamp studies between 24 and 96 h after plating.
Using the current method (11, 12) for isolation of ATII
cells, we were able to obtain an ATII cell population with 95% viability as confirmed by LIVE/DEAD Eukolight Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). The LIVE/DEAD assay kit provides a
two-color fluorescence cell viability assay that is based on the
simultaneous determination of live and dead cells with two probes,
calcein AM and ethidium homodimer, that measure two recognized parameters of cell viability: intracellular esterase activity and
plasma membrane integrity. The assay principles are applicable to most
eukaryotic cell types, including adherent cells. This fluorescence-based method of assessing cell viability can be used in
place of trypan blue exclusion. The cells were 95% pure [confirmed by
cell surface staining for surfactant protein C (SP-C) and aquaporin 5]. Contamination by ATI cells, macrophages, or fibroblasts was <10%. Cells were used for patch-clamp experiments during the first 96 h in culture while they maintained ATII cell phenotype (see previous paragraph) and function (surfactant production by
radiolabeled choline incorporation). The purity and viability of ATII
cells were similar when cells were cultured on glass submerged in media and on permeable supports exposed to air interface.
ATII, M1, and L2 cells were used to assay the specificity of
antibodies. The M1 cell line [American Type Culture Collection (ATCC)] was established from normal renal tissue taken from a mouse
transgenic for the SV40 early region. The cells retain many characteristics of cortical collecting duct (CCD) cells, including morphology and CCD antigens. When grown on permeable supports, the
cells develop a lumen-negative transepithelial potential difference (21, 53). L2 cells (ATCC) are an adult rat lung cell line with many similarities to ATII cells (2, 15-18, 50)
and can be grown in large numbers as a source of rat lung ENaC subunits.
Determination of surfactant synthesis by isolated type II cells
in vitro.
Freshly isolated cells were distributed onto 24-well culture plates and
incubated with 0.75 mCi/ml of
[methyl-3H]choline chloride for 20 h.
After the adherence period, the wells were washed, and the cells were
allowed to equilibrate in fresh medium without radioactivity for 30 min
at 37°C. In some experiments, phorbol 12-myristate 13-acetate (10 µM) was added after the 30-min equilibration period. After a 3-h
incubation, the medium was removed and saved. The adherent cells were
extracted with 70% methanol. Phospholipids from both the media and the
cells were then extracted with chloroform-methanol (1:1 vol/vol), and
the radioactivity of the organic phase was determined. The percentage
of cellular phospholipids secreted was calculated as
[cpmmedium/(cpmcells + cpmmedium)] × l00%. The percentage of surfactant
secreted during the 30-min equilibration period was subtracted from all
samples. The magnitude of the fractional release may be underestimated if a significant amount of phospholipid is trapped in the permeable supports; however, the support material is strongly hydrophilic, so the
amount trapped should correspond to no more than the pore volume of the
support (~60% of the support volume or ~15 µl for a
25-mm-diameter, 5-µm-thick support; i.e., ~0.002 of the
medium volume). An example of such a determination is shown in Fig.
1.

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Fig. 1.
Surfactant production by alveolar type II (ATII) cells in
culture. The basal rate of sufactant secretion by ATII cells in primary
culture increased by 1%/h. Phorbol 12-myristate 13-acetate (PMA)
caused a twofold increase in the basal surfactant secretion. Data are
means ± SD for 4 experiments. The mean of each group is
significantly different from that of any other group (P < 0.05). These data are in agreement with previously published data on
ATII cells.
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Cell markers for ATII and ATI phenotypes.
Other investigators have examined the presence of cell surface markers
for ATII and ATI phenotypes in cultured alveolar cells and found that
~95% of the cells cultured as described in the previous section had
ATII markers and that there was little conversion to an ATI phenotype
in the first 96 h after culture. Nonetheless, we reexamined the
issue under our culture conditions. Cells were cultured on glass (with
fluid interface) or permeable supports (with air interface) and, in
both groups, with or without dexamethasone. Cell surface staining for
SP-C and aquaporin 5 was performed at 24 and 96 h. The number of
cells staining for aquaporin 5 and SP-C was used to calculate the ratio
of ATI to ATII cells (Table 1) (14,
57). We found that ~5% of the cells stained for aquaporin 5 after 24 h, and a slightly larger number (although not
statistically significant) were present after 96 h (the longest
time we examined cells in our experiments). The balance of the cells
(~95%) stained for SP-C (Fig. 2). The
percentage of cells staining with a marker for ATII cells (SP-C
antibodies) and ATI cells (aquaporin 5 antibodies) showed that, during
the time (24-96 h) in which we performed patch-clamp experiments,
ATII cells began with and maintained an ATII phenotype for the duration
of our patch-clamp experiments. Because there was no significant
difference in cellular phenotype, we combined all patch data from the
period between 24 and 96 h.

