Modification of biophysical properties of lung epithelial
Na+ channels by dexamethasone
A.
Lazrak1,*,
A.
Samanta1,*,
K.
Venetsanou3,
P.
Barbry4, and
S.
Matalon1,2
Departments of 1 Anesthesiology and 2 Physiology and
Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
35233; 3 Intensive Care Unit, Athens Trauma Center, Kephesia,
Greece; and 4 Institut de Pharmacologie Moléculaire et
Cellulaire, Centre National de la Recherche Scientifique UPR411, 06560 Sophia Antipolis, France
 |
ABSTRACT |
There is
considerable interest in identifying the basic mechanisms by which
dexamethasone alters ion transport across the adult alveolar
epithelium. Herein, we incubated synchronized A549 cells, a human
alveolar epithelial cell line, with dexamethasone (1 µM) for
24-48 h. When normalized to HPRT (a housekeeping gene), A549
- and
-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR,
increased by 1.6- and 17-fold respectively, compared with control
values (P < 0.05). These changes were abolished by
actinomycin D, indicating transcriptional regulation. Western blotting
studies revealed that dexamethasone also increased expression of
-
and
-hENaC protein levels. In contrast,
-hENaC mRNA increased by
onefold (P > 0.05) and
-hENaC protein level was
unchanged. Incubation of A549 cells with dexamethasone increased their
whole cell amiloride-sensitive sodium currents twofold and decreased
the K0.5 for amiloride from 833 ± 69 to
22 ± 5.4 nM (mean ± SE; P < 0.01). Single
channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 ± 0.036 to
4.4 ± 0.027 pS (mean ± SE; P < 0.01). We
concluded that dexamethasone modulates the amiloride-sensitive
Na+ channels by differentially regulating the expression of
- and
-subunits at the mRNA and protein levels in the human A549
cell line, with little effect on
-hENaC subunit.
ENaC; lung; whole cell currents; single channels; A549 cells; amiloride; reverse transcription-polymerase chain reaction; Western
blots
 |
INTRODUCTION |
THE
AMILORIDE-SENSITIVE epithelial Na+ channels
(ENaC) are the main pathways for Na+ ion entry in a large
number of epithelial cells, including those in lung airways and
alveolar epithelium, the distal nephron, colon, and kidneys
(1, 13, 23, 31).
The channel, which belongs to a large family of ion channels, is a
heteromultimeric complex of at least three distinct but homologous
subunits, named
-,
-, and
-ENaC, which were first cloned from
the colon of salt-deprived rats and human lung tissue (3,
4, 20, 40). Experiments utilizing point mutations suggested that all three subunits were involved in the channel's pore formation (33). The
subunits are characterized by two short NH2 and COOH
termini, two short membrane-spanning segments, and a very large
extracellular loop domain (29). The expression of the
three subunits is necessary for maximal functional activity
(15). The exact stoichiometry is still debated, with
different groups reporting either four subunits (two
, one
, and
one
) (11) or nine subunits (three of each) in the
complex (35).
There is convincing evidence for the existence of active
Na+ transport across the lung alveolar epithelial type II
(ATII) cells. This active transport plays an important role both in the
regulation of the alveolar hypophase liquid volume and in the
reabsorption of edema fluid in pathological conditions
(25, 41). Expression of rat
-ENaC
(
-rENaC) mRNA in adult rat ATII cells was demonstrated by
Northern blot analysis (42), PCR (10), and by
in situ hybridization (10, 24,
42). The
- and
-mRNAs for rENaC were also detected in large and small airways, but they were less abundant in ATII cells
compared with
-rENaC (10).
In cultured lung and airway epithelia, glucocorticoid and
mineralocorticoid hormones have been shown to upregulate the
electrogenic Na+ absorption (6,
32). Recently, Sayegh et al. (32)
demonstrated that in a lung airway cell line (H441), dexamethasone
upregulated electrogenic Na+ transport and
-subunit
human ENaC (
-hENaC) expression via activation of a hormone
response element in the 5'-flanking regions of
-hENaC. This was
consistent with the previous report by Champigny et al. (6) that dexamethasone increases the functional expression of the amiloride-sensitive Na+ current by enhancing
transcription of all three subunits in fetal rat lung epithelial cells.
