Biophysical properties and molecular characterization of
amiloride-sensitive sodium channels in A549 cells
A.
Lazrak1,
A.
Samanta1, and
S.
Matalon1,2,3
Departments of 1 Anesthesiology,
2 Physiology and Biophysics, and
3 Comparative Medicine, University of Alabama at
Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
Amiloride-sensitive Na+ channels, present in
fetal and adult alveolar epithelial type II (ATII) cells, play a
critical role in the reabsorption of fetal fluid shortly after birth
and in limiting the extent of alveolar edema across the adult lung.
Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such
as A549 that derive from a human alveolar cell carcinoma. A549 cells
were shown to contain
-,
-, and
-epithelial Na+
channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na+; pipette, 145 mM K+), A549
cells exhibited inward Na+ currents reversibly inhibited by
amiloride, with an inhibition constant of 0.83 µM. Ion substitution
studies showed that these channels were moderately selective for
Na+ (Na+-to-K+ permeability ratio = 6:1). Inward Na+ currents were activated by forskolin (10 µM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in
cell-attached mode revealed the presence of an amiloride-sensitive
Na+ channel with a unitary conductance of 8.6 ± 0.04 (SE)
pS. Channel activity was increased by forskolin and decreased by nitric
oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na+ channels with
biophysical properties similar to those of ATII cells.
patch-clamp techniques; whole cell recordings; cell-attached mode; epithelial sodium channels; forskolin; adenosine
3',5'-cyclic monophosphate; guanosine
3',5'-cyclic monophosphate; human lung cells; alveolar type
II cells; alveolar epithelium
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INTRODUCTION |
AMILORIDE-SENSITIVE Na+ channels are the
primary pathway for the entry of Na+ into a large number of
epithelial cells (9, 10, 28, 38). Electrophysiological studies have
shown that Na+ channels display functional heterogeneity
regarding their biophysical and pharmacological properties (reviewed in
Ref. 10). Their classification is based according to their kinetics,
pharmacology, and single-channel conductance (10, 36, 42). Presently, these channels are thought to be composed of three different subunits, referred to as the
-,
-, and
-epithelial Na+
channels (ENaCs), cloned from the colon of salt-deprived rats and human
lung tissue by a number of investigators (2, 3, 24, 48). Experiments
utilizing point mutations suggest that all three subunits are involved
in pore formation (40), although the exact stoichiometry is
debated, with different groups reporting either four (7) or nine (43)
subunits in the complex.
Existing evidence indicates that active Na+ transport
across the adult alveolar epithelium plays an important role in
maintaining the alveolar space free of fluid, especially after lung
injury when alveolar permeability to plasma proteins has been increased (29, 34, 50, 51). Amiloride-sensitive Na+ channels, shown
to be present in both fetal (32, 35, 48) and adult (17, 27, 51, 52)
alveolar type II (ATII) cells, represent the major pathways for
Na+ entry across these cells, with other pathways such as
glucose and amino acid cotransporters accounting for only a minor
fraction of the total Na+ flux (33, 39, 50). Although both
fetal and adult ATII cells have been shown to contain mRNAs for the
various ENaC subunits (for a review, see Ref. 28), there is
considerable controversy as to whether the lung channels are composed
of the same subunits as channels found in the kidney and colon (28).
Because of the overall importance of Na+ transport in lung
fluid balance in both normal and pathological conditions, there is
considerable interest in identifying the basic mechanisms responsible for the regulation of Na+ channels in adult ATII cells.
These studies have been hampered by the fact that ATII cells are
difficult to isolate and maintain in primary culture. In addition,
during culture, ATII cells undergo dedifferentiation and lose their
ability to secrete surfactant and express Na+-channel
proteins (49). For these reasons, it is important to document the
presence of amiloride-sensitive Na+ channels in lung
epithelial cell lines and characterize their biophysical properties.
A number of these lines, including A549 cells that originated from a
human alveolar cell carcinoma and possess many characteristics of type
II cells including multilamellar cytoplasmic inclusion bodies and the
ability to synthesize surfactant phospholipids (22), are routinely used
for ion transport studies (19, 25). Herein we show that A549 cells
express a 8.6-pS amiloride-sensitive Na+ channel with
biophysical properties similar to those found in ATII cells in primary
culture. Furthermore, our results demonstrate that increases in A549
cell cAMP levels upregulate whole cell amiloride-sensitive currents by
increasing the product [channel activity
(NPo)] of the number of channels (N)
times their open probability (Po). Finally, nitric
oxide (· NO), in concentrations likely to be found in the
alveolar spaces of injured lungs, decreases whole cell current and
NPo by increasing A549 cell cGMP levels. These data
offer new insight into the cellular mechanisms responsible for the
regulation of Na+ transport across alveolar epithelial cells.
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MATERIALS AND METHODS |
Cell culture. A549 cells were purchased from American Type
Culture Collection (Manassas, VA) in the 76th passage. They were suspended in DMEM-F-12 medium (Cellgro) supplemented with 1%
penicillin-streptomycin and 10% fetal calf serum, plated on plastic
tissue culture flasks (Corning Glass Works, Corning, NY), and placed in
an incubator in 21% O2, 5% CO2, and balance
N2 at 37°C and 100% humidity. All measurements were
conducted in cells between the 78th and 97th passages.