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Fig. 2.
Cells began with and maintained an ATII phenotype for the duration
of our patch-clamp experiments. The percentage of cells staining with a
marker for ATII cells [surfactant protein C (SP-C) antibodies] and
ATI cells [aquaporin (AQP) 5 antibodies] showed that, during the time
(24-96 h) in which we performed patch-clamp experiments, ATII
cells retained their gross phenotype. Cells were cultured on glass
(with fluid interface) or permeable supports (with air interface), and,
in both groups, with (A) or without (B)
dexamethasone. Staining for SP-C and AQP5 was performed at 24 and
96 h. The number of cells staining for AQP5 and SP-C was used to
calculate the ratio of ATI to ATII cells. There was a significant
difference between the SP-C-positive and the AQP5-positive cells under
each condition. There was no significant difference in %SP-C under any
condition and no significant difference in AQP5-positive cells under
any condition.
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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. Bath and
pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4 with 2 N
NaOH. In the inside-out recordings, pipette solution was the same but
the bath solution was changed to (in mM) 5 NaCl, 140 KCl, 4 CaCl2, 5 EGTA, 1 MgCl2, and 10 HEPES, pH 7.4 with 2 N KOH. The contents of the bath and pipette solutions were
varied as appropriate for specific protocols. All chemicals were
obtained from Sigma except oligonucleotides, which were generated at
Emory University's microchemical facility (EUMF).
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, Sarasota, FL) 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 pipette
solution. We used the cell-attached configuration for most of our
studies. After formation of a high-resistance seal (>50 G
) between
the pipette and 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 a low-pass Bessel filter. Data
were recorded by 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. The open probability
of the channels was calculated using FETCHAN in pCLAMP 6. Single-channel conductance was determined using a linear regression of
unitary current amplitudes over the range of applied pipette potentials.
Voltage conventions.
For cell-attached patches, voltages are given as the negative of the
patch pipette potential (
Vpipette). This potential is the
displacement of the patch potential from the resting potential (about
40 mV for ATII cells), and positive potentials represent depolarizations, and negative potentials represent hyperpolarizations of the cell membrane away from the resting potential. For a HSC with a
sodium concentration gradient of 10 to 1 (outside to inside), the
reversal potential would be +60 mV. Therefore, it would require a
100-mV positive voltage displacement (
Vpipette = +100 mV) from the resting potential to reach the reversal potential.
Antibodies for Western blotting.
We had previously characterized several ENaC subunit-specific peptide
antibodies in Xenopus (x) epithelial cells
(52). We have now demonstrated that the same antibodies
recognize ENaC subunits in mammalian epithelial cells. The anti-
antibody is an affinity-purified rabbit polyclonal antibody raised
against a peptide corresponding to residues 614 (Asp) through 647 (Asn) in the
-xENaC sequence, which was conjugated to keyhole
limpet hemocyanin (KLH). The anti-
antibody is an affinity-purified polyclonal antibody against a KLH-conjugated peptide corresponding to
residues 608 (Gly) through 660 (Leu) in the
-xENaC sequence. An
anti-
-xENaC antibody was generated to a 26-amino acid peptide corresponding to residues 137 through 161 of
-xENaC conjugated to
KLH. Antibodies from rabbit serum were affinity purified using a
peptide affinity column according to standard protocol (Pierce). The
peptides were produced in the EUMF and sent to commercial 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. These antibodies have been
characterized extensively in published reports (49, 51,
62). Western blots included in our previous publications
(32) show that these antibodies specifically recognize rENaC subunits. Figure 3 shows that the
antibodies recognize proteins of the appropriate size in a mouse
epithelial cell line (M1), a lung epithelial cell line (L2), and ATII
cells and that the activity is competed by the antigenic peptide
(although there was not enough of the
-peptide to compete on the
ATII cells because the
-peptide is a 50mer, which is extremely
expensive to make). The antibodies also recognize competing bands of
the appropriate size in the human lung epithelial cell line (A549, data
not shown).

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Fig. 3.
