Nitric oxide inhibits lung sodium transport through a
cGMP-mediated inhibition of epithelial cation channels
Lucky
Jain1,
Xi-Juan
Chen1,
Lou Ann
Brown1, and
Douglas C.
Eaton2,3
Departments of 1 Pediatrics and
2 Physiology and
3 Center for Cell and Molecular
Signaling, Emory University School of Medicine, Atlanta, Georgia
30322
 |
ABSTRACT |
We used the patch-clamp technique to study the
effect of nitric oxide (NO) on a cation channel in rat type II
pneumocytes [alveolar type II (AT II) cells].
Single-channel recordings from the apical surface of AT II cells in
primary culture showed a predominant cation channel with a conductance
of 20.6 ± 1.1 (SE) pS (n = 9 cell-attached patches) and
Na+-to-K+
selectivity of 0.97 ± 0.07 (n = 7 cell-attached patches). An NO donor,
S-nitrosoglutathione (GSNO; 100 µM),
inhibited the basal cation-channel activity by 43% [open
probability (Po), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03;
P < 0.001;
n = 16 cell-attached patches],
with no significant change in the conductance. GSNO reduced the
Po by reducing
channel mean open and increasing mean closed times. GSNO inhibition was
reversed by washout. The inhibitory effect of NO was confirmed by using
a second donor of NO,
S-nitroso-N-acetylpenicillamine (100 µM; Po,
control 0.53 ± 0.05 vs.
S-nitroso-N-acetylpenicillamine 0.31 ± 0.04;
42%; P < 0.05;
n = 5 cell-attached patches). The GSNO
effect was blocked by methylene blue (a blocker of guanylyl cyclase;
100 µM), suggesting a role for cGMP. The permeable analog of cGMP,
8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner
similar to GSNO
(Po, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02;
P < 0.05;
n = 7 cell-attached patches).
Pretreatment of cells with 1 µM KT-5823 (a blocker of protein kinase
G) abolished the inhibitory effect of GSNO. The NO inhibition of
channels was not due to changes in cell viability. Intracellular cGMP
was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml;
P < 0.05;
n = 6 cell-attached patches). We
conclude that NO suppresses the activity of an
Na+-permeant cation channel on the
apical surface of AT II cells. This action appears to be mediated by a
cGMP-dependent protein kinase.
guanosine 3',5'-cyclic monophosphate; nonselective
cation channel; alveolar type II cells; S-nitrosoglutathione; sodium channel; single-channel recording; amiloride
 |
INTRODUCTION |
DISTAL LUNG EPITHELIUM plays a critical role in
maintaining normal alveolar fluid balance (1, 9, 19, 20)
and in the adaptation of newborn lungs to air breathing (9, 22). The
alveolar walls, lined by type I and type II cells, regulate the fluid
to keep the alveoli moist while avoiding excessive buildup of fluid.
Transepithelial fluid movement appears largely to be a result of active
salt transport, which drives the osmotic movement of water. Recent
patch-clamp studies show that Na+
channels, located on the apical surface of alveolar type II (AT II)
cells, allow vectorial transport of
Na+ from the alveolar space into
the cell, with subsequent extrusion into the interstitium by
Na+-K+-ATPase
located on the basolateral membrane (7, 17, 19, 20). The interstitial
fluid is then taken up by the flow vessels and lymphatics. The exact
mechanism by which the lung epithelial cells control fluid reabsorption
and prevent pulmonary edema is not clear, although disruption of this
process has been implicated in several disease states.
The use of inhaled nitric oxide (NO) is currently being evaluated in a
variety of lung disorders including pulmonary hypertension (25),
acute respiratory distress syndrome (27), and
high-altitude pulmonary edema (28). Studies done in vitro and in vivo
suggest that NO may have an effect on lung fluid dynamics, although the mechanism underlying the NO effect is largely unknown. Because alveolar
epithelial cells are exposed to high concentrations of NO during
inhaled NO treatment, it is possible that NO may alter lung epithelial
Na+ and water transport. In the
kidney, NO has been shown to inhibit Na+ reabsorption by cultured
cortical collecting duct cells (29). In the lung, Compeau et al. (4)
have shown that endotoxin-stimulated alveolar macrophages impair distal
lung epithelial ion transport by inactivating amiloride-sensitive,
nonselective cation (NSC) channels. This inhibition was dependent on NO
synthesis by the macrophage, suggesting that NO may promote lung edema
formation by inhibiting cation channels in the AT II cells. However,
other investigators have shown that NO prevents pulmonary edema
formation in the isolated rat lung (8) and in humans prone to
high-altitude pulmonary edema (28). The reasons for the discrepancy in
the findings of these investigators remain to be elucidated.