Patch-clamp recordings: whole cell measurements. In the first
series of experiments, macroscopic currents were recorded from A549
cells in the whole cell recording mode of the patch-clamp technique
(13). Twenty-four to thirty-six hours before any electrophysiological measurements, A549 cells were lifted from the tissue plates by treatment with 2.5% trypsin-EDTA (Sigma, St. Louis, MO) for 3-6 min at 37°C and then seeded on 12-mm-diameter glass coverslips in
DMEM-F-12 medium. Just before the start of the experiment, each
coverslip was rinsed with standard external solution (SES) with the
following ionic composition (in mM): 145 NaCl, 2.7 KCl, 1.8 CaCl2, 2 MgCl2, 5.5 glucose, and 10 HEPES, pH
7.4, 323 mosmol. The coverslip was then transferred to the recording
chamber and mounted on the stage of an inverted microscope (IMT-2
Olympus) for patch-clamp recordings.
Pipettes were made from LG16-type capillary glass (Dagan, Minneapolis,
MN) with a vertical puller (model PB-7, Narishige). They were
back-filled with standard internal solution (SIS; pH 7.2 at 22°C,
300 mosmol) with the following ionic composition (in mM): 135 potassium
methylsulfonic acid, 10 KCl, 6 NaCl, 1 Mg2ATP, 2 Na3ATP, 5.5 glucose, 10 HEPES, and 0.5 EGTA. The pipette resistance varied from 3 to 5 M
when filled with SIS. The pipette offset potential was corrected just before gigaseal formation. Series
resistance and capacitance transients were compensated for with the
patch-clamp amplifier (Axopatch 200, Axon). Junction potentials were
corrected as previously described (1).
The cell membrane potential was held at
40 mV during all whole
cell recordings. Inward and outward currents across the cell membrane
were elicited by altering the membrane potential from the holding value
(
40 mV) by
100 to + 100 mV in 10-mV increments of either
450 or 900 ms duration every 10 s with the Clampex program (pCLAMP,
Axon Instruments). Currents were digitized with a digital-to-analog and
analog-to-digital converter (DigiData 1200A, Axon Instruments), filtered through an internal four-pole Bessel filter at either 0.5 or 1 kHz, and sampled at 2 kHz. Current-voltage (I-V) curves were constructed by measuring the steady-state current values 300 ms
from the start of voltage pulses with the Clampfit Program (Axon
Instruments) and Origin Software (Microcal Software, Northampton, MA).
In some experiments designed to measure the relative permeability of
these channels to various ions, NaCl in SES was replaced with equivalent amounts of LiCl, KCl, or
N-methyl-D-glucamine chloride (NMDG); all other
components were maintained at their respective concentrations.
To test the extent to which whole cell currents were inhibited by
amiloride, we measured I-V relationships of cells in
the whole cell mode and then repeated the measurements after perfusing cells with SES containing amiloride in concentrations ranging from 1 nM
to 100 µM. We then calculated the amiloride-sensitive currents by
digitally subtracting the currents in the presence of amiloride from
its corresponding control value.
To study the short-term regulation of these Na+ channels by
cAMP and · NO, amiloride-sensitive I-V
relationships were measured before and after perfusion of the cells
with SES containing forskolin, 8-bromo-cGMP (8-BrcGMP; both from
Calbiochem, La Jolla, CA), or PAPA NONOate (Cayman Chemical, Ann Arbor,
MI), a · NO donor. PAPA NONOate stocks were prepared by
dissolving it in 43 mM phosphate buffer (pH 9) just before use.
Evolution of · NO in the SES medium (pH 7.4, 22°C) was
measured with an ISO-NO electrochemical probe (World Precision
Instruments, Sarasota, FL) connected to an IBM-compatible computer
equipped with an analog-to-digital converter. Mean · NO
concentration values were calculated as previously described (12). To
assess potential mechanisms by which · NO modulated whole
cell currents, A549 cells were incubated with
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson, St. Louis, MO), an inhibitor of guanylyl cyclase,
for 30 min and then perfused with SES containing PAPA NONOate as
described above.
Single channels. To further evaluate the biophysical properties
of these channels, we patched A549 cells in the cell-attached mode and
recorded single-channel currents. The ionic composition of the pipette
solution was (in mM) 145 sodium glutamate or sodium aspartate, 1 CaCl2, 5 MgCl2, 5.5 glucose, and 10 HEPES, pH
7.4. Cells were depolarized to 0 mV by perfusing them with the
following solution (in mM): 135 potassium glutamate, 10 KCl, 5 MgCl2, 10 HEPES, and 5.5 glucose, pH 7.4. Single-channel
currents were filtered at 1 kHz, sampled at 2 kHz, and analyzed with
the Fetchan and pStat programs (pCLAMP, Axon Instruments). The
amplitude and open probability (Po) were calculated
from all event histograms, constructed as previously described (21).
The product of the number of channels (N) times the
Po in a patch (NPo), which
reflects the activity of channels, was calculated from single-channel
recordings as follows
where
T is the total recording time, i is the number of open
channels, and ti is the recording time
during which i channels were open. Calculation of
NPo does not involve any assumptions about the
N in a patch or the Po of a single channel.