Subunit specific antibodies. Peptides were made that
corresponded to a unique antigenic sequence from each subunit. Each of
these antibodies was used to probe cell lysates from mouse epithelial
cells (M1), a lung epithelial cell line (L2), and ATII cells in the
presence (+) or absence ( ) of competing antigenic peptide. In each
case, the antibodies recognized one band of the appropriate molecular
weight (M.W.), and the band is competed by the antigenic peptide
(although there was not enough of the -peptide to compete on the
ATII cells because the -peptide is a 50mer, which is extremely
expensive to make). The antibodies also recognize competing bands of
appropriate size in the human lung epithelial cell line (A549, data not
shown). Details of antibody production are given in
METHODS.
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Preparation and use of antisense oligonucleotides.
We used previously described methods to inhibit expression of ENaC
subunits (32). Subunit-specific phosphorothioate
oligonucleotides directed against the sequences around the translation
start site of each subunit were produced by the EUMF according to the
sequences in Table 2. In a BLAST search
of GenBank, neither the sense nor the antisense oligonucleotides showed
any similarity to other known sequences, and the oligonucleotides for
each subunit had no significant similarity to the oligonucleotide
sequences for the other subunits. Cultured ATII cells were exposed to
medium containing 10 µM oligonucleotides overnight (16-18 h).
Our previous experiments have shown that freshly isolated ATII cells
take up oligonucleotides without the need for agents to enhance the
uptake. We have previously used Western blots (quantitative
densitometry) to determine that the antisense oligonucleotides do
reduce the subunit protein level and that the sense oligonucleotides
used as controls do not affect the protein levels (32);
however, in the current results, we provide additional evidence for the efficacy of the antisense oligonucleotides. Finally, patch-clamp techniques were used to determine the effect of oligonucleotides on the
expression of cation channels in apical membranes of ATII cells.
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Table 2.
Characteristics of the sense/antisense oligonucleotides used to inhibit
subunit protein production and the expression of NSC and HSC in
ATII cells
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Methods for statistical analysis.
Statistical analysis for the changes in open probability of channels
and the biochemical estimations were performed using SPSS for Windows.
Statistical significance between two groups was determined by paired or
unpaired t-tests, as appropriate. When the comparison
between more than one group was required, statistical significance was
determined by one-way analysis of variance (ANOVA) followed by pairwise
comparisons with the Student-Newman-Keuls test to determine significant
differences between groups. The significance of differences in the
observed frequency of channels between two groups of conditions was
determined using a z test for paired data or a
2 test for multiple groups. P values <0.05
were considered significant.
 |
RESULTS |
Culture support, growth medium, steroid treatment, and apical
interface influence the types of Na+
channels expressed in apical membranes of ATII cells in primary
culture.
To determine the effect of in vitro culture conditions on the
biophysical properties of Na+-permeant channels observed in
apical membranes of ATII cells, we cultured the cells under several
different conditions, including alternate media (standard vs.
enriched), type of culture surface (glass or plastic vs. permeable
support), and apical interface (liquid vs. air). Depending on the type
of culture condition, we observed two types of Na+
channels: a 21-pS NSC channel with Na+-to-K+
permeability (PNa/PK)
ratio ~1, and a HSC channel with single-channel characteristics
similar to those associated with the expression of ENaC subunits in
other tight epithelia. The frequency of observing these channels in
different culture conditions is shown in Table 3 and Fig.
4. There was a significant increase in
HSC channels (from 1 to 17%) when cells were cultured on permeable
supports instead of glass coverslips. Furthermore, when aldosterone or dexamethasone was added to the culture media, a high percentage of
patches had HSC channels whether the cells were grown on glass or on
permeable supports. Nonetheless, the highest expression of HSC channels
was observed when cells were cultured on permeable supports in the
presence of aldosterone with their apical surfaces exposed to an air
interface (HSC channels were recorded from 85.4% of 164 cell-attached
patches). Exposure to an air interface also led to a striking reduction
in NSC channels (69% in liquid interface vs. 20% in air interface).

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Fig. 4.
Effect of culture conditions on the relative
frequency of nonselective cation (NSC) and highly selective
Na+ (HSC) channels. We studied the effect of changes in
culture support, type of media, presence of steroids, and liquid vs.
air interface on the apical surface of cells. In all cases, cells were
incubated in 95% air-5% CO2 and used for patch-clamp
studies between 24 and 96 h after plating. To study the effect of
culture support, cells were seeded either on glass coverslips
(~2 × 105 cells/cm2) or a specialized
culture support optimized for patch-clamp recording, allowing the cells
to grow on a permeable support (Millipore) while submerged in medium.