The objective of this study was to examine the effect of NO on lung
epithelial Na+ transport and to
determine its mechanism of action. We used the patch-clamp technique to
study the effect of NO on an amiloride-sensitive, Na+-permeable cation channel on
the apical surface of rat AT II cells. Our results show that NO
inhibits these cation channels (and, presumably,
Na+ reabsorption) by AT II cells
and that this inhibition is mediated by intracellular cGMP acting
through a cGMP-dependent protein kinase (PK).
 |
METHODS AND PROCEDURES |
Type II pneumocyte isolation and
culture. AT II cells were isolated by enzymatic
digestion of lung tissue from adult Sprague-Dawley rats (200-250
g) with published techniques (2). Briefly, the rats were anesthetized
with pentobarbital sodium and heparinized (100 units/kg). AT II cells
were digested by tracheal installation of elastase (0.4 mg/ml). Lung
tissue was minced in DNase (1 mg/ml) and filtered sequentially through
100- and 20-µm nylon mesh. Purification was based on the differential
adherence of cells to dishes coated with rat IgG. Nonadherent AT II
cells were collected, centrifuged, and seeded onto glass coverslips
(~2 × 105
cells/cm2) in Dulbecco's
modified Eagle's medium-F-12 medium containing 5% FCS and
antimicrobial agents and supplemented with
L-glutamine and
Na+ bicarbonate. Cells were
incubated in 90% air-10% CO2 and
used for patch-clamp studies between 24 and 96 h after harvest. No significant difference in
Na+-channel activity was observed
within this time frame. Cell viability (90%) and purity (95%)
associated with this isolation procedure have been validated in our
laboratory (2).
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. The bath and pipette
solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl2, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4 with 2 N NaOH. In the inside-out recordings, the 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 bathing and pipette solutions were varied as
appropriate for specific protocols. All chemicals were obtained from
Sigma (St. Louis, MO) except 8-bromo-cGMP (8-BrcGMP) and KT-5823 that
were from Calbiochem.
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) 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 because, in
this configuration, the cytoplasmic constituents remain intact, thus
allowing us to study the role of cytoplasmic second messengers in the
regulation of ion-channel activity. Inside-out patches were also used
to determine the selectivity of the channel and to determine whether the effects of agents were directly on the channel or mediated by a
signaling cascade. After formation of a high-resistance seal (>50
G
) between the pipette and the 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 an eight-pole,
low-pass Bessel filter. Data were recorded by a computer with pCLAMP 6 software (Axon Instruments, Foster City, CA). Current-amplitude
histograms were made from stable continuously recorded data, and the
open and closed current levels were determined from least square fitted
Gaussian distributions. We used the product (NPo) of the
number of channels (N) times the
open probability (Po) as a
measure of the activity of the channels within a patch. This product
could be calculated from the single-channel record without making any
assumptions about the total N in a
patch or the Po
of a single channel
|
|
where
T is the total record time,
n is the number of channels open, and
tn is the record
time during which n channels are open.
Current-amplitude histograms provided the clearest demonstration of
multiple current levels. The total N
in a patch was estimated by observing the number of peaks in a
current-amplitude histogram over the entire duration of the recording
period. The Po of
the channels was calculated with FETCHAN in pCLAMP 6. Single-channel conductance was determined with a linear regression of unitary current
amplitudes over the range of applied pipette potentials.
To determine whether changes in
NPo were due to a
change in Po, the
mean open (
open) and closed
(
closed) times were
determined.
open and
closed are experimental
measures that can provide information as to the average duration in all
open and closed states. The mean
open and
closed for
N observed channels can be calculated from the following equations
where
n is the total number of transitions
between states during T and
N and
NPo were
calculated as described above. The mean open time calculated in this
manner is not the same as the mean open time for a single channel or,
for that matter, the mean time in any particular kinetic state of the
channel but is rather a reflection of the average open or closed time
for all channel states. As such, the mean open and closed times provide
a mechanism for distinguishing whether the effects of an experimental
maneuver that alters the
Po of multiple
channels are caused by a change in the duration of open intervals or a
change in the duration of closed intervals. Determinations of mean open
and closed times were made, and interval histograms were generated with
locally developed software (18).
Procedure for cGMP estimations.
Intracellular cGMP levels were measured in cultured AT II cells with an
enzyme immunoassay (Biotra EIA, Amersham, Arlington Heights, IL).
Briefly, cells were treated with
S-nitrosoglutathione (GSNO),
S-nitroso-N-acetylpenicillamine (SNAP), and carbachol for 20 min, and the reaction was stopped by
removal of the incubation medium and addition of 65% ice-cold ethyl
alcohol. The intracellular cGMP extracted into the supernatant was
measured by enzyme immunoassay in duplicate.
Methods for statistical analysis.
Statistical analysis for the changes in the
Po of channels
and the biochemical estimations were performed with 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 usually determined by one-way ANOVA followed by
pairwise comparisons with a Bonferroni t-test to determine significant
differences between each group. A P
value < 0.05 was regarded as significant. Because of variability in
the mean open and closed times of control cells, the effects of GSNO
were determined with a Kruskal-Wallis one-way ANOVA on ranks and
Dunn's method to determine statistically significant differences from
control values.
 |
RESULTS |
All cells used for the present experiments had lamellar bodies and
other phenotypic features of AT II cells. The predominant Na+-permeant channel seen in
apical cell-attached patches is shown in Fig.