RNA isolation and semiquantitative RT-PCR. A549 cells were
treated with a 2.5% trypsin-EDTA solution (Sigma) for 3-6 min at 37°C and seeded on 10-cm tissue culture plates for 24 h in
DMEM-F-12 medium. At that time, 1 ml of TRI Reagent (Molecular Research Center, Cincinnati, OH) was added to each plate, and RNA was isolated from ~2 million cells according to the protocol supplied by the manufacturer (Molecular Research Center) with the method of Chomczynski and Sacchi (4). 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, Bethesda, MD). In brief, 1 µl of
Superscript II (GIBCO BRL), 20 U of RNasin, 1 µl of random hexamer, 2 µl of 100 mM dithiothreitol, and 1 µl of 10 mM deoxynucleotide triphosphate mixture 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 RT. Two
microliters of the reaction mixture were used for PCR amplification in
a Robocycler (Stratagene) with 1 µl (20 pmol) each of upstream and
downstream primers specific for the
-,
-, and
-subunits of the
human ENaC (hENaC) gene and the housekeeping gene
hypoxanthine phosphoribosyltransferase (HPRT; Table
1). Each primer was added into
a 50-µl mixture containing 1 µl of 10 mM deoxynucleotide
triphosphate mixture, 5 µl of 10× PCR buffer, 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, 72°C for 1 min, and 95°C for 1 min for a total of 35 cycles and final extension for 7 min at 72°C. Twenty microliters of
the final amplified product were electrophoresed on a 1.2% agarose gel, and DNA was visualized after ethidium bromide staining.
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RESULTS |
Amiloride effect on whole cell currents in A549 cells. In the
whole cell mode, A549 cells exhibited inward rectifying currents carried by Na+ (Fig. 1).
Perfusion of the cells with SES containing 10 µM amiloride rapidly
reduced the inward (Na+) but not the outward
(K+) currents (Fig. 1, B-D). The
amiloride-sensitive current reversed at +47 mV. Amiloride dose-response
relationships of inward currents measured at
100 mV are shown in
Fig. 2. The amiloride inhibition constant
(Ki), calculated as described in the legend of Fig.
2, was 0.83 ± 0.07 (SE) µM (n = 6 cells). When
the ionic gradients were reversed (i.e., bath, 145 mM K+;
pipette, 145 mM Na+), perfusion with SES containing
amiloride (10 µM) reduced the outward (Na+) but not the
inward (K+) currents (data not shown).

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Fig. 1.
Representative recordings (A-C) and mean values
(D) of whole cell currents in A549 cells. A: cell was
held at a potential of 40 mV. Currents were elicited by applying
voltage steps between 100 and +100 mV in 10-mV steps lasting 450 ms every 10 s. Pipette was filled with standard internal solution
(SIS), and cell was perfused with standard external solution (SES; see
MATERIALS AND METHODS for details). B: currents
were recorded after perfusion of cell with SES solution containing 10 µM amiloride. C: amiloride-sensitive currents obtained by
subtracting residual current in B from total current in
A. D: current-voltage (I-V)
relationships of total and amiloride (Amil)-sensitive currents. Values
are means ± SE; n = 30 cells.
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Fig. 2.
Inhibition of whole cell Na+ currents by amiloride. For
each cell, inward Na+ currents recorded at 100-mV
voltage step were measured during perfusion with SES. Cells were then
perfused with SES containing indicated concentrations of amiloride
until a steady state was reached, usually within 3-5 min from
onset of perfusion. Normalized amiloride-sensitive currents were
calculated as follows: I = [1 (I0 Ix)/(I0 I10)] · 100, where
I0 is control value, Ix
is steady-state current during perfusion with concentration x,
and I10 is current measured during perfusion with
10 µM amiloride. Values are means ± SE; n = 6 cells.
Inhibition constant (Ki) was calculated by best fit
of data points to the following equation: I = {1 [1/(1 + Ki/x)]} · Imax,
where Imax is maximum current.
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Replacement of Na+ in the bath with NMDG+ or
K+ greatly decreased the inward amiloride-sensitive
Na+ current (Fig. 3A).
On the other hand, substitution of external Na+ with
Li+ had no appreciable effect on the amiloride-sensitive
currents. The relative permeabilities of Na+ to
K+ (PNa/PK) and
Na+ to Li+
(PNa/PLi), calculated from the
constant-field equation with the reversal potentials of the
amiloride-sensitive currents, were 6:1 and 1:1.2, respectively.

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Fig. 3.
A: cation selectivity of A549 ion channels. For each cell,
amiloride-sensitive currents were computed by subtracting value of
inward whole cell current obtained at 100-mV voltage step in
presence of 10 µM amiloride from corresponding total current.
Measurements were conducted with SES containing Na+,
N-methyl-D-glucamine (NMDG+),
K+, or Li+. Values are means ± SE; N,
no. of cells. B: dependence of amiloride-sensitive currents on
bath Na+ concentration
([Na+]out). Normalized
amiloride-sensitive currents were computed by dividing
amiloride-sensitive current at 100-mV voltage step at indicated
[Na+]out by corresponding value at
145 mM Na+. Each point represents mean ± SE of 3 experiments; n = 6 cells. Solid line, Michaelis-Menten equation
fitted through these data (half-saturation constant = 37 ± 3.5 mM).
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Effect of extracellular Na+
concentration on the whole cell current. Values of normalized
amiloride-sensitive Na+ currents at
100-mV voltage
step (i.e., ratio of the amiloride-sensitive current measured at a
known external Na+ concentration divided by the
corresponding value at 145 mM Na+) were plotted against the
Na+ concentration in the bath and fitted to the
Michaelis-Menten equation (Fig. 3B). The half-saturation
constant (Km) was found to be 37 ± 3.5 (SE) mM
(n = 6 cells).
Amiloride-sensitive single Na+
channels in A549 cells. Single-channel activity was seen in ~20%
of 144 successful cell-attached patches. A characteristic recording is
shown in Fig. 4A. Two current levels are seen. The single-channel conductance was determined with the
histogram distribution fitted to a Gaussian equation (Fig. 4B).
