Departments of Pediatrics and Physiology and The Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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We investigated the mechanism by which
cAMP increases sodium transport in lung epithelial cells. Alveolar type
II (ATII) cells have two types of amiloride-sensitive, cation channels:
a nonselective cation channel (NSC) and a highly selective channel
(HSC). Exposure of ATII cells to cAMP, -adrenergic agonists, or
other agents that increase adenylyl cyclase activity increased activity
of both channel types, albeit by different mechanisms. NSC open
probability (Po) increased severalfold when
exposed to terbutaline, isoproterenol, forskolin, or cAMP analogs
without any change in NSC number. In contrast, terbutaline increased
HSC number with no significant change in HSC Po.
For both channels, the effect of terbutaline was blocked by propranolol
and H-89, suggesting a protein kinase A (PKA) requirement for
-adrenergic-induced changes in channel activity. Terbutaline
increased cAMP levels in ATII cells, but intracellular calcium also
increased. Calcium sequestration with BAPTA blocked
-adrenergic-induced increases in NSC Po but
did not alter HSC activity. These observations suggest that
-adrenergic stimulation increases intracellular cAMP and activates
PKA. PKA increases HSC number and increases intracellular calcium. The increase in calcium increases NSC Po. Thus
increased cAMP levels are likely to increase lung sodium transport
regardless of which channel type is present.
single channel recording; ion transport; epithelial sodium
channels; amiloride; adenosine 3',5'-cyclic monophosphate; -adrenergic agents; alveolar type II cells
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INTRODUCTION |
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AMILORIDE-SENSITIVE
SODIUM absorption across the alveolar epithelium is believed to
be enhanced by cAMP and by agents such as -adrenergic agonists that
stimulate adenylyl cyclase. In the perinatal period, a rise in
endogenous catecholamines correlates with enhanced clearance of fetal
lung fluid. In postnatal life,
-adrenergic agents have a potential
use in limiting pulmonary edema that accompanies a variety of lung
conditions.
-Adrenergic agonists increase sodium reabsorption in
anesthetized fetal and adult animals (5, 13, 30, 40),
isolated rat lungs (10, 20, 37, 38), and cultured type-II
cells (9). The effects of
-adrenergic agonists may be
mediated by increases in intracellular cAMP, since other agents that
increase cAMP produce effects similar to
-adrenergic agonists
(9). However, Nakahari and Marunaka (28) have
proposed that the action of
-adrenergic agonists is mediated by a
cAMP-dependent increase in intracellular calcium that activates
calcium-dependent nonselective cation channels (NSC).
Despite this difference, the exact mechanism by which cAMP influences sodium transport in vivo is disputed, as is evident from
recent editorials and review articles from experts in this area
(21, 32, 43). Lazrak et al. (23, 24) have
postulated that
-adrenergic agents stimulate sodium absorption by
increasing the membrane conductance to sodium
(GNa). They based their opinion on single
channel studies, which showed that terbutaline increases the open
probability (Po) of a 25-pS moderately selective
cation channel (21). However, O'Grady et al.
(32) propose that the terbutaline-induced increase in
sodium transport is secondary to an increase in chloride conductance,
which in turn changes the apical membrane potential
(Va). In support of their hypothesis, O'Grady
et al. (32) cite studies with cultured alveolar epithelial cell monolayers that show increases in apical membrane chloride conductance in the absence of any change in sodium conductance after
stimulation by
-adrenergic agonists. However, as pointed out by
Widdicombe (42), neither hypothesis is adequately
substantiated, and additional studies need to be done to test both
hypotheses. Such studies are critical for lung biologists because of
the widespread use of
-adrenergic agonists in clinical medicine and
the potential therapeutic role for selective adrenergic agents in
several pathological states accompanied by pulmonary edema.