However, there are no studies correlating dexamethasone-induced changes
in ENaC proteins with changes in functional properties of
amiloride-sensitive Na+ channels in lung epithelial cells.
Herein we demonstrate that treatment of synchronized A549 cells, a cell
line derived from human lung epithelial carcinoma cells with many of
the characteristic functions of ATII cells (19), with
dexamethasone resulted in a large upregulation of the
-hENaC mRNA,
together with a modest upregulation of
-hENaC mRNA. In addition,
both
- and
-hENaC protein levels were increased. Interestingly,
-hENaC mRNA and protein levels were not significantly different from
control. Biophysical studies at the single channel level revealed that
the treatment of synchronized A549 cells with dexamethasone increased
the open probability (Po) and mean open time of
their amiloride-sensitive Na+ channel and decreased
its unitary conductance. Also, dexamethasone-treated A549 cells had
larger amiloride-sensitive whole cell currents that were more sensitive
to amiloride than the corresponding currents in control cells. Our data
provide evidence for the upregulation of the amiloride-sensitive
currents in an epithelial cell line in the absence of significant
changes in
-hENaC.
 |
MATERIALS AND METHODS |
Cell culture.
A549 cells were purchased from American Type Culture Collection
(Rockville, MD) in the 76th passage. They were cultured using a 50/50
mixture of DMEM and F-12 (DMEM-F12, Cellgro), supplemented with 1%
penicillin-streptomycin and 10% fetal calf serum, plated on plastic
tissue culture flasks (Corning Glass Works, Corning, NY), and incubated
in a humidified atmosphere of 5% CO2-95% air at 37°C.
All measurements were conducted on cells synchronized by serum
deprivation, prepared as follows. One million A549 cells were plated on
100 × 20-mm Falcon Primaria tissue culture dishes (Becton-Dickinson, Lincoln Park, NJ) for 24 h, using the
above-mentioned medium. They were then washed with 10 ml of PBS and
synchronized by incubation at 37°C in DMEM-F12 containing 0.2%
charcoal-stripped calf serum (Hyclone) for 48 h. At that time,
they were washed and incubated with fresh serum-free medium
supplemented with 0, 10, 100, or 1,000 nM dexamethasone (Sigma, MO) for
24 or 48 h.
RNA isolation and RT-PCR.
A549 cells were washed with cold PBS, and RNA was isolated according to
the protocol supplied by the manufacturer (MRC, Cincinnati, OH) using
the modified method of Chomcyznski and Sacchi (7). One
microgram of total RNA in a total volume of 12 µl was denatured at
70°C for 10 min. Denatured RNA was chilled for 2 min in ice and used
for reverse transcription as described by the standard protocol (GIBCO
BRL; Life Technologies, Bethesda, MD). In brief, 1 µl
Superscript II (GIBCO BRL), 20 U RNasin, 1 µl random hexamer, 2 µl
100 mM dithiothreitol, and 1 µl of 10 mM each of four dNTPs mixed in a total volume of 20 µl were added to the sample, mixed well, and incubated for 1 h at 42°C. The reaction mixture was heated to 70°C for 15 min to inactivate reverse transcriptase. Two
microliters of the reaction mixture was used for PCR
amplification in a Robocycler (Stratagene) using 1 µl (20 pmol) each
of upstream and downstream primers specific for the
-,
-, and
-subunits of human ENaC (hENaC) genes and the housekeeping gene HPRT
(18). The HPRT gene was used
to ensure the RNA integrity and concentration (14,
22, 28).