At a patch potential of
100 mV, the single-channel conductance
was 8.6 ± 0.04 pS (n = 411 events). Single-channel open time
for this record ranged from a few milliseconds to hundreds of
milliseconds (395 ± 18 ms), with an Po of 0.68 ± 0.02 for amplitude level 1 and 0.19 ± 0.02 for
amplitude level 2. When amiloride (10 µM) was included in the
pipette solution, no channel activity was seen in 12 cell-attached
patches (Fig. 4C).

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Fig. 4.
Single-channel currents in A549 cells. Single-channel currents recorded
in a cell-attached patch from an A549 cell at a holding potential of
100 mV. Pipette was filled with 145 mM Na+; cell was
perfused with a solution containing 145 mM K+. Recording
(A) and amplitude distribution (B), constructed from a
384-s recording, show 2 active channels with amplitude of 0.86 ± 0.004 pA. Single-channel conductance was 8.6 ± 0.04 (SE) pS
(n = 411 events). No single-channel activity was seen when 10 µM amiloride was present in pipette solution (C). Results are
from typical experiments that were repeated at least 6 times with
different cells.
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Modulation of whole cell current and single-channel activity by
cAMP. Perfusion of A549 cells patched in the whole cell mode with
SES containing 10 µM forskolin resulted in a large increase in the
inward (Na+) current, which was rapidly reversed by
amiloride. Mean values of the inward currents as a function of time
after forskolin perfusion are shown in Fig.
5.

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Fig. 5.
Effect of forskolin on A549 whole cell inward current as shown by
continuous recording of inward Na+ currents across A549
cells elicited by 100-mV voltage pulse applied to cells every 10 s from a holding potential of 40 mV. Pipette was filled with
SIS, and cell was perfused with SES. Perfusion of cells with SES
containing 10 µM forskolin induced a significant increase in inward
current; >90% of forskolin-induced current was inhibited by
amiloride. Data points are means ± SE; n = 5 cells. Currents
were normalized by dividing current in each patch by measured value of
current 2 min from onset of perfusion with forskolin.
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In the cell-attached mode, forskolin increased single-channel activity
and NPo of single channels without affecting their amplitude (Fig. 6, A and
B). Forskolin increased NPo in all
patches. Mean NPo values measured in five
different cells with low spontaneous single-channel activity were 0.01 ± 0.04 for control cells and 1.5 ± 0.2 for forskolin-treated cells
(n = 5). The fact that the effect was immediate in the
cell-attached mode suggests that the delay observed in whole cell
measurements was not due to a slow perfusion rate but rather to the
dilution of necessary intracellular components by the intrapipette
solution. When 10 µM amiloride was included in the pipette solution,
no single-channel activity was seen in six separate patches after
perfusion of the cells with forskolin. A typical record is shown in
Fig. 6C.

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Fig. 6.
Effects of forskolin on A549 single-channel currents recorded in a
cell-attached patch from an A549 cell at a holding potential of
100 mV just before and during perfusion with forskolin. Pipette
was filled with 145 mM Na+; cell was perfused with a
solution containing 145 mM K+. Recording (A) and
amplitude distribution (B) show at least 5 different channels.
Effect of forskolin was totally abolished by presence of 10 µM
amiloride in pipette (C). Results are from typical experiments
that were repeated 6 times with different cells.
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Effect of · NO and cGMP on whole cell and single-channel
currents. Addition of 100 µM PAPA NONOate into the SES solution
resulted in a rapid release of ~4 µM · NO, which
persisted for >20 min. Addition of oxyhemoglobin (20 µM), a potent
scavenger of · NO, into the bath solution decreased
· NO release to zero. As shown in Fig.
7, perfusion of A549 cells
with SES containing 100 µM PAPA NONOate inhibited the inward but not
the outward whole cell currents in a rapid and reversible fashion;
furthermore, the inward current returned to the baseline value when the
cells (n = 6) were reperfused with SES alone. The reversal
potential of the · NO-sensitive currents (obtained by
subtracting the current remaining after perfusion with PAPA NONOate
from the total current) was similar to that of the amiloride-sensitive
current (+47 mV). Similar effects were achieved with considerably lower
concentrations of PAPA NONOate (10 µM), releasing 300 nM
· NO (results not shown). Preincubation of A549 cells with 3 µM ODQ (a potent inhibitor of soluble guanyl cyclase) for 30 min
before perfusion with PAPA NONOate totally prevented the reduction of
the inward Na+ currents (Fig.
8). Furthermore, perfusion of A549 cells
with 100 µM 8-BrcGMP (n = 4) markedly inhibited the inward
but not the outward currents (Fig. 9).
Finally, 100 µM PAPA NONOate markedly decreased single-channel
activity in the cell-attached patches of A549 cells (Fig
10).

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Fig. 7.
Effects of nitric oxide (· NO) on whole cell A549 currents.
A: time course recording of whole cell inward (Na+)
current evoked by 100-mV voltage pulses every 10 s before,
during (solid line), and after perfusion of an A549 cell with SES
containing 100 µM PAPA NONOate. Pipette was filled with SIS.
B and C: whole cell I-V relationships
before and 5 min after, respectively, PAPA NONOate perfusion when
steady-state currents were seen. Whole cell current inhibited by
· NO (· NO sensitive) was calculated by digitally
subtracting currents at steady-state effect of · NO (as shown
in C) from current before perfusion with · NO
containing SES (as shown in B). D: I-V
relationships for total and · NO-sensitive (sensit) currents.
Values are means ± SE; n = 6 cells.
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Fig. 8.