1A. This
channel had a linear current-voltage
(I-V)
relationship (Fig. 1B) with a
conductance of 20.6 ± 1.1 pS (n = 9 cell-attached patches) with 140 mM NaCl in the bath and pipette. No
rectification of the
I-V
curve was observed (Fig. 1B). The
pipette potential at which current polarity reversed was estimated to
be
37 mV. Because the resting membrane potential of alveolar
epithelium has previously been shown to be approximately
30 to
40 mV, the reversal potential appears to be close to 0 mV, which
would be expected for an NSC channel (20). Ion selectivity was
determined with inside-out recording and solutions of varying ionic
compositions. The channel had a similar permeability to
Na+ and
K+
(Na+-to-K+
permeability = 0.97 ± 0.07; n = 7 cell-attached patches). The channel
Po was decreased
by amiloride (0.1-1 µM) applied to the extracellular side (i.e.,
in the micropipette;
Po, control 0.31 ± 0.01 vs. amiloride 0.03 ± 0.01;
P < 0.01;
n = 7 cell-attached patches). More
than one current level was observed in 85.7% of active patches. Thus
this channel was very similar in characteristics to the NSC channel
described by Orser et al. (24) and Marunaka (17).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Characteristics of the cation channel.
A: raw single-channel recordings from
a cell-attached patch in alveolar type II (AT II) cell apical membrane
at different applied voltages. Arrows, closed state. Downward
deflection, inward current across patch membrane. Voltages, holding
potentials applied to membrane through micropipette.
B: single-channel current-voltage
(I-V)
relationship for channel shown in A.
In this plot, voltage used in micropipette [compared with voltage
in bath (0 mV)] is shown. , Means ± SE of single-channel
currents.
|
|
NO reduces the Po of apical NSC channels.
To investigate the acute effects of NO on NSC channel activity, we used
two agents that are known to release NO. GSNO (100 µM) was applied to
the bath solution after apical cell-attached recording was established.
The GSNO stock solution was freshly prepared just before
it was added to the bath. Figure
2A
shows the typical time course of NSC channel activity after exposure to
GSNO in the bath. With each cell-attached patch acting as its own
control, channel activity, measured as
Po, consistently
decreased from a mean control value of 0.28 ± 0.05 to a mean
treated value of 0.16 ± 0.03 (
43%;
P < 0.001;
n = 16 cell-attached patches; Fig.
2B). The effect was immediate in
onset and was sustained for up to 30 min of recording in stable
patches. It was reversible, with a return to control levels after
washout (Po,
control 0.16 ± 0.05 vs. 100 µM GSNO 0.062 ± 0.03 vs. washout
0.21 ± 0.06; Fig. 2C). There was
no change in the conductance of the channel. An alternate donor of NO,
SNAP (100 µM), caused a decrease similar to that of GSNO in the
Po of the channel
(control 0.53 ± 0.05 vs. SNAP 0.31 ± 0.04;
42%;
P < 0.05;
n = 5 cell-attached patches; Fig.
3). This effect was not seen when GSH, the
carrier of NO in GSNO, was used in a 100 µM concentration
[Po,
control 0.35 ± 0.11 vs. GSH 0.27 ± 0.07;
P = not significant (NS);
n = 8 cell-attached patches;
Fig. 4]. Taken together,
these experiments indicate that NO released by the NO donors suppresses
basal NSC channel activity in apical cell-attached patches in AT II
cells.

View larger version (22K):
[in this window]
[in a new window]

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Acute exposure of AT II cells to
S-nitrosoglutathione (GSNO) inhibits
nonselective cation (NSC) channel activity in apical cell-attached
patches. GSNO was added to bath after 3 min of stable recording.
A: typical single-channel recordings
showing channel activity before and after addition of 100 µM GSNO to
bath and after washout. Arrows, closed state.
B: summary of results (means ± SE;
on left and
right) from 16 cell-attached patch
experiments. Channel activity was measured as open probability
(Po) before and
2 min after addition of 100 µM GSNO to bath. Each
Po was calculated
from at least 3 min of consecutive recording. * GSNO decreased
Po by 43% from
control level, P < 0.001. C: summary of results (means ± SE;
on left and
right) from 7 cell-attached patch
experiments showing reversibility of effect of GSNO after washout. Each
symbol represents a different patch; lines connect data points from the
same patch. * GSNO decreased Po from control
level, P < 0.01. ** Washout of GSNO resulted in
reversal of GSNO-induced suppression of Po,
P < 0.01.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of 100 µM
S-nitroso-N-acetylpenicillamine
(SNAP) on cation channel. A: acute
exposure to SNAP causes inhibition of channel as seen with GSNO.
Arrows, closed state. B: summary of
results (means ± SE; on left
and right) from 5 cell-attached
patch experiments. Each symbol represents a different patch; lines
connect data points from the same patch. * SNAP decreased
Po by 42% from
control level, P < 0.05.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Exposure of cells to 100 µM glutathione (GSH) did not have a
significant effect on channel activity.
A: single-channel recordings show no
change in channel activity. Arrows, closed state.
B: summary of results (means ± SE;
on left and
right) from 8 cell-attached patch
experiments shows no significant change in
Po after exposure
to GSH. Each symbol represents a different patch; lines connect data
points from the same patch. Each cell-attached patch served as its own
control.
|
|
NO reduces mean open time and increases mean closed
time of NSC channels. NO could reduce
Po either by
reducing the mean open time of the channels or by increasing the mean
closed time. The difficulty in examining changes in channel kinetics is
the large variability from cell to cell in the mean open and closed
times. Nonetheless, by using each cell as its own control, we were able to make appropriate comparisons. GSNO usually caused a decrease in mean
open time (from 157 ± 48.2 ms in untreated cells to 49.3 ± 20.2 ms after GSNO; n = 19 cell-attached
patches) and always caused an increase in mean closed time (from 495 ± 334 ms in untreated cells to 2,080 ± 639.2 ms after GSNO;
n = 19 cell-attached patches; Fig.