Each primer was added into a 50-µl mixture containing 1 µl of 10 mM
4 dNTP mix, 5 µl of 10× PCR buffer (containing 200 mM Tris · HCl, pH 8.4, and 500 mM KCl), 2 mM MgCl2 and 2.5 U of
Taq polymerase. The cycle parameters were initial
denaturation at 95°C for 5 min, 62°C for 1 min for annealing,
72°C for 1 min for extension, 95°C for 1 min for denaturation,
number of cycles used 35 (which was within the linear range of
amplification), and final extension for 7 min at 72°C. Twenty
microliters of the final amplified product was electrophoresed in 1.2%
agarose gel, and the DNA was visualized after ethidium bromide staining.
In some experiments, synchronized cells were incubated with 1 µM
actinomycin D (100 mM stock in DMSO; GIBCO BRL) for 24 h. The
cells were then washed, and the medium was replaced with serum-free medium containing dexamethasone and actinomycin D for an additional 24 h and then processed for RNA extraction.
Western blotting studies.
Levels of
-,
-, and
-hENaC in synchronized A549 cells prior to
and following dexamethasone treatment were assessed by Western blotting
studies. In brief, A549 cells were washed with cold PBS, incubated with
1 ml of 10 mM Tris-Cl (pH 7.4) containing 250 mM sucrose, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin,
pepstatin, leupeptin, and trypsin inhibitor (buffer A; all
chemicals from Sigma), and scraped using a cell scraper. The resulting
suspension was sonicated for 10 s in ice and allowed to cool for
30 s; this procedure was repeated five times. More than 95% of
the cells were broken as observed with light microscopy.
Sonicated cells were centrifuged at 2,000 g for 10 min at
4°C to remove nuclei, unbroken cells, and cell debris. The
supernatant was collected and centrifuged at 45,000 g for
1 h. The membrane pellet was lysed in buffer A,
containing 1% Nonidet P-40 (Sigma) and 150 mM NaCl and the
above-mentioned protease inhibitors, for 1 h at 4°C with
rotation. The suspension was centrifuged at 45,000 g for 30 min. The supernatant was then carefully removed, and its protein
concentration was measured by the bicinchoninic acid (BCA) method
(Pierce) using BSA as standard. Fifty micrograms of the membrane lysate
was denatured in SDS-sample lysis buffer (2% SDS, 62.5 mM
Tris-Cl, pH 6.8, 5%
-mercaptoethanol, and 0.1% bromophenol blue)
by boiling for 5 min. Samples were separated in 7% SDS-PAGE and were
transferred to Sequi-Blot polyvinyldidene difluoride (PVDF) membranes
(Bio-Rad). After blocking with 2% BSA, the membranes were probed with
an anti-
-rENaC antibody, generated by using a synthetic peptide
corresponding to residues 44-57 within the
NH2-terminal intracellular domain of
-rENaC as
previously described (34, 43), and then with
anti-rabbit-conjugated horseradish peroxidase (HRP) and
developed by enhanced chemiluminescence (ECL) using the kit provided by
Pierce. In control experiments, membranes were probed with the
anti-
-rENaC antibody and the synthetic peptide, followed by the
secondary antibody. The anti-
-rENaC antibody and the immunizing
peptide were kindly provided to us by Dr. Peter R. Smith of the
Department of Physiology and Biophysics, University of Alabama at Birmingham.
To assess levels of
- and
-hENaC, A549 proteins were separated in
7% SDS-PAGE as outlined above and transferred to Sequi-Blot PVDF
membrane (Bio-Rad) in the absence of reducing agents. After blocking,
the membranes were probed with polyclonal antisera against
-rENaC or
-rENaC, followed by the secondary antibody (anti-rabbit-conjugated HRP) and developed by ECL using the kit from Pierce. The polyclonal antisera were raised as previously described and have been
characterized extensively (12, 16,
30). In control studies, membranes were incubated with
preimmune sera. All other procedures were as described above.
Patch clamp recordings: whole cell measurements.