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ) prevents decrease of inward current by PAPA NONOate as shown by
time-course recording of whole cell inward (Na+) current
evoked by 100-mV voltage pulses every 10 s in an A549 cell
incubated for 30 min with ODQ (an inhibitor of soluble guanylyl
cyclase). Typical experiment was repeated 3 different times. Note that
in this case PAPA NONOate did not decrease current.
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Fig. 9.
Effect of 8-bromo-cGMP (100 µM) on whole cell currents of an A549
cell. Cell was held at a potential of 40 mV. Currents were
elicited by applying voltage steps between 100 and + 100 mV in
10-mV steps lasting 900 ms every 10 s. Pipette was filled with SIS, and
cell was perfused with SES (see MATERIALS AND METHODS for
details). A: control whole cell currents. B: currents
after perfusion with 8-bromo-cGMP (100 µM) for 4 min when a
steady-state effect was seen. C: cGMP-inhibitable whole
currents obtained by digitally subtracting records in B from
those in A. D: total and cGMP-sensitive
I-V relationships. Values are means ± SE; n = 4 cells.
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Fig. 10.
Effects of PAPA NONOate on single-channel currents. Single-channel
currents were recorded in cell-attached mode at 100 mV. Cell was
perfused with depolarizing K+ solution (see MATERIALS
AND METHODS) containing 100 µM PAPA NONOate. Note gradual
inhibition and complete cessation of channel activity within 40 s from
onset of perfusion with PAPA NONOate solution. Results are from a
typical experiment that was repeated 6 times in different cells.
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Functional relationship with ENaC gene expression. RT-PCR of
total RNA obtained from rapidly growing A549 cells was performed with
-,
-, and
-subunit-specific upstream and downstream primers of
the hENaC gene (Table 1). These primers amplified the expected size for each subunit message of the hENaC genes (440 bp for
, 542 bp for
, 633 bp for
; Fig.
11). Two
-bands were seen. The origin
of the minor lower band has not been identified. A very faint
-band
was occasionally seen but is not present in Fig. 11. No signal was seen
when RT was omitted during the first-strand cDNA synthesis, indicating
no cellular DNA contamination in the RNA preparations. The results
indicate that A549 cells contain at least
- and
-ENaC and
possibly
-ENaC mRNAs.

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Fig. 11.
RT-PCR showing presence of - and -subunit mRNAs of epithelial
Na+ channel (ENaC) genes and a housekeeping gene
[hypoxanthine phosphoribosyltransferase (HPRT)] in
A549 cells (see MATERIALS AND METHODS for detail). A faint
-band was seen occasionally but is not clear here. This is a typical
result that was reproduced 3 times.
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DISCUSSION |
The presence of amiloride-inhibitable 22Na+
uptake across A549 cells was previously documented (25), and these
cells have been used as a model of ion transport for ATII cells. To our
knowledge, this is the first study characterizing the
amiloride-sensitive pathway in the human A549 cell line. We have
demonstrated the presence of the message for the three subunits of
the ENaC (
,
, and
) with the RT-PCR technique and the presence
of an amiloride-sensitive channel with a unitary conductance of 8.6 pS
by patch-clamp techniques, which is moderately selective for
Na+ over K+
(PNa/PK = 6:1). The single
channel identified in the cell-attached mode mediates the inward whole
cell current recorded in the presence of Na+ in the outside
medium. Na+ channels with a similar unitary conductance
were found in the A6 kidney cell line (8.4 pS) (14) and in rat
macrophages (10.2 pS) (31)
A variety of channels have been described in freshly isolated and
cultured adult and fetal ATII cells (for a review, see Ref. 28). Yue et
al. (52) identified a 25-pS channel with
PNa/PK = 6:1 in inside-out
membrane patches of ATII cells in primary culture. This channel was
inhibited by both amiloride and ethylisopropylamiloride. The same
biophysical properties were seen in channels expressed by a putative
Na+-channel protein isolated from ATII cells and
reconstituted in planer lipid bilayers (41). Jain et al. (17) reported
the existence of a 20-pS, cGMP-inhibited, nonselective cation channel in cell-attached patches of ATII cells cultured for 24-96 h.
Marunaka et al. (26) found two channels in fetal ATII cells: a
nonselective Ca2+-activated channel of 25 pS and a highly
selective channel of 11.2 pS. A 25-pS, nonselective,
Ca2+-activated Na+ channel has also been
reported in cultured ATII cells (6). However, Voilley et al. (48) found
a highly selective 4.4-pS channel in outside-out patches of fetal rat
ATII cells. ATII cells have been shown to contain rat
-ENaC
(
-rENaC) mRNA (51) and
and
mRNAs, although these last two
subunits exist in very low abundance compared with
-rENaC (5).
Because the biophysical properties of ATII cell Na+
channels differ from those of ENaCs, there is a controversy as to
whether ENaC is the major channel in fetal and adult alveolar epithelial cells (28). However, a recent study (18) demonstrated that
treatment of ATII cells with antisense oligonucleotides targeting
-rENaC resulted in decreased density of the nonselective 20-pS channel. Interestingly, inhibition of
- or
-rENaC had no effect on channel density.
Results shown herein indicate that the amiloride-sensitive whole cell
Na+ currents in A549 cells are inwardly rectifying and
exhibit no time or voltage dependence. When A549 cells were internally
dialyzed with high Na+, the current became outwardly
rectifying (results not shown). Similar modulation of
amiloride-sensitive Na+ currents was observed in
Madin-Darby canine kidney (MDCK) cells transfected with rENaC (15) and,
in principle, cells of kidney collecting ducts (8). In contrast,
Na+ channels in ATII cells have linear I-V
relationships (52). This may be due to different stoichiometry of the
Na+-channel subunit proteins. Oocytes transfected with
various combinations of rENaC subunits (30) exhibited
amiloride-sensitive Na+ channels with various unitary
conductances and biophysical properties.