5). For three patches that, based on their
amplitude histograms, only had single channels, we generated interval
histograms (Fig. 6). Examination
of the histograms suggests that there is one predominant open state of
the channel (with a mean duration of 10.0 ± 0.861 ms), although
there are occasional long openings that might represent a second long
open state (with a mean duration of 313 ± 29.5 ms). On the other
hand, the closed interval histogram clearly consists of at least two
populations of events: short closures (with a mean duration of 2.15 ± 0.507 ms) and long closures (with a mean duration of 381 ± 48.6 ms). To emphasize the effects of GSNO, we superimposed the open
and closed interval histograms obtained in the absence and presence of
GSNO. An examination of the histograms shows that there are two
predominant effects of GSNO. First, GSNO increases the number and
causes an almost sevenfold reduction in the mean duration of short open
events (from a mean of 10.0 ± 0.861 ms in untreated cells to 1.45 ± 0.583 ms after GSNO), with little effect on the number or
duration of long open events (from a mean of 313 ± 29.5 ms in
untreated cells to 143 ± 21.3 ms after GSNO). Second, GSNO
increases the number and causes more than a threefold increase in the
mean duration of long closed events (from a mean of 381 ± 48.6 ms
in untreated cells to 1,130 ± 199 ms after GSNO), with no effect on
the number or duration of short closed events (from a mean of 2.15 ± 0.507 ms in untreated cells to 2.56 ± 0.300 ms after GSNO).
Thus the effect of GSNO is to change the kinetics of the channels in
such a way as to favor less time in open states and more time in closed
states.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
GSNO reduces mean open time and increases mean closed time. In 7 cell-attached patch experiments, GSNO usually produced a decrease in
mean open time (A). Significant
decrease between control and GSNO, P < 0.05. In the 3 cases in which GSNO was washed off cell, mean open
time always increased, and there was no statistically significant
difference between control patches and patches after GSNO was washed
off. In the same patches, there was always an increase in mean closed
time in GSNO and a decrease when GSNO was subsequently washed off
(B). There was a significant
GSNO-induced increase in mean closed time compared with control
level (P < 0.05) but no
significant difference after washout.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
GSNO alters both open (A) and closed
(B) interval histograms. Solid bars,
untreated cells; open bars, after GSNO. There is a significant shift of
short open events to a population of even shorter duration events. At
the same time, GSNO induces a change in closed interval histogram.
Untreated cells have populations of both short-duration events and
long-duration events. After GSNO, there is a major increase only in
population of long-duration events.
|
|
NO acts via a cGMP-mediated pathway.
We next tried to elucidate the mechanism of action of NO. To see
whether NO was acting via a cGMP-mediated pathway, we studied the
effect of methylene blue (MeB; an inhibitor of guanylyl cyclase) added
to the bath before application of GSNO. MeB (100 µM) resulted in an
increase in basal channel activity
(Po, control 0.17 ± 0.03 vs. MeB 0.34 ± 0.04; P < 0.04; n = 7 cell-attached
patches). GSNO (100 µM) was then added to the bath. MeB (100 µM)
blocked the effect of 100 µM GSNO
(Po, MeB 0.36 ± 0.07 vs. MeB±GSNO 0.37 ± 0.07;
P = NS;
n = 6 cell-attached patches),
suggesting a role for cGMP (Fig. 7). This
was further confirmed by application of a permeable analog of cGMP,
8-BrcGMP (1 mM), to the bath solution; 8-BrcGMP decreased the
Po of channels in
cell-attached apical patches.
(Po, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02;
76%;
P < 0.05;
n = 7 cell-attached patches; Fig.
8).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Methylene blue (MeB) blocks GSNO-induced inhibition of NSC channels. AT
II cells were pretreated with MeB before exposure to GSNO.
A: single-channel recordings show no
change in channel activity in patches pretreated with MeB when 100 µM
GSNO was added. Arrows, closed state.
B: summary of results (means ± SE;
on left and
right) from 6 cell-attached patch
experiments shows no significant change in channel activity. Each
symbol represents a different patch; lines connect data points from the
same patch. Each cell-attached patch served as its own control.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 8.
cGMP inhibits NSC channel. A:
single-channel recordings show inhibition of channel activity by
addition of 1 mM 8-bromo-cGMP (8-BrcGMP) to bath solution. Arrows,
closed state. B: summary of results
(means ± SE; on left and
right) from 6 cell-attached patch
experiments after exposure to 100 mM 8-BrcGMP. Each symbol represents a
different patch; lines connect data points from the same patch. cGMP
decreased the Po
by 76% from control level, P < 0.05.
|
|
cGMP action is mediated via a PK. To
determine whether cGMP action on NSC channels is direct or is mediated
via a PK, we pretreated cells with KT-5823 (a PKG inhibitor; 1 µM)
before applying 100 µM GSNO to the bath. Prior application of KT-5823
did not alter basal channel activity
(Po, control
0.303 ± 0.05 vs. KT-5823 0.35 ± 0.04;
P = NS;
n = 13 cell-attached patches).
However, KT-5823 blocked the action of GSNO
(Po, KT-5823
0.41 ± 0.06 vs. KT-5823+GSNO 0.49 ± 0.06;
P = NS;
n = 5 cell-attached patches),
suggesting that PKG was necessary for the action of NO (Fig.