For electrophysiological measurements, cells were seeded on glass cover
slips, synchronized by incubation in serum-free medium, and exposed to
dexamethasone as described above. Just before the start of experiments,
coverslips were rinsed with standard external solution (SES) containing
(in mM) 135 sodium methanesulfonate, 10 NaCl, 2.7 KCl, 1.8 CaCl2, 2 MgCl2, 5.5 glucose, and 10 HEPES, pH
7.4. They then were transferred to the recording chamber mounted on the
stage of an inverted microscope (model IMT-2, Olympus) for patch clamp
recordings. The recording chamber was continuously perfused with SES at
a rate of 0.5 ml/s using a gravity-driven perfusion system.
The patch pipettes were made from Kimax50 type capillary glass tubing
(Kimax) using a vertical puller (model PB-7; Narishige). They were
filled with the standard internal solution (SIS; pH 7.2 at 22°C, 300 mosmol) containing (in mM) 120 potassium methylsulfonic acid, 15 KCl, 6 NaCl, 1 dimagnesium ATP, 2 trisodium ATP, 10 HEPES, and 0.5 EGTA. Only
pipettes with a resistance in the 3-5 M
range, when filled with
SIS, were used for whole cell recordings. The pipette offset potential
was corrected just prior to the gigaseal formation, and series
resistance and capacitance transients were compensated using the patch
clamp amplifier (Axopatch 200A; Axon).
The cell membrane potential was held at
40 mV during all recordings,
unless stated otherwise. Inward and outward currents were elicited by
voltage protocols (from
100 to + 100 mV in 10 mV steps for 450 ms), using the Clampex program (PCLAMP, Axon). The whole cell currents
were digitized using a digital-to-analog and analog-to-digital
converter (Digidata 1200A; Axon), sampled at 2-5 kHz, and filtered
through an internal four-pole Bessel filter at 1 kHz. Current-voltage
relationships were constructed from steady-state currents measured at
300 ms from the start of voltage pulses using the Clampfit program
(Axon Instruments) and Origin software (Microcal Software, Northampton, MA).
To test the extent to which whole cell currents were inhibited by
amiloride, we measured current-voltage relationships before and during
perfusion of cells with SES containing amiloride (Calbiochem, La Jolla,
CA), in concentrations ranging from 1 nM to 100 µM. The
amiloride-sensitive currents were calculated by digitally subtracting
the currents in the presence of amiloride from controls.
Single channel recording.
Single channels were recorded from serum-deprived,
dexamethasone-treated A549 cells patched in cell-attached mode, using
pipettes with a resistance in the 5-10 M
range. The pipettes
were coated with a thin layer of Sylgard and filled with a solution
containing (in mM) 145 sodium methanesulfonate, 5 MgCl2,
5.5 glucose, 40 mannitol, and 10 HEPES, pH 7.4. The Sylgard coating was
necessary to reduce the noise and enhance the recording resolution.
Prior to seal formation, cells were depolarized to 0 mV by bathing them in the following solution (mM): 145 potassium methanesulfonate, 5 MgCl2, 40 mannitol, 10 HEPES, and 5.5 glucose, pH 7.4. Single channel activity was sampled at 2-5 kHz and filtered at 1 kHz. The data were analyzed using the Fetchan and pStat programs
(PCLAMP, Axon Instruments). Single channel conductance was calculated
from all event histograms, constructed as previously described
(17).
Statistics.
Results were expressed as means ± 1 SE. The Student
t-test was used for statistical analysis between two group
means. Statistical differences among multiple groups with equal
variance were determined using the one-way analysis of variance
(ANOVA). If the equal variance test failed, then data were analyzed
using ANOVA on ranks. If the H value was statistically significant
(P < 0.05), we used the Dunnett method to test the
significance of each mean value against the control value.
 |
RESULTS |
RT-PCR.
Results shown in Fig. 1A
indicate the presence of
- and
-hENaC mRNAs in control A549
cells. A very small amount of
-hENaC mRNA was detected in one of the
three preparations. Exposure of A549 cells to dexamethasone
(0.01-1 µM) induced dose-dependent increases in
- and
-hENaC mRNA expression. For each experiment, values were normalized
to the corresponding HPRT mRNA. Mean values (±1 SE) are shown in Fig.