To study the effect of culture media, culture medium was either DMEM
(F-12 medium) containing 5% fetal calf serum and antimicrobial agents
and supplemented with L-glutamine and Na+
bicarbonate or enriched medium (3 parts Coon's modification of Ham's
F-12 and 7 parts Liebovitz's L-15). To study the effect of apical
surface interface, some cells were grown on permeable supports, and
after they had attached to the culture surface (this usually required
2-4 h), medium was drained from the apical surface, and cells were
allowed to grow with medium on the basolateral surface and air on the
apical side. Alternatively, cells were cultured in an identical fashion
but without the medium being drained, so that cells remained submerged
in medium. To study the effect of steroids, aldosterone or
dexamethasone was added to the culture medium. +, Presence; ,
absence.
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Because addition of aldosterone caused an increase in HSC
channels, we further studied whether this effect was observable in cells grown with an air interface on permeable supports and whether
the effect was mediated through mineralocorticoid or glucocorticoid receptors. Cells were first cultured on permeable supports with an air
interface under three conditions: 1) in the presence of 300 nM aldosterone and 300 nM RU-486 (to block glucocorticoid receptors),
2) with 300 nM dexamethasone and 300 nM spironolactone (to
block mineralocorticoid receptors), and 3) with 300 nM
RU-486 and 300 nM spironolactone but with no steroids added. The
results of these experiments are shown in Table
4. Stimulation of either mineralocorticoid or glucocoticoid receptors resulted in statistically significant increases in the frequency of HSC channels and decreases in
NSC. However, when both types of steroid receptors were blocked, type
II cells still had relatively high frequencies of HSC compared with NSC
channels. Therefore, steroids appear to increase the frequency of
channels, but exposure to an air interface and growth on permeable
supports by itself is sufficient to promote expression of functional
HSC channels.
Single-channel characteristics of channels recorded from apical
membrane patches of ATII cells.
The distribution of single-channel conductance of the two types of
channels recorded from ATII cells can best be appreciated by comparing
the channels observed under the two most extreme differences in culture
conditions: cells grown on glass, under liquid, and with no steroids
vs. cells grown on permeable supports, with an air interface, and with
steroids. The results of this comparison are shown in Fig.
5. Details of the single-channel characteristics of the high-conductance NSC channels have been published by us previously (30, 31). In the current study, we describe the electrophysiological characteristics of the
low-conductance HSC channels that were best seen when cells were
cultured on permeable supports in the presence of enriched medium and
apical air interface. Figure 6 shows a
recording of a single channel in a cell-attached membrane patch along
with a representative current-voltage plot for this channel. At
pipette potentials more negative than +40 mV, the channel had a
conductance of 6.6 ± 3.4 pS (n = 94) with 140 mM
NaCl in bath and pipette. Ion selectivity was determined using excised
inside-out patches and solutions of varying ionic compositions. In
inside-out mode (bath 140 mM potassium gluconate; pipette 140 mM sodium
gluconate), patches exhibited nonlinear current voltage relationships,
with a reversal of current at approximately +115 mV. For this
reversal potential, calculation with the Goldman equation
(25) gives a permeability of the channel for
Na+ >80 times that of K+
(PNa/PK > 80). The channel is sensitive to submicromolar concentrations of
amiloride, with 37 nM amiloride producing a 50% decrease in channel
open probability (Fig. 7). The kinetics
of this channel were slow, with mean open and closed times in the
range of seconds. More than one current level was observed in
98% of active patches. Thus this channel is very similar in its
single-channel characteristics to the ENaC observed in other
Na+-transporting epithelia and to the one described from
fetal lung epithelia by Voilley et al. (58).

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Fig. 5.
Distribution of single-channel conductances of observed
channels cultured under significantly different culture conditions.
When cells were grown on glass coverslips submerged in media devoid of
steroids, the predominant channels seen are NSC channels with high
conductance (filled bars), but when grown on permeable filters with
high concentration of aldosterone (10 µM) and air interface, the
predominant channels are HSC channels with low conductance (open bars).
Data are combined from all cells recorded between 24 and 96 h
after culture. Mean conductances from the two groups are significantly
different (P < 0.001).
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Fig. 6.
Single-channel characteristics of HSC channels.
A: single-channel recordings from a cell-attached patch on
the apical membrane of ATII cell cultured with air interface.
B: current-voltage (I-V ) relationship showing
that conductance of the channel is ~6 pS. Vp, pipette
potential. C: amplitude histogram of the patch shown in
A. There was no difference in channel characteristics for
cells between 24 and 96 h after initial culture.