9).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 9.
Inhibition of protein kinase G with KT-5823 blocked effect of GSNO.
Cells were pretreated with KT-5823 for 15 min before exposure to GSNO.
A: single-channel recordings show no
effect of 100 µM GSNO on cells pretreated with 1 µM KT-5823.
Arrows, closed state. B: summary of
results (means ± SE; on left
and right) from 5 cell-attached
patch experiments. Each symbol represents a different patch; lines
connect data points from the same patch.
|
|
cGMP is elevated in cells treated with
NO. Figure 10 shows the
results of intracellular cGMP levels in cells treated with NO. cGMP
measurements by ELISA 20 min after exposure of cells to two donors of
NO (100 µM GSNO and 100 µM SNAP) and to 100 µM carbachol (positive control) showed that AT II cells were capable of increasing cGMP levels in response to NO.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 10.
AT II cells respond to nitric oxide donors (100 µM GSNO and 100 µM
SNAP) with a rise in cGMP as measured by ELISA 20 min after exposure in
6 cell-attached patch experiments. Carbachol (100 µM) served as a
positive control. prot, Protein. * Significant difference between
control and each treatment group, P < 0.05.
|
|
 |
DISCUSSION |
Research during the past several years utilizing a variety of
approaches has underscored the physiological importance of active Na+ transport by alveolar
epithelium. Disruption of this process has been implicated in a number
of disease states. Because several pharmacological agents, especially
those applied topically to the lung epithelium, have the potential for
altering epithelial ion and water transport, their effect on lung fluid
balance warrants detailed study before they are inducted into the
clinical armamentarium. The major findings of this study are that NO
inhibits an NSC channel in AT II cells and that this action is mediated
by a cGMP-activated PK. This is the first study to report the effect of
NO on cation channels in the distal lung epithelia. We believe that
this effect may be beneficial in some situations and may be harmful in
others. A classic example of the former situation is cystic fibrosis in which increased Na+-channel
activity results in viscid secretions because of excessive salt and
water reabsorption from the alveolar spaces. Agents inhibiting Na+ transport by the lung
epithelium would have a beneficial role in cystic fibrosis, and
aerosolized amiloride has been employed with some benefit. Our
observation that GSNO inhibits amiloride-sensitive Na+ transport in the lung points
to a novel role for this compound with its potential bronchodilator,
antimicrobial, and vasoregulatory properties. However, our study
suggests that lung conditions accompanied by pulmonary edema could
potentially be worsened by NO treatment. This is especially important
if the lung edema is not associated with pulmonary hypertension.
Because cation channels with characteristics similar to those reported
in this paper have been reported from a variety of tissues, as has the
ability to produce NO locally, our results could have a greater general
significance.
Lung epithelial cation channels. A
complete understanding of the role of NSC channels in the physiology of
lung water has yet to be achieved. Our study examined a 20.6-pS NSC
channel recorded from apical cell-attached patches of AT II cells in
primary culture. When grown under the conditions described in
METHODS AND PROCEDURES, this was the
predominant cation-permeable channel in AT II cells. The presence of
NSC channels in alveolar epithelial cells has been shown by several
investigators (7, 18, 24). Orser et al. (24) studied fetal distal lung
epithelial cells from 20-day-gestation rat fetuses cultured on
collagen-coated coverslips. Using symmetrical solutions and inside-out
recording, the investigators observed single channels with a
conductance of 23 ± 1.1 pS and an
Na+-to-K+
permeability of 0.9. These channels were blocked by amiloride applied
to the apical side of the membrane. Marunaka (17) described an NSC
channel with a linear
I-V
relationship and a single-channel conductance of 26.9 ± 0.8 pS in
the fetal distal lung epithelium. Feng et al. (7) recently described a
similar NSC channel observed in apical cell-attached and inside-out
patches from rat AT II cells. Like the channels observed by us, these
channels are nonselective (Na+-to-K+
permeability = 1), voltage independent, and inhibited by amiloride. A
wide variety of single-channel properties has been reported for
amiloride-sensitive cation channels, including single-channel conductances ranging from 1 to over 50 pS (7, 11). It has been proposed
that different combinations of the various subunits comprising the
channel (
,
, and
) could produce channels with varying unitary
conductances (3, 11, 31). Kizer et al. (13) recently showed that
expression of the
-subunit of the epithelial
Na+ channels from osteoblasts into
a null cell line (LM TK
)
resulted in an NSC channel
(Na+-to-K+
permeability = 1.1 ± 0.1) and a conductance of 24.2 ± 1.0 pS. Alternatively, the conductance could reflect the ionic conditions and
membrane composition in the tissue, which determine the physical state
of the membrane (11). We have also observed considerable variability in
the Po of these
channels. Such variabilty has also been observed in single-channel
recordings of amiloride-sensitive channels in cultured
Xenopus renal cells, human
lymphocytes, rat osteoclasts, and rat colonic epithelial cells. The
exact physiological role for these NSC channels is unclear, although
Tohda et al. (30) showed that these channels may play a role in the
increased reabsorption of fluid by alveolar epithelia in response to
-agonist stimulation.
Effect of NO on apical NSC channels.