It is known that Na+ absorption via the amiloride-sensitive
channels located in the apical membrane of epithelia is controlled by
the fluctuations of the external Na+ concentration (10).
However, the exact mechanisms involved have not been elucidated. Data
presented herein show that the amiloride-sensitive whole cell current
recorded in A549 cells saturate, with a Km of 36.93 mM. It is unlikely that the saturation of the whole cell
Na+ current reported here is due to a large modification of
the intracellular ionic composition because the cytosol was dialyzed
with a pipette solution with a well-defined ionic composition. Palmer
and Frindt (37) described saturation of the ENaC at the single-channel level in the collecting duct, with a Km of 25 mM, a
value that is not very much different from our whole cell measurement.
Two saturation values were reported for rENaC heterologously expressed in oocytes (30) and MDCK cells (15). The value reported in oocytes (4.9 mM) is different from the value reported here (37 mM) and from rENaC
expressed in MDCK cells (24.4 mM) (15) and the collecting duct (25 mM)
(37). This discrepancy could be due to the different experimental
approaches used because the intracellular ionic composition cannot be
controlled in oocytes with only two microelectrodes.
The exact mechanism by which single-channel activity is regulated by
Na+ was not identified in this study. However, we can
assume that single channels will saturate at the same rate as the whole
cell current because the macroscopic current equals the
NPo. Direct evidence of ENaC regulation by
Na+ was reported by Ishikawa et al. (15) in MDCK cells, in
mouse submandibular duct cells (20), and by Ismailov et al. (16) using
immunopurified bovine renal papillary Na+ channels
reconstituted in planar lipid bilayers, although in this case, channel
activation required the presence of Ca2+ in the bath.
The experimental data reported here demonstrate that the
amiloride-sensitive Na+ channel in the human A549 cell line
is inhibited by · NO in concentrations likely to be present
in inflamed tissues. · NO has complex biological reactivity,
and its physiological effects depend on its concentration and redox
state, the nature of the target molecules, and the presence of other
free radicals (44). There is evidence to indicate that · NO
modulates cation-channel activity by increasing cGMP levels. Light et
al. (23) demonstrated the presence of a 28-pS cation channel in rat
renal inner medullary collecting duct cells, the activity of which was
decreased both by cGMP per se and via cGMP kinase-induced
phosphorylation. · NO released from bradykinin-stimulated endothelial cells or spermine NONOate decreased net
22Na+ flux across isolated perfused cortical
collecting ducts (47) and decreased Na+ short-circuit
current across a cortical collecting duct (CCD) cell line while
increasing their cGMP content (45, 46). Selective permeabilization of
the apical membranes of the CCD cells with nystatin reversed the
inhibition of short-circuit current. Based on these findings, it was
concluded that · NO inhibited CCD apical Na+
channels by increasing their cGMP content (46). Jain et al. (17)
reported that S-nitrosoglutathione and
S-nitroso-N-acetylpenicillamine increased ATII cell
cGMP content and significantly reduced the Po of a
20-pS nonselective channel in cell-attached patches; pretreatment with
a protein kinase G inhibitor prevented the inhibitory effects of
S-nitrosoglutathione on this channel; incubation of ATII cells with a cell-permeable analog of cGMP (8-BrcCMP) also decreased the
Po. They concluded that · NO decreased
the activity of this channel by activating a cGMP-dependent protein
kinase. Our results show that ODQ suppressed the inhibitory effect of
exogenous · NO on the amiloride-sensitive channels and that
the incubation of A549 cells with 8-BrcGMP mimicked the effects of
· NO and provided evidence that the inhibitory effect of
· NO is mediated by an increase in cGMP. On the other hand,
in a previous study, Guo et al. (11) showed that · NO
decreased short-circuit currents across cultured ATII monolayers by
inhibiting both the amiloride-sensitive Na+ channels and
Na+-K+-ATPase through cGMP-independent
mechanisms. Thus it is possible that · NO could modulate ion
channels by a variety of mechanisms.
In summary, our results indicate that A549 cells contain mRNAs for
-,
-, and
-hENaC and express Na+ channels in their
plasma membranes. In agreement with what has been reported in ATII
cells, the biophysical properties of these moderately selective
Na+ channels differ from those expressed when the three
ENaC subunits are expressed in oocytes and are regulated by both cAMP
and cGMP. Although there are many differences between A549 and ATII
cells, these studies help establish the A549 cells as a model to
investigate regulation of alveolar epithelial Na+ channel
by second messengers and reactive species.
 |
ACKNOWLEDGEMENTS |
We thank Carpantato Myles and Glenda Davis for technical assistance.
 |
FOOTNOTES |
This project was supported by National Heart, Lung, and Blood Institute
Grants HL-31197 and HL-51173 and Office of Naval Research Grant
N00014-97-1-0309.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St. S., THT 940, Birmingham, AL 35249 (E-mail:
Sadis.Matalon{at}ccc.uab.edu).
Received 4 August 1999; accepted in final form 16 November 1999.
 |
REFERENCES |
1.
Barry, PH,
and
Lynch JW.
Liquid junction potentials and small cell effects in patch-clamp analysis.
J Membr Biol
121:
101-117,
1991[ISI][Medline]. [Corrigenda. J Membr Biol 125: February 1992, p. 286.]