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Fig. 7.
Dose-response curve for amiloride. Application of 37 nM
amiloride to the extracellular side (i.e., in the micropipette) caused
a 50% reduction in the open probability of the HSC channels. Data were
combined from all cells recorded between 24 and 96 h after
culture.
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Effect of oxygen tension on channel expression.
In our experiments evaluating the effect of apical surface
interface on expression and activity of ion channels, we found that the greatest increase in highly selective cation channels was seen
when cells were cultured with their apical surface exposed to air
interface. Because this effect could be mediated by increased availability of oxygen at the cell surface, we cultured cells overnight
with an air interface on permeable supports with steroids (like group 7 in Table 3) but with only 5% oxygen. Under
these conditions, the predominant channels seen in apical patches were nonselective (Fig. 8). However, when
cells cultured in 5% oxygen for 24 h were exposed to 95% oxygen
for 2 h, apical membrane patches returned to their previous ratio
of predominantly HSC channels (Fig. 8).

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Fig. 8.
Effect of reduced O2 tension on the frequency
of HSC and NSC channels in cells grown on permeable supports with an
air interface, and the effect of O2 on the incidence of NSC
and HSC channels in cells grown on permeable supports in the presence
of steroids. Exposure to 5% O2 increased the frequency of
NSC channels and decreased the frequency of HSC channels. In high
O2, HSC comprised almost 100% of all observable channels.
Data were combined from all cells recorded between 24 and 96 h
after culture.
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Relationship of the observed channels to the cloned ENaC.
We have previously shown (32) that NSC channels recorded
from ATII cells cultured with fluid interface belong to the ENaC family
of Na+ channels and are formed from
-subunit protein
alone or in combination with some subunit other than
and
. This
conclusion was based on our studies in which we used antisense
oligonucleotides directed against the sequences around the start codon
of each subunit and used patch-clamp techniques to evaluate
single-channel activity in ATII cells treated with oligonucleotides. We
now report results from similar experiments with both the NSC and HSC
channels observed when cells are cultured under different conditions.
Testing the efficacy of the antisense oligonucleotides required a large
amount of ENaC protein; therefore, we used a rat alveolar cell line
(L2) with phenotypic characteristics similar to those of native ATII cells (23, 27), including expression of rat ENaC subunits (Fig. 3). Figure 9 shows that, in ATII
cells treated with antisense oligonucleotides (vs. cells treated with
sense oligonucleotides), the expression of each subunit was
dramatically reduced (although not to undetectable levels). The ATII
cells were cultured with an air interface on permeable supports with
aldosterone and were exposed to oligonucleotides for 16-18 h.
Exposure to antisense oligonucleotides against any of the three
subunits of ENaC (
,
, and
) led to a significant reduction in
apical membrane patches with HSC channels (Table
5) compared with those treated with sense
oligonucleotides (Fig. 10). These
frequencies are significantly different (P < 0.01),
indicating that inhibition of
-,
-, or
-subunits inhibits the
expression of HSC channels. Treatment with oligonucleotides did not
cause any significant changes in open probability or conductance of HSC
channels (Table 5).

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Fig. 9.
Effect of antisense oligonucleotides on relative amounts
of subunit proteins in alveolus-derived cells in continuous culture. L2
cells grown on permeable supports with dexamethasone were exposed to
either sense or antisense oligonucleotides specific for the translation
start site of each subunit (see METHODS for details of
oligonucleotide composition and preparation). After 24 h, cells
were lysed, and equal amounts of lysate protein were run on a gel. Gels
were probed with subunit-specific antibody. Long exposure after
enhanced chemiluminescence shows that antisense treatment does not
reduce subunit concentration to zero, but for each subunit the
reduction is substantial.
|
|
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|
Table 5.
Effect of ENaC sense and antisense oligonucleotide treatment on
frequency of HSC and NSC channels in apical membrane patches
|
|

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Fig. 10.