1B. Exposure of A549 cells to 1 µM dexamethasone increased
the relative expressions of
- and
-hENaC mRNA by 1.6- and 17-fold
from control values, respectively (P < 0.05).
-hENaC mRNA increased by onefold, but this change was not
statistically significant. The dexamethasone-induced increases in
-
and
-hENaC mRNA expression were due to increased transcription,
since they were not seen in the presence of actinomycin D (Fig.
2).

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Fig. 1.
A: effect of dexamethasone (Dex) concentration on the
expression of hENaC subunit messages in synchronized A549 cells. One
microgram of total RNA from synchronized A549 cells treated with the
indicated concentrations of dexamethasone was used for reverse
transcription (RT). For amplification conditions a high stringency of
primer annealing (62°C) was used. The amount of starting RNA for RT
and the number of amplification cycles used were within the linear
range of amplification. Results of a typical experiment reproduced at
least three times. B: increase in hENaC subunits amplified
DNA levels following dexamethasone treatment of synchronized A549
cells. The dexamethasone concentrations (nM) used are shown as the
bottom axis values. The amplified hENaC bands shown in A
were scanned by a densitometer. The obtained values for each band were
normalized to the readings for the amplified housekeeping (HPRT) gene
at each dexamethasone concentration. Values are means ± 1 SE;
n = 3. *P < 0.05 compared with the
corresponding control value. Data were analyzed using ANOVA on ranks.
Since H value was statistically significant (P < 0.05), we used the Dunnett method to test the significance of each mean
value against its corresponding control. hENaC, human
amiloride-sensitive sodium channel.
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Fig. 2.
Transcriptional regulation of dexamethasone (Dex) in A549
cells. Synchronized A549 cells were treated with the indicated
concentrations of dexamethasone in the presence (+) or in the absence
( ) of actinomycin D (Act D, 1 µM). Total RNA (1 µg) extracted
from A549 cells was used for reverse transcription, and 2 µl each of
the 1st strand reaction mixture were used for amplification with human
ENaC (hENaC) and also with the HPRT-gene-specific upstream and
downstream primers (see text for details). Typical records were
reproduced three times.
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In Western blotting studies, the antibody against
-rENaC recognized
a single 75-kDa band in both control and dexamethasone-treated A549
extracted proteins under reducing conditions (Fig.
3A). This signal was blocked completely by the immunizing peptide (data not
shown).
-hENaC levels in A549 cells did not increase significantly following dexamethasone treatment. We were unable to detect
- or
-hENaC in control membranes of synchronized A549 cells. However, following dexamethasone treatment, antibodies against
- and
-hENaC recognized bands in nonreduced gels at around 100 and 90 kDa, respectively (Fig. 3, B and C). No signal was
seen when the
-hENaC antibody was substituted by preimmune serum
(data not shown). Thus dexamethasone treatment increased the abundance
of
- and
- but not
-hENaC proteins. This is consistent with
the RT-PCR data shown above.

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Fig. 3.
Effect of dexamethasone on -ENaC protein expression
in A549 cells. Fifty micrograms of membrane lysate form synchronized
and dexamethasone-treated or untreated A549 cells were separated in 7%
SDS-PAGE, transferred to polyvinyldidene difluoride membranes, followed
by blocking and probing with anti- -rENaC (A),
anti- -rENaC (B), and anti- -rENaC primary antibodies
(C) and anti-rabbit horseradish peroxidase (HRP) conjugate
as secondary antibody, and finally developed by enhanced
chemiluminescence (ECL) reagents. Cont, control; MW, molecular mass.
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Electrophysiology: single channel recordings.
Single channel activity was seen in 23 dexamethasone-treated and 19 control cell-attached patches. A characteristic recording at
100 mV
of a control cell is shown in Fig.