One explanation for such disparate results may lie in the differences in the experimental protocols followed by various investigators, especially culture conditions. Our studies have shown that culture conditions have a profound effect on the type of sodium conductance expressed on the apical surface of cultured alveolar epithelial cells (17). Specifically, when alveolar type II (ATII) cells are cultured under conditions when oxygen delivery to the cells is reduced or in the absence of steroid hormones, the predominant channel is a 21-pS NSC with a sodium-to-potassium selectivity ratio of 1:1. However, when ATII cells are grown on permeable supports in the presence of steroids with an air interface, the predominant channel is a low-conductance (6-pS), highly sodium-selective channel (HSC) with an sodium-to-potassium selectivity ratio >80:1. Both channels are amiloride sensitive so that, even though the half-maximal inhibition constant values are different, at a whole tissue level, the two channel types are difficult to distinguish based on amiloride sensitivity alone. Nonetheless, HSC, because of their high selectivity for sodium, should be much more effective as sodium transport pathways than NSC.
We hypothesized that signal transduction pathways mediated by cAMP and
protein kinase A (PKA) regulate the expression and activity of both HSC
and NSC, and the relative abundance of one channel over the other would
determine the net ability of ATII cells to transport sodium. To address
these issues, we used single channel studies to evaluate the effect of
-adrenergic agonists and cAMP on both NSC and HSC. One reason to
make single channel measurements is to be sure which channels are
responsible for transepithelial currents, especially because these
agents are known to stimulate chloride channels also. Our results show
that
-adrenergic agonists and cAMP increase the activity of both
types of channels, but increases in intracellular cAMP appear to
increase the Po of the 21-pS NSC, with no change
in the density of these channels, whereas cAMP produces no change in
the Po of HSC but does increase the density of
channels. In fact, cAMP appears to cause insertion of clusters of new
HSC in the apical membrane patches.
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MATERIALS AND METHODS |
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ATII cell isolation and culture. ATII cells were isolated by enzymatic digestion of lung tissue from adult Sprague-Dawley rats (200-250 g) using previously described methods (17). Briefly, the rats were anesthetized with pentobarbital sodium and heparinized (100 U/kg). ATII cells were digested by tracheal instillation of elastase (0.4 mg/ml). Lung tissue purification was based on differential adherence of cells to dishes coated with rat IgG. Nonadherent ATII cells were collected, centrifuged, and seeded on permeable supports in a highly enriched medium (3 parts Coon's modification of Ham's F-12 and 7 parts Liebovitz's L-15 with 1.5 µM aldosterone). After isolation, some cells were allowed to grow on glass coverslips, and others were allowed to attach to a specialized culture support (24), which is optimized for patch-clamp recording and allows the cells to grow on a permeable support (Millipore) while they are submerged in medium. After the cells had attached to the culture surface (this usually required 2-4 h), medium was drained from the apical surface, and cells were allowed to grow with medium on the basolateral surface and air on the apical side. Alternatively, cells were cultured in an identical fashion but without draining the medium so that cells remained submerged. Cells were incubated in 95% air and 5% CO2 and were used for patch-clamp studies between 24 and 96 h after plating.
Using this method for isolation of ATII cells, we were able to obtain an ATII cell population with 95% viability (confirmed by Live/Dead Eukolight Viability/Cytotoxicity Kit 1-3224; Molecular Probes, Eugene, OR) and 95% purity (confirmed by staining for surfactant proteins A and B; see Ref. 17). Contamination by macrophages and fibroblasts was <5%. Cells were used for patch-clamp experiments during the first 24-96 h in culture while they maintained ATII cell phenotype (light microscopy) and function (surfactant production using radiolabeled choline incorporation; see Ref. 17). The purity and viability of ATII cells were similar when cells were cultured on glass submerged in media and on a permeable support exposed to an air interface. ATII cells used in these experiments had lamellar bodies and other phenotypic features of type II cells and secreted surfactant (17). Bath and pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4, with 2 N NaOH. In the inside-out recordings, pipette solution was the same, but the bath solution was changed to (in mM) 5 NaCl, 140 KCl, 4, CaCl2, 5 EGTA, 1 MgCl2, and 10 HEPES, pH 7.4, with 2 N KOH. The contents of bathing and pipette solutions were varied as appropriate for specific protocols. All chemicals were obtained from Sigma (St. Louis, MO). All treatments (adrenergic agents, cAMP, etc.) were applied to the basolateral side of the cells, i.e., the bottom of the filter support.Single channel recording.