2.
Canessa, CM,
Horisberger JD,
and
Rossier BC.
Epithelial sodium channel related to proteins involved in neurodegeneration.
Nature
361:
467-470,
1993[ISI][Medline].
3.
Canessa, CM,
Schild L,
Buell G,
Thorens B,
Gautschi I,
Horisberger JD,
and
Rossier BC.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-467,
1994[ISI][Medline].
4.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
5.
Farman, N,
Talbot CR,
Boucher R,
Fay M,
Canessa C,
Rossier B,
and
Bonvalet JP.
Noncoordinated expression of
-,
-, and
-subunit mRNAs of epithelial Na+ channel along rat respiratory tract.
Am J Physiol Cell Physiol
272:
C131-C141,
1997[Abstract/Free Full Text].
6.
Feng, ZP,
Clark RB,
and
Berthiaume Y.
Identification of nonselective cation channels in cultured adult rat alveolar type II cells.
Am J Respir Cell Mol Biol
9:
248-254,
1993[ISI][Medline].
7.
Firsov, D,
Gautschi I,
Merillat AM,
Rossier BC,
and
Schild L.
The heterotetrameric architecture of the epithelial sodium channel (ENaC).
EMBO J
17:
344-352,
1998[Abstract/Free Full Text].
8.
Frindt, G,
Sackin H,
and
Palmer LG.
Whole cell currents in rat cortical collecting tubule: low-Na diet increases amiloride-sensitive conductance.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F562-F567,
1990[Abstract/Free Full Text].
9.
Garty, H,
and
Benos DJ.
Characteristics and regulatory mechanisms of the amiloride-blockable Na+ channel.
Physiol Rev
68:
309-373,
1988[Abstract/Free Full Text].
10.
Garty, H,
and
Palmer LG.
Epithelial sodium channels: function, structure, and regulation.
Physiol Rev
77:
359-396,
1997[Abstract/Free Full Text].
11.
Guo, Y,
Duvall MD,
Crow JP,
and
Matalon S.
Nitric oxide inhibits Na+ absorption across cultured alveolar type II monolayers.
Am J Physiol Lung Cell Mol Physiol
274:
L369-L377,
1998[Abstract/Free Full Text].
12.
Haddad, IY,
Zhu S,
Crow J,
Barefield E,
Gadilhe T,
and
Matalon S.
Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide.
Am J Physiol Lung Cell Mol Physiol
270:
L898-L906,
1996[Abstract/Free Full Text].
13.
Hamill, OP,
Marty A,
Neher E,
Sackman B,
and
Sigworth FJ.
Improved patch-clamp technique for high-resolution current recording from cells and cell-free patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
14.
Hamilton, KL,
and
Eaton DC.
Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels.
Membr Biochem
6:
149-171,
1986[ISI][Medline].
15.
Ishikawa, T,
Marunaka Y,
and
Rotin D.
Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+.
J Gen Physiol
111:
825-846,
1998[Abstract/Free Full Text].
16.
Ismailov, II,
Berdiev BK,
and
Benos DJ.
Biochemical status of renal epithelial Na+ channels determines apparent channel conductance, ion selectivity, and amiloride sensitivity.
Biophys J
69:
1789-1800,
1995[Abstract].
17.
Jain, L,
Chen XJ,
Brown LA,
and
Eaton DC.
Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels.
Am J Physiol Lung Cell Mol Physiol
274:
L475-L484,
1998[Abstract/Free Full Text].
18.
Jain, L,
Chen XJ,
Malik B,
Al-Khalili O,
and
Eaton DC.
Antisense oligonucleotides against the
-subunit of ENaC decrease lung epithelial cation-channel activity.
Am J Physiol Lung Cell Mol Physiol
276:
L1046-L1051,
1999[Abstract/Free Full Text].
19.
Kleinzeller, A,
Dodia C,
Chander A,
and
Fisher AB.
Na+-dependent and Na+-independent systems of choline transport by plasma membrane vesicles of A549 cell line.
Am J Physiol Cell Physiol
267:
C1279-C1287,
1994[Abstract/Free Full Text].
20.
Komwatana, P,
Dinudom A,
Young JA,
and
Cook DI.
Cytosolic Na+ controls an epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.
Proc Natl Acad Sci USA
93:
8107-8111,
1996[Abstract/Free Full Text].
21.
Lazrak, A,
and
Peracchia C.
Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells.
Biophys J
65:
2002-2012,
1993[Abstract].
22.
Lieber, M,
Smith B,
Szakal A,
Nelson-Rees W,
and
Todaro G.
A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.
Int J Cancer
17:
62-70,
1976[ISI][Medline].
23.
Light, DB,
Corbin JD,
and
Stanton BA.
Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.
Nature
344:
336-339,
1990[ISI][Medline].
24.
Lingueglia, E,
Renard S,
Voilley N,
Waldmann R,
Chassande O,
Lazdunski M,
and
Barbry P.
Molecular cloning and functional expression of different molecular forms of rat amiloride-binding proteins.
Eur J Biochem
216:
679-687,
1993[Abstract].
25.
Mairbaurl, H,
Wodopia R,
Eckes S,
Schulz S,
and
Bartsch P.
Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia.
Am J Physiol Lung Cell Mol Physiol
273:
L797-L806,
1997[ISI][Medline].
26.
Marunaka, Y,
Tohda H,
Hagiwara N,
and
O'Brodovich H.
Cytosolic Ca2+-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and beta agonist action in fetal lung epithelium.