4A. The open time ranged from
a few milliseconds to hundreds of milliseconds (395 ± 15 ms;
mean ± SE), with an open probability, Po,
of 0.69 ± 0.016 for amplitude level 1 and 0.21 ± 0.02 for
amplitude level 2. The mean Po for the whole
recording shown in Fig. 4A (combining the values of both
levels) was 0.39 ± 0.05. Channel conductance was determined using
the histogram distribution fitted to a Gaussian equation (Fig.
4B). At the patch potential of
100 mV and pipette Na+ concentration of 145 mM, the single channel conductance
was 8.63 ± 0.036 pS (mean ± SE).

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Fig. 4.
Single channel currents in A549 cells. Characteristic
single channel currents recorded in a cell-attached patch from a
control synchronized A549 cell at a holding potential of 100 mV. The
pipette was filled with 145 mM sodium methanesulfonate, and the cell
was perfused with a solution containing 145 mM potassium
methanesulfonate (see MATERIALS AND METHODS for more
details). The recording (A) and the conductance histogram
(B) show the presence of an 8.6-pS channel in these cells.
The peak at 17.2 pS resulted from the simultaneous opening of two
channels rather than a new and different channel. The horizontal arrow
in A indicates the closed (C) state.
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Figure 5, A and B,
shows single channel activity in cell-attached patches of synchronized
A549 cells treated with 1 µM dexamethasone for 24 h. A single
channel with unitary conductance of 4.4 ± 0.027 pS (mean ± SE) was found. The mean unitary conductance, mean open time, and
Po before and after dexamethasone are summarized
in Table 1. Dexamethasone treatment shifted the single channel reversal potential from + 47 mV in control cells to + 66 mV after
dexamethasone (Fig. 6B). This
shift is consistent with an increase in the selectivity of the channel
to Na+ ions.

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Fig. 5.
Effects of dexamethasone treatment of A549 cells on
single channel unitary conductance: single channel currents recorded in
cell-attached mode at 100 mV. The cells were synchronized and treated
with 1 µM dexamethasone for 24 h. The pipette was filled with
145 mM Na+, and the cell was perfused with a solution
containing 135 mM K+ (see MATERIALS AND METHODS
for the ionic composition). The recording (A) and histogram
(B) show the presence of a channel with unitary conductance
of 4.4 pS. The horizontal arrow in A indicates the closed
(C) state of the channel.
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Table 1.
Single channel measurements in cell-attached patches of synchronized
A549 cells treated with or without dexamethasone (1 µM) for 24 h
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Fig. 6.
Na+ single channel current-voltage
(I-V) relationships before and after
dexamethasone. A: single channel activity recorded from
synchronized A549 cells after treatment with dexamethasone for 24 h. The cell membrane potential was depolarized to 0 mV by bathing the
cells in a solution containing 135 mM potassium in the absence of
calcium ions. B: illustrates the I-V
relationships before and after treatment with dexamethasone; numbers
are means ± 1 SE (n = 3). The dexamethasone
treatments caused a shift of the I-V relation to
positive potential values, close to sodium reversal potential, from
~47 (before dexamethasone) to ~66 mV after dexamethasone.
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Whole cell currents.
Synchronized control and dexamethasone-treated A549 cells exhibited
inward-rectifying currents (Fig. 7). We
have previously shown that these currents are carried by
Na+ ions (18). Perfusion of the cells with the
SES containing 10 µM amiloride inhibited the inward (Na+)
currents rapidly. The observed effect of amiloride was totally reversible upon washout (not shown). A549 cells treated with 1 µM
dexamethasone for 24 h showed significantly larger total and amiloride-sensitive whole cell currents compared with the corresponding values in control cells (Fig. 8). In
addition, an increased sensitivity of the channels to amiloride was
observed. Following incubation of the cells with dexamethasone, the
inhibition constant, K0.5, for amiloride
decreased from 833 ± 69 nM to 22 ± 5.4 nM
(P < 0.01) (Fig. 9).

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Fig. 7.