Patch-clamp experiments were carried out at room temperature. The
pipettes were pulled from filamented borosilicate glass capillaries
(TW-150; World Precision Instruments) 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 since, in this
configuration, the cytoplasmic constituents remain intact, thus
allowing us to study the role of cytoplasmic second messengers in
regulation of ion channel activity. After formation of a
high-resistance seal (>50 G
) between the pipette and cell membrane,
channel currents were sampled at 5 kHz with a patch-clamp amplifier
(Axopatch 200A; Axon Instruments, Foster City, CA) and were filtered at
1 kHz with a low-pass Bessel filter. Data were recorded by a computer
with pCLAMP 6 software (Axon Instruments). Current amplitude histograms
were made from stable, continuously recorded data, and the open and
closed current levels were determined from least-square fitted Gaussian
distributions. The Po of the channels was
calculated using FETCHAN in pCLAMP 6. Single-channel conductance was
determined using a linear regression of unitary current amplitudes over
the range of applied pipette potentials.
Single channel analysis.
We used the product of the number of channels (N) times the
single Po as a measure of channel activity
within a patch. This product was calculated without making any
assumptions about the total numbers of channels in a patch or the
Po of a single channel
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Measurement of intracellular cAMP levels. The intracellular cAMP level in ATII cells was measured by a Biotrak cAMP enzyme immunoassay (EIA) system (Amersham Life Science). After treatment with experimental agents, ATII cells were placed on ice, scraped, and homogenized and then 65% of ice-cold ethanol was used to extract cAMP from the cells. cAMP EIA is based on the competition between unlabeled cAMP and a fixed quantity of peroxidase-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody. With fixed amounts of antibody and peroxidase-labeled cAMP, the amount of peroxidase-labeled ligand bound by the antibody is inversely proportional to the concentration of added unlabeled ligand. The amount of peroxidase-labeled cAMP bound to the antibody is determined by addition of a tetramethylbenzidine/hydrogen peroxide single pot substrate. The reaction is stopped by addition of an acid solution, and the resultant color is read at 450 nm in a microtiter plate spectrophotometer. A standard curve was made for a range of cAMP concentrations from 12.5 to 3,200 fmol. The concentrations of unknown samples were determined by comparison with the standard curve. The cellular levels of cAMP were normalized to total cellular protein. Protein concentration was measured against a BSA standard using a Bradford dye-binding assay.
Measurement of intracellular calcium. To measure intracellular calcium levels, indo 1 fluorescence was measured with ATII cells grown on glass coverslips or filter supports. Cells were preincubated with 5 µM indo 1-AM (Molecular Probes) for 30 min at room temperature in darkness and then were washed with saline solution three times. Supports were then placed in a chamber secured to the stage of a Meridian ACAS 570/Ultima laser scanning confocal microscope (Laser Cytofluorimeter Working Station; Meridian Instruments). Cells loaded with indo 1-AM were excited by ultraviolet light at 351-364 nm, and the cytoplasmic free calcium signal was read as the fluorescence ratio at 405- and 530-nm wave lengths. Intracellular calcium concentrations were calculated by using the standard working curves of the fluorescence ratio vs. free calcium in calcium-EGTA buffer.
Statistical analysis. Statistical analysis for the changes in Po of channels and the biochemical estimations were performed using SPSS or SigmaStat for windows. Statistical significance between two groups was determined by paired or unpaired tests, as appropriate. When the comparison between more than one group was required, statistical significance was determined by one-way ANOVA followed by comparison of treated with untreated cells using Dunnett's test or pairwise comparisons with the Student-Newman-Keul's test to determine significant differences. Values < 0.05 were considered significant.
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RESULTS |
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Channel characteristics.
We have previously shown (15-17) that, when ATII
cells are under conditions of reduced oxygen delivery or in the absence
of steroids, the predominant sodium-permeant cation channel is a 21-pS
NSC with an sodium-to-potassium selectivity of 1:1, a linear current-voltage relationship, and a reversal potential near 0 mV.
However, when grown on permeable supports in the presence of steroids
with an air interface, the predominant channel is a 6-pS HSC with an
sodium-to-potassium selectivity of >80:1, moderate inward
rectification, and a reversal potential near +100 mV. Both channels are
amiloride sensitive. Details of single channel characteristics have
been described previously (17). In this study, we have focused our attention on the effect of -adrenergic agonists and the
cAMP-PKA pathway on these channels.