The inhibition of NSC channels by NO suggests that NO may play a role
in the regulation of alveolar fluid and edema formation. The inhibitory
effect of NO on NSC channels is consistent with studies by Stoos et al. (29), who showed that NO inhibits
Na+ reabsorption in the isolated
cortical collecting duct, and Koivisto and Nedergaard
(14), who found that NO donors block NSC channel activity in rat brown
adipose tissue. Compeau et al. (4) showed that endotoxin-stimulated
alveolar macrophages impair distal lung epithelial ion transport by
inactivating amiloride-sensitive NSC channels. These investigators
showed a 60% reduction in amiloride-sensitive short-circuit current
and a 60% decrease in the density of 25-pS NSC channels on the apical
membrane of epithelium exposed to endotoxin and macrophages. This
effect was blocked by
NG-monomethyl-L-arginine,
suggesting an NO effect. These studies are in contrast to studies by
Guidot et al. (8), who used isolated perfused rat lungs to show that
inhaled NO prevents a neutrophil-mediated, oxygen radical-dependent
leak in isolated perfused rat lungs. The investigators reported a
modest reduction in pulmonary arterial pressure 30 min after NO
exposure but felt that NO prevented an oxygen radical-dependent leak in
the lungs. The question of whether NO increases or decreases the
propensity for pulmonary edema is yet to be resolved. It is possible
that in in vivo studies where pulmonary hypertension is contributing to
pulmonary edema formation, inhaled NO may act by reducing the
hydrostatic pressure and hence alveolar fluid formation. In situations
where pulmonary vasoconstriction is not a major player and in vitro, NO
appears to worsen pulmonary edema by impeding epithelial ion transport.
NO may also have an effect on
Na+-K+-ATPase,
but we have not addressed this in our study. The answer to these
questions is important because inhaled NO is currently undergoing
clinical trials in a variety of lung disorders.
The effects of GSNO and SNAP are generally attributed to the release of
NO. The fact that two biochemically different but specific NO-releasing
compounds, GSNO and SNAP, were equipotent in their effect on the NSC
channel suggests a common mechanism of action. In this study, GSH
(carrier of NO in GSNO) did not affect the NSC channels, suggesting
that GSNO was acting via release of NO (3). We were able to demonstrate
that the GSNO effect can be reversed by washout. One puzzling
observation was the apparent stimulating effect of washout on patches
previously exposed to GSNO. Possible mechanisms for this phenomenon may
include suppression of endogenous NO and/or cGMP production by
exogenous GSNO. Once the inhibitory effect of GSNO was washout, there
was an increase in channel activity attributable to the lower level of
endogenous inhibition.
The concentration of NO donors used in this study is higher than the
range of NO concentrations encountered in the physiological state (10).
However, because NO has a short half-life and needs to diffuse inside
the cell for its action, the actual effective concentration of NO
inside the cell may have been lower than the donor concentration used.
Ichimori et al. (10) found that 100 µM SNAP generated a stable
concentration of 0.1 µM NO at 25°C, a concentration that is well
within the physiological range.
The interval histograms are consistent with a minimum model of the NSC
channel that has one short-duration and one long-duration open state
and two similar closed states. Such a model can be represented by the
following kinetic scheme
The
simplest interpretation of the results is that GSNO produces an overall
change in the rate constants of the model above that favors a shift in
the equilibrium toward the closed states on the left side of the
equation. This idea is consistent with the decrease in the number of
long-duration open events, an increase in the number of short-duration
open events, and a significant increase in the long-duration closed
events (Fig. 6).
NO acts via cGMP-dependent activation of a
PK. Guanylate cyclase stimulation is believed to be
responsible for many of the physiological and pathological effects of
NO (21). We hypothesized that NO was acting on NSC channels via a
guanylate cyclase-mediated increase in cGMP. We found that a permeable
analog of cGMP (8-BrcGMP) had a virtually identical effect on NSC
channels as did GSNO and SNAP. To further examine the role of cGMP in
the inhibition of NSC channels, we utilized MeB to block soluble
guanylate cyclase. We found that MeB abolished the effect
of NO, suggesting that the inhibitory effect of GSNO on NSC channels in
AT II cells is largely mediated by cGMP.
These studies show that NO acts on NSC channels via a cGMP-dependent
mechanism. This is clear from the fact that cGMP analogs mimic and MeB
blocks the action of NO. Furthermore, AT II cells respond to NO by
production of cGMP. These findings are consistent with other studies
(4, 5, 31) that have suggested a role for intracellular second
messengers as modulators for ion-channel activity. Rocha and Kudo (26)
showed that, in the kidney, hormones such as atrial natriuretic factor
that increase cGMP levels result in inhibition of
Na+ reabsorption. Light et al.
(16) confirmed this with patch-clamp studies in which they showed that
cGMP inhibits cation channels both directly and through a
cGMP-dependent PK. We have also shown that cGMP acts via activation of
PKG. It is possible that NO may have additional effects through the
tyrosine kinase pathway, or the G protein-coupled receptor, via release
of cytokines or other second messengers. The physiological regulation
of epithelial Na+ channels appears
to be complex because, in addition to the pathway discussed, channel
activity is also modulated by methylation, arachidonic metabolites, and
interactions with the cytoskeleton (15, 16).