Effect of epithelial Na+ channel (ENaC)
antisense and sense oligonucleotide treatment of ATII cells. Antisense
oligonucleotides directed against any of the 3 ENaC subunits ( , ,
and ) resulted in a statistically significant reduction in the
number of apical cell-attached patches having 1 open channels vs.
those treated with sense oligonucleotide for the corresponding subunit
(control). Data were combined from all cells recorded between 24 and
96 h after culture. * P < 0.01.
|
|
 |
DISCUSSION |
We found that when in vitro culture conditions simulated
conditions expected in vivo, the apical membranes of ATII cells express HSC channels with electrophysiological properties similar to
channels expected from coexpression of ENaC subunits (conductance
6.6 pS, sensitivity to amiloride with K0.5 = 37 nM, PNa/PK > 80, slow kinetics). In particular, the presence of steroid hormones,
permeable supports, and an air interface was essential for high-level
expression of HSC channels. Our experiments with the use of antisense
oligonucleotides to inhibit individual subunits of ENaC show that these
channels are formed by at least all three subunits of ENaC (
,
,
and
). Taken together, these studies provide strong evidence for the presence in ATII cells in vitro of HSC channels similar to those observed when all three ENaC subunits are expressed in heterologous expression systems.
The presence of amiloride-sensitive Na+ transport in
alveolar epithelia has been demonstrated by numerous studies using
macroscopic measures of Na+ movement (26, 39, 40,
43). Studies have also demonstrated the functional importance of
epithelial Na+ absorption in clearance of alveolar fluid in
the developing and mature lungs (28). There is
molecular and functional evidence that ENaC is present in the
lung epithelial cells and that absence of
-ENaC results in failure
of absorption of fetal lung fluid. The perinatal period is
characterized by a rapid increase in the capacity for
amiloride-sensitive Na+ transport in the lung
(44), and there is a concomitant increase in expression of
ENaC genes (54, 56). However, except for one study by
Voilley et al. (58) in fetal lung cells (in which the
frequency of HSC channels was not given and in which the presence of
NSC channels was not reported), patch-clamp studies have failed to
identify HSC channels expected from ENaC expression in apical membrane
patches from type II cells. Instead, the predominant Na+-permeant channels recorded from type II cells of both
fetal and adult origin are mostly NSC channels that vary considerably
in their single-channel characteristics from HSC channels
(41). The physiological role of the NSC channels has been
questioned because of the reported high intracellular Ca2+
concentration required to activate this channel (37).
There is recent evidence that maturation and assembly of the ENaC
subunits occur slowly and that the lifetime and number of assembled
ENaC channels in the plasma membrane are substantially different from the lifetime of ENaC subunits in the preapical or cytosolic pool of
proteins. Therefore, detection of increased ENaC gene expression as
increased mRNA or as increased total cellular ENaC subunit protein may
not correlate with the expression of functional ENaC in the plasma
membrane (55).
Our studies show that, depending on culture conditions, two different
types of amiloride-sensitive Na+-permeant channels can be
observed in apical membrane patches from both fetal and adult type II
cells. The first channel is a NSC channel, observed most frequently
when ATII cells are cultured in the absence of steroids on glass and
submerged in culture medium. This channel has a unitary conductance of
21 pS and a Na+-to-K+ selectivity ratio close
to 1. The second channel is a HSC channel, observed most frequently
when cells are cultured on permeable supports with an air interface on
their apical surface. This channel has low conductance (6.6 pS) and is
highly selective for Na+ over K+.
The effect of culture conditions.
As aforementioned, culture conditions are the primary determinant of
the frequency with which we observe the two channel types; however, the
culture conditions appear to have somewhat different effects on HSC and
NSC channels. An examination of Fig. 4 and Table 3 shows that the
frequency of HSC channels is equally sensitive to steroids (regardless
of other culture conditions) and air interface (regardless of the
presence of steroids). In fact, the frequency of HSC channels in cells
grown under three conditions (group 2: with steroids,
on glass, under liquid; group 6: without steroids, on
permeable supports, with air interface; or group 7: with
steroids, with air interface, on permeable supports) are not
statistically different from one another (for the number of patches we
examined) but are significantly different from all other conditions
(P < 0.05). On the other hand, the highest frequency
of NSC channels was observed when cells were grown on glass
(statistically different from all other conditions, P < 0.05). Addition of any combination of permeable supports, steroids,
or air interface significantly decreases the frequency of NSC channels,
with the lowest frequency occurring when all three conditions are
present (group 7). Only change of the medium type
appears to have no significant effect on the frequency of either NSC or
HSC channels.
Molecular basis for HSC and NSC channels.
We have shown that despite the large differences in biophysical
properties of HSC and NSC channels, both are related to the ENaC family
of channels. The NSC channel is formed by the
-subunit (alone or in
combination with subunits other than
and
), whereas the HSC
ENaC-like channel is formed by at least
-,
-, and
-subunits.