-Adrenergic agonists increase cellular cAMP levels in ATII
cells.
We measured the intracellular cAMP levels in ATII cells before and
after 15 min of exposure to agents known to increase cAMP levels in
other epithelial cells and found, as expected, that
-adrenergic
agents and forskolin increase cAMP levels in ATII cells from 111.8 ± 51.3 fmol/mg protein in untreated cells to 630.8 ± 275.2 fmol/mg protein in 20 µM terbutaline, 358.4 ± 121.6 fmol/mg
protein in 20 µM isoproterenol, and 323.6 ± 108 fmol/mg protein
in 20 µM forskolin (cAMP levels for each of the treatments was
significantly different from untreated cells; mean ± SD , n = 6 for all conditions). We added no
phosphodiesterase inhibitors in any of the experiments described in
this study, although addition almost surely would have increased the
magnitude of our responses. Thus the levels of cAMP and the responses
of sodium channels are what might be expected from normal physiological responses.
-Adrenergic agonists increase intracellular calcium levels in
ATII cells.
Although
-adrenergic agents are usually thought to act by increasing
cAMP with subsequent activation of PKA, there are several reports that
suggest that an additional effect of
-adrenergic agents is to
increase intracellular calcium in a variety of target cells (27,
28, 30). Therefore, we measured changes in intracellular calcium
in ATII cells in response to the addition of terbutaline (20 µM) and
found that terbutaline produced a significant increase in intracellular
calcium from basal levels of 118 ± 49.7 nM to a maximum
stimulated level of 909 ± 97.9 nM (peak calcium level after
terbutaline was significantly different from the basal level; mean ± SD, n = 6). The steady-state level in the continued
presence of terbutaline was 2.5- to 3-fold higher than baseline levels (293 ± 60.5 nM; Fig. 1).
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-Adrenergic agents increase the number of HSC without changing
the Po.
As mentioned above, when ATII cells are cultured on permeable supports
with an apical air interface, they express HSC in their apical
membranes. Under these conditions, examination of single channel
records from untreated ATII cells and cells treated with the
-agonist terbutaline showed that 20 µM terbutaline produced a
large increase in the number of channels observable in a typical cell-attached patch (Fig. 2). In 45 untreated cell-attached patches, we observed 2.6 ± 0.18 channels/patch (mean ± SE); after 10 min of exposure to 20 µM
terbutaline the mean number of channels per patch in 43 patches
increased to 6.0 ± 0.34. After 1 h of exposure, the mean
number of channels was lower but still elevated (4.7 ± 0.36, n = 45). Both values were significantly larger than the number of channels in untreated patches. In contrast,
Po of the channels in terbutaline-treated cells
(0.224 ± 0.0269 after 10 min and 0.188 ± 0.0268 after
1 h) did not change significantly from the
Po of untreated cells (0.182 ± 0.0202).
These results are summarized in Fig. 3.
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Effect of terbutaline on HSC is mediated by -adrenergic
receptors.
Terbutaline is usually considered to be a
-adrenergic agonist. To
test this, we applied 20 µM propranolol (a
-adrenergic receptor
antagonist) in the bath solution. Propranolol blocked the
terbutaline-mediated increase in channel number with no significant effect on channel Po. [The number of channels
per patch in 45 patches on untreated cells was 2.6 ± 0.175 and
increased to 6.0 ± 0.340 in 45 patches after 20 µM terbutaline
but was only 2.5 ± 0.143 when 25 patches were pretreated for 30 min with 20 µM propranolol and then treated with 20 µM terbutaline
(Table 1). The number of channels in
propranolol-treated patches is not different from untreated patches
(P > 0.05).]
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Increases in intracellular cAMP produce the same effect on HSC as
-adrenergic agonists.