There is considerable indirect evidence that cGMP action on renal
epithelial Na+ channels is
mediated via activation of PKG, leading to phosphorylation of cation
channel or some related protein (5, 6). In the present study, we used
KT-5823, a blocker of PKG (12), to study whether the NO-cGMP-induced
inhibition of the channels was mediated by PKG. We found that KT-5823
blocked the effect of NO, suggesting a role for PKG in the observed
effect of NO on the cation channels. It is possible that higher
concentrations of KT-5823 may inhibit other PKs, which can then
affect the channel under study or other related proteins
(32).
Other mechanisms may contribute, in part, to the observed inhibition of
NSC channels. These include a direct cGMP effect on the membrane and a
phosphodiesterase (PDE)-mediated fall in cAMP levels because cAMP is
known to stimulate epithelial Na+
channels (23). To invoke this mechanism, one would assume that elevated
cGMP levels lead to an increase in PDE concentration that then lowers
the cellular cAMP concentration. Whether there is a role for PDE in the
NO-mediated inhibition of NSC channels is not clear.
Exposure of AT II cells to NO does not cause cell
death. It is possible that at the concentrations used
in this study, NO may be toxic to epithelial cells. We did not find any
difference in the viability of the cells after exposure of AT II cells
to be NO donors under the conditions used in our patch-clamp protocols. Furthermore, reversibility of NO effect after washout confirms that the
inhibition of NSC channels was not related to any permanent changes in
the cell and/or due to spontaneous "rundown" of channels in the patches being examined.
In summary, our study suggests that NO inhibits cation channels on the
apical surface of AT II cells via a cGMP-mediated action. This suggests
that NO may have a regulatory role in lung epithelial Na+ transport. We speculate that
pharmacological modalities, which act via this mechanism, may affect
lung Na+ and water transport.
Although NO or its donors may have a therapeutic role in patients with
cystic fibrosis, patients with preexisting lung edema need to be
closely monitored for worsening of edema when being treated with
inhaled NO.
 |
ACKNOWLEDGEMENTS |
We are grateful to B. Reynolds for assistance with preparation of
this manuscript.
 |
FOOTNOTES |
Support was provided by American Lung Association Award RG-133-N (to L. Jain) and National Institute of Diabetes and Digestive and Kidney
Diseases Grant DK-37963 (to D. C. Eaton).
Preliminary results were presented at the Society for Pediatric
Research meeting in Washington, DC, in May 1997 and the American Thoracic Society meeting in San Francisco, CA, in May 1997.
Address for reprint requests: L. Jain, Dept. of Pediatrics, Emory Univ.
School of Medicine, 2040 Ridgewood Dr., NE, Atlanta, GA 30322.
Received 14 April 1997; accepted in final form 11 December 1997.
 |
REFERENCES |
1.
Bland, R. D.,
and
C. A. Boyd.
Cation transport in lung epithelial cells derived from fetal, newborn, and adult rabbits.
J. Appl. Physiol.
61:
507-515,
1986[Abstract/Free Full Text].
2.
Brown, L. A. S.,
C. Bai,
and
D. P. Jones.
Glutathione protection in alveolar type II cells from fetal and neonatal rabbits.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L305-L312,
1992[Abstract/Free Full Text].
3.
Canessa, C. M.,
L. Schild,
G. Buell,
B. Thorens,
I. Gautschi,
J. D. Horisberger,
and
B. C. Rossier.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-467,
1994[Medline].
4.
Compeau, C. G.,
O. D. Rotstein,
H. Tohda,
Y. Marunaka,
B. Rafii,
A. S. Slutsky,
and
H. O'Brodovich.
Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1330-C1341,
1994[Abstract/Free Full Text].
5.
Eaton, D. C.,
A. Becchetti,
H. Ma,
and
B. N. Ling.
Cellular regulation of amiloride blockable Na+ channels.
Biomed. Res. (Tokyo)
12:
31-35,
1991.
6.
Eaton, D. C.,
A. Becchetti,
H. Ma,
and
B. N. Ling.
Renal sodium channels: regulation and single channel properties.
Kidney Int.
48:
941-949,
1995[Medline].
7.
Feng, Z.,
R. B. Clark,
and
Y. Berthiaume.
Identification of nonselective cation channels in cultured adult rat alveolar type II cells.
Am. J. Respir. Cell Mol. Biol.
9:
248-254,
1993[Medline].
8.
Guidot, D. M.,
M. J. Repine,
B. M. Hybertson,
and
J. E Repine.
Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs.
Am. J. Physiol
269 (Lung Cell. Mol. Physiol. 13):
L2-L5,
1995[Abstract/Free Full Text].
9.
Hummler, E.,
P. Barker,
J. Gatzy,
F. Beerman,
C. Verdumo,
A. Schmidt,
R. Boucher,
and
B. C. Rossier.
Early death due to defective neonatal lung liquid clearance in
-ENaC-deficient mice.
Nat. Genet.
12:
325-328,
1996[Medline].
10.
Ichimori, K.,
H. Ishida,
M. Fukahori,
H. Nakazawa,
and
E. Burahami.
Practical nitric oxide measurement employing a nitric oxide-selective electrode.
Rev. Sci. Instrum.
65:
1-5,
1994.
11.
Ismailov, I. I.,
M. S. Awayda,
B. K. Berdiev,
J. K. Bubien,
J. E. Lucas,
C. M. Fuller,
and
D. J. Benos.
Triple barrel organization of EnaC, a cloned epithelial Na+ channel.