Whole cell currents recorded from A549 cells before and after
dexamethasone. Synchronized A549 cells were treated with dexamethasone
(1 µM) for 24 h as described in the MATERIALS AND
METHODS section. A: whole cell currents recorded from
a control cell. The membrane potential was held at -40 mV between
voltage steps (from 100 to 100 mV in 10-mV increments every 10 s
for 450 ms). The pipette was filled with the standard internal
solution, and the cell was perfused with standard external solution
(MATERIALS AND METHODS). B: the cell was
perfused with standard external solution (SES) containing 10 µM
amiloride. C: the amiloride-sensitive current is obtained by
digitally subtracting the residual current (B) from the
total current (A). Left
(D-F): whole cell currents when the same
protocol was applied to a cell treated with 1 µM dexamethasone for
24 h.
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Fig. 8.
Amiloride-sensitive I-V relationships in
control and dexamethasone-treated A549 cells. Whole cell
amiloride-sensitive currents were calculated as in Fig. 7 and plotted
against the voltage steps applied to the cell. Data points are
means ± SE of 9 experiments in control cells and 13 experiments
after dexamethasone treatment.
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Fig. 9.
Effect of dexamethasone on the whole cell current
sensitivity to amiloride. Whole cell currents were recorded from
synchronized and synchronized dexamethasone-treated (1 µM for 24 h) A549 cells, and the amiloride-sensitive currents were extracted as
shown in Fig. 7 for concentrations of amiloride ranging from 1 nM to
100 µM. Normalized amiloride-sensitive currents (y-axis)
were calculated as follows: I = [1 (I0 Ix)/(I0 I10)] × 100, where I0
is the total current, Ix is the current recorded
at the steady-state effect of the amiloride concentration x,
and I10 is the current recorded at the
steady-state effect of 10 µM amiloride. Values are means ± SE;
n = 6 (control) and 5 (dexamethasone). Values for the
inhibition constants (K0.5) were calculated by
the best fit of the data points to the following equation:
I = (1 [1/(1 + K0.5/x)]) × Imax, where Imax is the
maximum current, and x is the concentration of
amiloride.
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 |
DISCUSSION |
Data shown herein are consistent with the view that dexamethasone
differentially regulates hENaC expression. RT-PCR and Western blotting
studies demonstrated that treatment of A549 cells with dexamethasone
for 24 h resulted in upregulation of
- and
-hENaC mRNA and
protein levels. The inhibition of the observed changes by actinomycin
D, an RNA polymerase II inhibitor, indicates that dexamethasone
treatment increased transcription of
- and
-hENaC genes without
altering the stability of their mRNAs. Both transcriptional and
posttranscriptional mechanisms have been previously evoked in
explaining the action of dexamethasone on surfactant gene regulation in
fetal ATII cells (37).
The somewhat surprising finding was the absence of a significant effect
of dexamethasone on the expression of the
-hENaC subunit. Exposure
of confluent monolayers of an airway cell line (H441) to dexamethasone
resulted in a fivefold increase in the amiloride-sensitive
short-circuit current, which correlated with a fivefold increase in
-hENaC mRNA levels (32). The same authors showed the
presence of a glucocorticoid response element in the 5'-flanking region
of the
-hENaC subunit gene. In addition, Venkatesh and
Katzberg (38) reported that the exposure of fetal
and adult human lung explants to dexamethasone for 8-48 h
increased all three hENaC mRNA levels by two- to threefold. Tchepichev
et al. (36) showed that administration of corticosteroids
to pregnant rats caused large increases in
-rENaC mRNA of alveolar
epithelial cells harvested from the fetuses. In contrast, the
- and
-rENaC mRNA expression remained unchanged. Finally, Renard et al.
(30) reported that dexamethasone did not alter the
expression of rENaC proteins in rat lung; the same authors have shown
that the transcription of all three subunits was increased by
glucocorticoids in primary cultures of rat fetal lung epithelial cells
(6, 39). The same investigators also showed
that injection of dexamethasone into rats upregulated
- and
-rENaC mRNA levels in the colon but had no effect on
-rENaC mRNA,
which was already high under control conditions. From that perspective,
the behavior observed in A549 cells fits best with the behavior
described by Renard et al. (30) in rat distal colon. Taken
together, all the experimental data mentioned above is added evidence
and a clear indication of a wide spectrum of responses to
dexamethasone, depending probably on factors specific to each tissue.