One of the ways in which
-agonists produce their effect is through
activation of adenylyl cyclase with a subsequent increase in
intracellular cAMP. Indeed, we demonstrated that
-adrenergic agonists increase intracellular cAMP and that the same agonists increase channel density (Fig. 1). Therefore, we examined the effect of
cAMP on HSC activity. Application of a membrane-permeable analog of
cAMP, 8-(4-chlorophenylthio)cAMP (cpt-cAMP), produces the same increase
in HSC density as terbutaline, with no significant change in
Po. [The number of channels per patch in 26 patches on cells treated with 100 µM cpt-cAMP for 15 min was 5.5 ± 0.267 compared with 6.0 ± 0.340 in 45 patches after 10 min
exposure to 20 µM terbutaline (Table 1). The number of channels in
cpt-cAMP-treated patches is not different from terbutaline-treated
patches (P > 0.05).] These results imply that the
increase in HSC density depends on the
-adrenergic-mediated increase
in intracellular cAMP.
Increases in intracellular calcium are not associated with the
-adrenergic-induced increase in HSC density.
We show that
-adrenergic agonists increase intracellular cAMP. On
the other hand,
-adrenergic agonists also increase intracellular calcium (Fig. 1) so that the increase in channel density could also
depend on the increase in calcium. We tested this by adding the
membrane-permeable calcium chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM to inhibit the increase in intracellular calcium. Incubation with 1 mM BAPTA-AM for 30 min reduced intracellular calcium
in 30 cells to 78 ± 23.4 nM (compared with 118 ± 49.7 nM in
untreated cells), but addition of terbutaline increased calcium only to
108 ± 38.5 nM (compared with 909 ± 97.9 nM in untreated
cells). Despite the effect of BAPTA on intracellular calcium,
incubation with BAPTA-AM did not change the terbutaline-induced increase in HSC density [in 22 patches on cells pretreated with 1 mM
BAPTA-AM for 30 min, terbutaline still increased channel density to
5.8 ± 0.45 channels/patch, comparable to patches treated with
terbutaline alone and significantly greater than patches from untreated
cells (Table 1)].
PKA activity is necessary for the -adrenergic-induced increase
in HSC density.
The effects of increases in intracellular cAMP are often associated
with an activation of PKA. Therefore, we examined whether blocking PKA
activity reduced the effect of terbutaline on HSC density. If PKA was
blocked with the A kinase blocker H-89, terbutaline was no longer
capable of producing an increase in the density of HSC [in 19 patches
on cells pretreated with 5 µM H-89 for 30 min, terbutaline treatment
produced only 2.6 ± 0.16 channels/patch, which is not
significantly different from 45 untreated patches that had 2.6 ± 0.18 channels/patch (Table 1)]. These results taken together imply
that terbutaline increases the activity of HSC by increasing the number
of channels per unit area of membrane through a PKA-dependent process.
-Adrenergic agents increase the Po of NSC but do not
change the channel number.
-Adrenergic agonists also increased the activity of NSC in ATII
cells, but the mechanism was quite different from the effect on HSC.
Figure 4 shows, in a continuous recording
from a single cell-attached patch, that application of 20 µM
terbutaline dramatically alters the Po of NSC
with no change in the number of channels. In 17 cell-attached patches,
the Po before addition of terbutaline was
0.207 ± 0.0281 (mean ± SE), but after 15 min of exposure to 20 µM terbutaline, Po increased to 0.455 ± 0.0667, which is significantly larger than the
Po before terbutaline. In contrast, the number of channels per patch in terbutaline-treated cells (3.1 ± 0.90) did not change significantly from the number of untreated cells (3.3 ± 0.70). The results of these experiments are summarized in
Fig. 5. As expected, isoproterenol, a
drug known to activate both
1- and
2-receptors, also increased channel activity by increasing Po from 0.269 ± 0.0302 before
application to 0.515 ± 0.111 (n = 17, P < 0.01; Table 2).
There was no change in the number of channels in cell-attached patches
treated with isoproterenol (2.8 ± 0.29 in untreated cells vs.
2.7 ± 0.56 after isoproterenol), and single channel conductance
remained unchanged.
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Effect of terbutaline on NSC is mediated by -adrenergic
receptors.