Biochem Biophys Res Commun
187:
648-656,
1992[ISI][Medline].
27.
Matalon, S,
Kirk KL,
Bubien JK,
Oh Y,
Hu P,
Yue G,
Shoemaker R,
Cragoe EJ, Jr,
and
Benos DJ.
Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes.
Am J Physiol Cell Physiol
262:
C1228-C1238,
1992[Abstract/Free Full Text].
28.
Matalon, S,
and
O'Brodovich H.
Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance.
Annu Rev Physiol
61:
627-661,
1999[ISI][Medline].
29.
Matthay, MA,
Folkesson HG,
and
Verkman AS.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am J Physiol Lung Cell Mol Physiol
270:
L487-L503,
1996[Abstract/Free Full Text].
30.
McNicholas, CM,
and
Canessa CM.
Diversity of channels generated by different combinations of epithelial sodium channel subunits.
J Gen Physiol
109:
681-692,
1997[Abstract/Free Full Text].
31.
Negulyaev, YA,
and
Vedernikova EA.
Sodium-selective channels in membranes of rat macrophages.
J Membr Biol
138:
37-45,
1994[ISI][Medline].
32.
O'Brodovich, H.
Epithelial ion transport in the fetal and perinatal lung.
Am J Physiol Cell Physiol
261:
C555-C564,
1991[Abstract/Free Full Text].
33.
O'Brodovich, H,
Hannam V,
and
Rafii B.
Sodium channel but neither Na(+)-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance.
Am J Respir Cell Mol Biol
5:
377-384,
1991[ISI][Medline].
34.
Olivera, W,
Ridge K,
Wood LD,
and
Sznajder JI.
Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats.
Am J Physiol Lung Cell Mol Physiol
266:
L577-L584,
1994[Abstract/Free Full Text].
35.
Orser, BA,
Bertlik M,
Fedorko L,
and
O'Brodovich H.
Cation selective channel in fetal alveolar type II epithelium.
Biochim Biophys Acta
1094:
19-26,
1991[ISI][Medline].
36.
Palmer, LG.
Epithelial Na channels: function and diversity.
Annu Rev Physiol
54:
51-66,
1992[ISI][Medline].
37.
Palmer, LG,
and
Frindt G.
Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li.
J Gen Physiol
92:
121-138,
1988[Abstract].
38.
Rossier, BC,
Canessa CM,
Schild L,
and
Horisberger JD.
Epithelial sodium channels.
Curr Opin Nephrol Hypertens
3:
487-496,
1994[Medline].
39.
Russo, RM,
Lubman RL,
and
Crandall ED.
Evidence for amiloride-sensitive sodium channels in alveolar epithelial cells.
Am J Physiol Lung Cell Mol Physiol
262:
L405-L411,
1992[Abstract/Free Full Text].
40.
Schild, L,
Schneeberger E,
Gautschi I,
and
Firsov D.
Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.
J Gen Physiol
109:
15-26,
1997[Abstract/Free Full Text].
41.
Senyk, O,
Ismailov I,
Bradford AL,
Baker RR,
Matalon S,
and
Benos DJ.
Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers.
Am J Physiol Cell Physiol
268:
C1148-C1156,
1995[Abstract/Free Full Text].
42.
Smith, PR,
and
Benos DJ.
Epithelial Na+ channels.
Annu Rev Physiol
53:
509-530,
1991[ISI][Medline].
43.
Snyder, PM,
Cheng C,
Prince LS,
Rogers JC,
and
Welsh MJ.
Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.
J Biol Chem
273:
681-684,
1998[Abstract/Free Full Text].
44.
Stamler, JS.
Redox signaling: nitrosylation and related target interactions of nitric oxide.
Cell
78:
931-936,
1994[ISI][Medline].
45.
Stoos, BA,
Carretero OA,
Farhy RD,
Scicli G,
and
Garvin JL.
Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells.
J Clin Invest
89:
761-765,
1992[ISI][Medline].
46.
Stoos, BA,
Carretero OA,
and
Garvin JL.
Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.
J Am Soc Nephrol
4:
1855-1860,
1994[Abstract].
47.
Stoos, BA,
Garcia NH,
and
Garvin JL.
Nitric oxide inhibits sodium reabsorption in the isolated perfused cortical collecting duct.
J Am Soc Nephrol
6:
89-94,
1995[Abstract].
48.
Voilley, N,
Lingueglia E,
Champigny G,
Mattei MG,
Waldmann R,
Lazdunski M,
and
Barbry P.
The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning.
Proc Natl Acad Sci USA
91:
247-251,
1994[Abstract].
49.
Yue, G,
Hu P,
Oh Y,
Jilling T,
Shoemaker RL,
Benos DJ,
Cragoe EJ, Jr,
and
Matalon S.
Culture-induced alterations in alveolar type II cell Na+ conductance.
Am J Physiol Cell Physiol
265:
C630-C640,
1993[Abstract/Free Full Text].
50.
Yue, G,
and
Matalon S.
Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats.
Am J Physiol Lung Cell Mol Physiol
272:
L407-L412,
1997[Abstract/Free Full Text].
51.
Yue, G,
Russell WJ,
Benos DJ,
Jackson RM,
Olman MA,
and
Matalon S.
Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.
Proc Natl Acad Sci USA
92:
8418-8422,
1995[Abstract].
52.
Yue, G,
Shoemaker RL,
and
Matalon S.
Regulation of low-amiloride-affinity sodium channels in alveolar type II cells.
Am J Physiol Lung Cell Mol Physiol
267:
L94-L100,
1994[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 278(4):L848-L857
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