J. Biol. Chem.
271:
807-816,
1996[Abstract/Free Full Text].
12.
Kase, H.
New inhibitors of protein kinases from microbial sources.
In: Biology of Actinomycetes 88: Proceedings of Seventh International Symposium on Biology of Actinomycetes, edited by Y. Okami,
T. Bappu,
and H. Ogawara. Tokyo: Japan Scientific Societies Press, 1988, p. 159-164.
13.
Kizer, N.,
X.-L. Guo,
and
K. Kruska.
Reconstitution of stretch activated cation channels by expression of the alpha subunit of the epithelial sodium channel cloned from osteoblasts.
Proc. Natl. Acad. Sci. USA
94:
1013-1018,
1997[Abstract/Free Full Text].
14.
Koivisto, A.,
and
J. Nedergaard.
Modulation of calcium activated non-selective cation channel by nitric oxide in rat brown adipose tissue.
J. Physiol. (Lond.)
486:
59-65,
1995[Abstract].
15.
Kokko, K. E.,
P. S. Matsumoto,
B. N. Ling,
and
D. C. Eaton.
Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron line (A6).
Am. J. Physiol.
267 (Cell Physiol. 36):
C1414-C1425,
1994[Abstract/Free Full Text].
16.
Light, D. B.,
J. D. Corbin,
and
B. A. Stanton.
Dual ion channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.
Nature
344:
336-339,
1990[Medline].
17.
Marunaka, Y.
Amiloride-blockable Ca2+-activated Na+-permeant channels in the fetal distal lung epithelium.
Pflügers Arch.
431:
748-756,
1996[Medline].
18.
Marunaka, Y.,
and
D. C. Eaton.
Chloride channels in the apical membrane of a distal nephron A6 cell line.
Am. J. Physiol.
258 (Cell Physiol. 27):
C352-C368,
1990[Abstract/Free Full Text].
19.
Matalon, S.
Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes.
Am. J. Physiol.
261 (Cell Physiol. 30):
C727-C738,
1991[Abstract/Free Full Text].
20.
Matthay, M. A.,
H. G. Folkesson,
and
A. S. Verkman.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L487-L503,
1996[Abstract/Free Full Text].
21.
Moncada, S.,
R. M. J. Palmer,
and
E. A. Higgs.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol. Rev.
43:
109-142,
1991[Medline].
22.
O'Brodovich, H.,
V. Hannam,
M. Seear,
and
J. B. M. Mullen.
Amiloride impairs lung water clearance in newborn guinea pigs.
J. Appl. Physiol.
68:
1758-1762,
1990[Abstract/Free Full Text].
23.
O'Brodovich, H.,
B. Rafii,
and
P. Perlon.
Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium.
Pediatr. Res.
31:
318-322,
1992[Abstract].
24.
Orser, B. A.,
L. Bertlik,
L. Fedorko,
and
H. O'Brodovich.
Cation selective channel in fetal alveolar type II epithelium.
Biochim. Biophys. Acta
1094:
19-26,
1991[Medline].
25.
Pepke-Zaba, J.,
T. W. Higgenbottam,
A. T. Dinh-Zuan,
D. Stone,
and
J. Wallwork.
Inhaled nitric oxide causes selective pulmonary vasodilatation in patients with pulmonary hypertension.
Lancet
338:
1173-1174,
1991[Medline].
26.
Rocha, A. S.,
and
L. H. Kudo.
Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F258-F268,
1990[Abstract/Free Full Text].
27.
Rossiant, R.,
K. S. Falke,
F. Lopez,
K. Slama,
U. Pison,
and
W. M. Zapol.
Inhaled nitric oxide for the adult respiratory distress syndrome.
N. Engl. J. Med.
328:
399-405,
1993[Abstract/Free Full Text].
28.
Scherrer, U.,
L. Vollenweider,
A. Delabays,
M. Savcic,
U. Eichenberger,
G. R. Kleger,
A. Fikrle,
P. E. Ballmer,
P. Nicod,
and
P. Bartsch.
Inhaled nitric oxide for high altitude pulmonary edema.
N. Engl. J. Med.
334:
624-629,
1996[Abstract/Free Full Text].
29.
Stoos, B. A.,
N. H. Garcia,
and
J. L. Garvin.
Nitric oxide inhibits Na+ reabsorption in the isolated perfused cortical collecting duct.
J. Am. Soc. Nephrol.
6:
89-94,
1995[Abstract].
30.
Tohda, H.,
J. K. Foskett,
H. O'Brodovich,
and
Y. Marunaka.
Cl
regulation of a Ca2+-activated nonselective cation channel in
-agonist-treated fetal lung epithelium.
Am. J. Physiol.
266 (Cell Physiol. 35):
C104-C109,
1994[Abstract/Free Full Text].
31.
Voilley, N. E.,
E. Lingueglia,
and
G. Champigny.
The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, autogenesis, and molecular cloning.
Proc. Natl. Acad. Sci. USA
91:
247-251,
1994[Abstract].
32.
Wahler, G. M.,
and
S. J. Dollinger.
Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase.
Am. J. Physiol.
268 (Cell Physiol. 37):
C45-C54,
1995[Abstract/Free Full Text].
AJP Lung Cell Mol Physiol 274(4):L475-L484
1040-0605/98 $5.00
Copyright © 1998 the American Physiological Society