The differential regulation of ENaC genes indicates that the mechanisms
involved could be more complex than the binding of dexamethasone to its
receptor and the activation of a glucocorticoid response element
(GRE). Instead, the presence of various tissue-specific
cofactors may be necessary for the activation to occur and may affect
the response to the hormone.
The novel aspect of this study is the demonstration that the
dexamethasone-induced changes in
- and
-hENaC mRNA and protein levels altered the function of the amiloride-sensitive Na+
channel in A549 cells. Champigny et al. (6) showed that
treatment of fetal lung epithelial cells with dexamethasone resulted in depolarization of their membranes and large increases in the amplitude of the amiloride-induced hyperpolarization, suggesting an increase in
the amiloride-sensitive currents. Sayegh et al. (32)
showed that treatment of H441 cells with dexamethasone increased the magnitude of the amiloride-sensitive short-circuit current. Herein we
demonstrate for the first time that treatment of a human cell line,
A549 cells, with dexamethasone for 24 h increased the
amiloride-sensitive Na+ whole cell currents with only a
slight increase in
-hENaC mRNA and protein levels. Single-channel
measurements showed a significant increase in Po
and open time of amiloride-sensitive currents (which may account for
the increase in whole cell current), a shift in conductance from 8 to 4 pS, and a shift in the reversal potential of single channel
current-voltage relationships toward positive values.
The observed modification of the biophysical and pharmacological
properties of the amiloride-sensitive Na+ channel could
have resulted from alterations in the stoichiometry of the
-,
-,
and
-subunits. The heterologous expression of rENaC subunits in
Xenopus oocytes and in MDCK cells showed that the presence
of all three subunits of the ENaC was a necessary condition for the
optimal function of the channels (15, 27). Both methods of expression showed that the properties and the function
of the channels were profoundly altered in absence of either the
-
or
-rENaC. The
-subunit was found to form channels (21, 27) by itself but with little or no
function. Therefore the role of
- and
-subunits seems to be
regulatory, which is in the line of our findings described herein.
Previous studies have shown that injection of dexamethasone into
newborn pigs ameliorated lung injury caused by hyperoxia and
barotrauma (8, 9). It has been thought that
the protective effects of dexamethasone were due to its ability to
downregulate the production of inflammatory mediators. However, other
studies have shown that treatment of fetal and adult ATII cells with
dexamethasone also resulted in increased expression of the
1- and
1-Na-K-ATPase mRNA and protein
levels (2, 5). These findings and our data lead us to speculate that dexamethasone may also decrease lung injury
by upregulating Na+ transport across the injured alveolar
epithelium, thus limiting the amount of lung edema (26,
42).
In conclusion, this work reports for the first time the modification of
the biophysical properties of amiloride-sensitive Na+
channels in A549 cells following treatment with a
corticosteroid. In addition, this work establishes the A549 cell line
as a model for studying ion transport in human distal lung at the
cellular level.
 |
ACKNOWLEDGEMENTS |
This project was supported by National Heart, Lung, and Blood
Institute Grants HL-31197 and HL-51173 and by Office of Naval Research
Grant N00014-97-1-0309. P. Barbry is supported by grants from the
Centre National de la Recherche Scientifique and the French Research Ministry.
 |
FOOTNOTES |
*
A. Lazrak and A. Samanta contributed equally to this work
and are listed in alphabetical order.
Address for reprint requests and other correspondence: S. Matalon,
Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St.
S, THT 940, Birmingham, AL 35233 (E-mail:
Sadis.Matalon{at}ccc.uab.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. §1734 solely to indicate this fact.
Received 29 October 1999; accepted in final form 30 March 2000.
 |
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