Several other investigators have suggested that different combinations
of various subunits of ENaC can form Na+ channels with
different biophysical characteristics. Fyfe and Canessa
(24) have recently shown that subunit composition of ENaC
is a main determinant of channel kinetics. Canessa et al. (8), Ismailov et al. (29), and Kizer et al.
(35) have all shown in whole cell measurements that
-subunit alone can apparently form amiloride-sensitive cation
channels, although in oocytes, currents produced by these channels are
small (only ~1% of
-,
-, and
-channels). Although addition
of
- and
-subunits alters channel properties, these subunits are,
by themselves, unable to form functional channels either alone or in
combination. Canessa et al. (8) showed that coexpression
of
- and
- or
- and
- but not
- and
-subunits induces
whole cell currents but that these reach only 10-20% of the
magnitude obtained when
,
, and
are expressed together. The
pivotal role for
-subunit is further confirmed by the studies
conducted by Hummler et al. (28), in which they showed
that genetically engineered mice that lacked
-subunit were unable to
clear fetal lung fluid and died in the early perinatal period. In
contrast, newborn mice in which the
- or
-subunit had been
deleted were able to clear fetal lung fluid, albeit more slowly, and
died from hyperkalemia instead (5). These results suggest
that, in the absence of
- or
-subunits, channels from
-subunit
alone or from
- and
- or
-subunits can reabsorb sodium and
water from lungs at birth, although this process is less efficient. Our
inhibition of ENaC subunit expression supports the idea that NSC
channels can be formed from
-subunits alone, whereas HSC channels
require all three subunits. This effect can most easily be seen in
antisense-treated cells grown under conditions (permeable supports, air
interface, and added steroid) when the predominant channel is HSC
(>70%). In Fig. 11, data from Table 5
are replotted as the change in both types of channels and the number of
patches with no activity relative to the sense-treated cells (i.e., a
value of zero would mean no difference between sense- and
antisense-treated cells,
4 would indicate a 4-fold decrease produced
by antisense, and +4 would indicate a 4-fold increase). As
expected, the number of HSC channels decreases after treatment with any
antisense oligonucleotide. However, treatment with
-antisense
reduces the frequency of all channel types, leading to a large increase
in the percentage of patches in which no channel can be observed. In
contrast, treatment with
- or
-antisense actually increases the
number of NSC channels, with an additional moderate increase in patches
with no activity. This is exactly the pattern that is expected if
-subunits are required to form any channel, but
and
can
combine with
to form HSC channels, and
alone can form NSC
channels.

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Fig. 11.
Effect of antisense oligonucleotides on relative numbers of HSC
and NSC channels after treatment with antisense oligonucleotides to
different ENaC subunits. Treatment with -antisense (A)
significantly reduced the frequency of all types of channels
(P < 0.05) but greatly increased the frequency of
observing patches with no channel activity (P < 0.05).
Treatment with - (B) or -antisense (C) both
significantly reduced (P < 0.05) the frequency of
observing HSC channels but actually increased the frequency of
observing NSC channels (P < 0.05). Both also increased
the frequency of observing patches with no activity (P < 0.05). These results were most consistent with a model in which
-subunits were required for both channel types but in which and
were absolutely required only for HSC channels. Cells were grown on
permeable supports with an air interface, steroid hormone, and enriched
medium. Data were combined from all cells recorded between 24 and
96 h after culture and are expressed as relative degree of
change ± SE.
|
|
Role of steroids and alveolar air in promoting lung fluid
reabsorption.
What role does the alveolar environment play in the composition and
function of lung epithelial Na+ channels, and what
physiological changes trigger the change in fetal lung epithelia from
secretory to reabsorptive mode in the perinatal period? Although
several agents have been postulated to play a role in fetal pulmonary
transition (including steroids, catecholamines, vasopressin, prolactin,
and lung inflation), none explains this switch completely. Studies
evaluating the role of these agents in other tissues need to be viewed
with caution because regulation of Na+ channels by these
agents may occur in a tissue-specific manner (3). Our
culture technique employed high concentrations of steroids in the
culture medium. There is considerable evidence that steroids increase
ENaC expression in several tight epithelia (5, 6, 9, 10, 29,
39). Glucocorticoids are believed to have a greater effect on
the lungs than mineralocorticoids and have been shown to increase
-ENaC mRNA in rat and human fetal alveolar epithelial cells
(9, 54) and
- and
-ENaC mRNA in A549 cells
(36). In the case of A549 cells, Lazrak et al. (36) showed that addition of dexamethasone reduced
the conductance of the most frequently observed channel from ~21 to 6 pS and increased the selectivity of the channel from essentially
nonselective to highly selective. Thus their work essentially parallels
our observations. The interesting difference is that whereas they could
detect little if any
- and
-ENaC protein before
addition of dexamethasone, we saw significant amounts of total cellular
levels of protein for all three subunits, even when cells were grown
under conditions when the most common channels were NSC channels,
presumably consisting of only
-subunits. Our interpretation of these
results is that steroids may be altering the production of subunits,
but their primary function is to promote proper assembly and delivery
of HSC channels to the membrane. In fact, in preliminary work, we have
shown that inhibitors of protein trafficking can inhibit the appearance
of HSC channels in the membrane (e.g., brefeldin A blocks the increase
in HSC channels after increases in oxygen tension). Although
aldosterone can also stimulate ENaC expression, this effect may be
mediated via glucocorticoid receptors (9). Indeed, our
studies show that aldosterone and dexamethasone are both effective in
increasing the expression of HSC channels.