Like HSC, the effect of terbutaline on NSC was also mediated through
-adrenergic receptors. Propranolol (20 µM) added to the
basolateral surface of a cell on which a cell-attached patch containing
NSC blocked the effect of terbutaline, but it did not change the basal
channel activity (the Po of untreated channels in nine patches is 0.248 ± 0.0160, 0.243 ± 0.0217 after 20 µM propranolol, and 0.235 ± 0.0164 after 20 µM
propranolol and 20 µM terbutaline together). The
Po of all of the treatments are not
significantly different, and the number of channels per patch are also
not different.
cAMP increases Po of NSC in ATII cells. In a cell-attached patch with NSC activity, addition of 100 µM cpt-cAMP caused a significant increase in NSC Po [increase from 0.139 ± 0.0411 to 0.5826 ± 0.0471, n = 10, P < 0.01 (Table 2)] with no significant change in channel density (untreated 2.9 ± 0.433 vs. 2.7 ± 0.47 after cpt-cAMP). Similar results were obtained with 1 mM 8-bromo-cAMP [8-Br-cAMP; Po significantly increased from 0.130 ± 0.0339 to 0.554 ± 0.0574, n = 9, P < 0.005 (Table 2)]. In another set of experiments, we pretreated ATII cells with 1 mM 8-Br-cAMP for 5 min before patch-clamp recordings, with a group of untreated cells serving as a control. Here also, pretreatment of cells with 8-Br-cAMP resulted in a significant increase in the number of channels times the Po (NPo; Table 2), with all of the increase resulting from an increase in Po (control NPo 1.053 ± 0.157 vs. 1 mM 8-Br-cAMP 1.819 ± 0.185, +73%, n = 24, P < 0.005). Further confirmation of the cAMP effect was obtained by the use of forskolin, an agent known to increase cellular cAMP by direct stimulation of adenylate cyclase. Treatment of cells with 20 µM forskolin added to the bath after stable cell-attached patches were obtained resulted in a significant increase in NSC Po [untreated 0.167 ± 0.0324 to 0.564 ± 0.0455 after treatment with 20 µM forskolin, n = 16, P < 0.001 (Table 2)]. These results suggest that an increase in cellular cAMP level stimulates NSC activity in apical membranes of ATII cells.
cAMP stimulation of NSC is mediated by PKA. To determine whether cAMP action on NSC is mediated via PKA, we pretreated cells with 5 µM H-89 for 5 min after we acquired control channel activity but before using any reagents to induce an increase in cAMP. We found (Table 2) that H-89 blocked the effect of 20 µM terbutaline to increase NSC Po. H-89 itself did not cause any significant change in the basal channel activity (untreated Po was 0.239 ± 0.0138 vs. H-89 0.227 ± 0.00919 vs. H-89 + terbutaline 0.213 ± 0.00635, n = 14, no significant differences between either treatments or control). In addition, 5 µM H-89 blocked the effect of 20 µM forskolin on NSC (untreated Po was 0.232 ± 0.0188 vs. H-89 0.228 ± 0.0132 vs. H-89 + forskolin 0.233 ± 0.0115, n = 14, no significant differences between either treatments or control). These results suggest that the effect of cAMP (and agents that increase cAMP) on NSC is mediated by PKA.
Increases in intracellular calcium are necessary for the
-adrenergic-induced increase in NSC Po.
We previously showed that
-adrenergic agonists increase
intracellular cAMP and that
-adrenergic agonists also increase
intracellular calcium (Fig. 1). However, an increase in the number of
HSC only requires an increase in cAMP and not an increase in calcium.
The increase in NSC activity is different; the increase in NSC
Po is associated with an increase in cAMP but
also depends on an increase in intracellular calcium. Incubation of
with 1 mM BAPTA-AM for 30 min (Fig. 6)
prevented the terbutaline-induced increase in NSC
Po (in 12 patches on cells pretreated with 1 mM
BAPTA-AM for 30 min the Po was 0.0926 ± 0.0333 and after application of terbutaline Po
was 0.115 ± 0.0519, which is not significantly different from
BAPTA alone). The terbutaline-induced increase in NSC
Po could require both an increase in
intracellular cAMP and calcium, or the increase in calcium could be a
response to the increase in cAMP, and the change in
Po could finally only require a change in
calcium. To investigate this question, we excised patches of membranes
containing NSC in a saline that mimicked the intracellular composition
(Fig. 6). In particular, it had 100 nM calcium but no cAMP. We then
increased the calcium to 1,000 nM and found that the
Po of the NSC increased substantially (in 19 cell-free, excised patches exposed to 100 nM calcium on their cytosolic
face the Po was 0.112 ± 0.0304 and after
application of 1,000 nM calcium Po was
0.459 ± 0.0504, which is significantly different from 100 nM
calcium, P < 0.001). We interpret these results to
mean that NSC are activated by intracellular calcium but that the
increase in intracellular calcium is dependent on an increase in
intracellular cAMP.