However, our study shows that cells grown on permeable supports in the
presence of steroids have maximum expression of HSC channels when
exposed to air interface. There are several possible ways in which the
presence of air interface may bring about this change. These include
changes in physical forces affecting the apical membrane of the cells
or an increase in the amount of available oxygen. Awayda and
Subramanyam (1) and Ji et al. (33) have addressed the issue of mechanosensitivity of ENaC, but it is not clear
whether ENaC function is altered in vivo by mechanical perturbations. As for the role of oxygen, several recent studies have shown that exposure of rat alveolar cells to high oxygen concentration results in
upregulation of Na+ channel activity (4).
Pitkanen et al. (47) have shown that oxygen exposure
alters the bioelectric properties of fetal distal lung epithelium.
There is also evidence to show that hypoxia suppresses Na+
channel expression and activity (48). Thus oxygen may be
playing a significant role in fetal lung transition at birth. Other
investigators have shown that exposure of ATII cells to air is
important for ATII cell function and may promote Na+
transport. Dobbs et al. (14) found that ATII cells
cultured on collagen gels with culture medium containing fetal calf
serum and with an apical surface exposed to air retain morphological characteristics of ATII cells for a longer duration. These cells express surfactant proteins and their mRNAs and express a plasma membrane marker specific for ATII cells. Yamaya et al.
(59) showed that expression of differentiated
characteristics by these cells was inversely correlated to the depth of
liquid overlying the apical surface of the cells. Johnson et al.
(34) proposed that apical air interface promotes aerobic
metabolism in ATII cells and decreases lactate levels and found
increased Na+ transport in these cells. These studies have
yet to be undertaken with fetal cells. It is possible that fetal
alveoli, with a considerable amount of fetal lung fluid bathing the
apical surface of alveolar epithelial cells, have low HSC channel
activity but high chloride secretion into the alveoli. However, at
birth, distension of alveoli with air facilitates an increase in
Na+ transport through activation and/or increased
expression of HSC channels. This exposure to air, coupled with a rise
in endogenous catecholamines and steroids, which are also known to
increase epithelial Na+ transport and HSC channel activity,
would be expected to increase Na+ and fluid absorption.
Little is known about other factors that influence this change at
birth, and the speculations offered here are, at best, a small fraction
of what is likely to be a very complex process.
In summary, our study suggests that under specific culture conditions,
type II cells express highly selective sodium channels and that
presence of
-,
-, and
-ENaC is necessary for the expression of
these channels. Previously, there had been no electrophysiological evidence that ENaC-like HSC channels were present in ATII cells, raising questions about whether such channels could contribute to
alveolar sodium transport. We have now shown that, at least under
appropriate conditions, ENaC-like channels are the predominant channels
in in vitro ATII cells. This observation must at least raise the
possibility that ENaC-like channels could be present in vivo.
 |
ACKNOWLEDGEMENTS |
We thank B. J. Duke and Frank L. Harris for excellent
technical support.
 |
FOOTNOTES |
This research was supported by American Heart Association Grant
9950875-V, American Lung Association Grant 6-38665, Children's Research Center at Emory University Grant 2-56036 to L. Jain, National
Institutes of Health Grants R01-DK-37963 and P05-DK-50268 to D. C. Eaton, and the Center for Cell and Molecular Signaling.
Address for reprint requests and other correspondence: L. Jain,
Dept. of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Dr., Atlanta, GA 30322 (E-mail:
ljain{at}emory.edu).
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. Section 1734 solely to indicate this fact.
Received 27 July 2000; accepted in final form 9 November 2000.
 |
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