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DISCUSSION |
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In vivo and in vitro studies that use whole tissue measurements of
sodium transport indicate that -adrenergic agonists and cAMP
increase alveolar salt and water reabsorption. However, the underlying
mechanism is unclear and is the subject of an ongoing debate. In this
study, we have used single channel measurements to evaluate the effect
of
-adrenergic agonists and cAMP on the sodium channels in ATII
cells. This approach allows us to unambiguously identify the effect of
-adrenergic agonists on specific channels in ATII cells. As
previously reported, we have identified two different types of
amiloride-sensitive sodium-permeable channels in ATII cells
(15-17), a 20-pS NSC and a 6-pS HSC. The single
channel characteristics of the NSC are comparable to those described in both adult and fetal lung cells by several other research groups (11, 25, 35, 38). The HSC has single channel
characteristics that are similar to those of channels seen when
-,
-, and
-epithelial sodium channel subunits are reconstituted in
Xenopus oocytes (6) and that are observed in
other sodium-transporting epithelial cells (for a review, see Ref.
12). Our studies show that the
-adrenergic-cAMP-PKA
pathway has a direct regulatory role for sodium channels in ATII cells,
although the specific mechanism by which channel activity is increased
varies with the channel type. An increase in intracellular cAMP causes
an increase in Po of individual NSC in ATII cell
apical membranes without affecting their numbers; on the other hand,
cAMP increases the channel density or number of HSC without affecting
their Po. The exact role of these two channels
in different physiological and pathological states has yet to be
completely understood. However, our studies show that agents that
increase intracellular cAMP are likely to be effective in increasing
alveolar sodium transport regardless of which of these two channels is
present. Our results also clearly differ from those reported by Jiang
et al. (20), who could find no effect of
-adrenergic
agents on sodium transport in ATII cells and, indeed, could find no
sodium channels even though their cells were grown under conditions
similar to ours. We are somewhat at a loss to reconcile their results
with ours.
-Adrenergic agents and cAMP enhance active sodium absorption
across alveolar epithelium.
Vectorial transport of solutes between the alveolar surface and the
interstitial space is believed to play a key role in regulation of lung
salt and fluid balance. Several investigators, using different experimental approaches, have shown that this process can be stimulated by agents that increase cellular cAMP. Using an in vivo sheep model,
Berthiaume et al. (4) have shown that
-adrenergic
agents or cAMP analogs stimulate the removal of fluid from
saline-filled lungs, and this increase in fluid clearance can be
inhibited by amiloride. Similar results were obtained by Olver et al.
(33, 34) in the fetal lamb model. In monolayers of
alveolar epithelial cells, terbutaline has similarly been shown to
enhance amiloride-sensitive short-circuit current
(Isc), and an increase in transepithelial fluxes
of 22Na (9, 31). Even though the first of
these studies was published more than a decade ago, the mechanism of
action of
-agonists continues to be a subject of debate (23,
32). There are at least two broad mechanisms that have been
considered (21, 32, 43). In general, because the net
inward movement of sodium across the apical membrane is dependent on
GNa and the driving force for entry of sodium
into the cell (Va
ENa, where ENa is the equilibrium potential for sodium; see Ref. 42), cAMP could
be working by affecting GNa (24) or
Va. O'Grady et al. (32) have proposed that, instead of a direct effect on sodium channels, cAMP
works by increasing chloride conductance. This increase in cAMP-dependent inward movement of chloride leads to hyperpolarization of the apical membrane and generates a driving force
(Va) for movement of sodium from the alveolar
space into the cell. Their conclusion is based on studies involving
Isc and whole cell patch-clamp measurements
(18-20) in which they permeabilized basolateral
membranes of alveolar epithelial cells in monolayers using amphotericin B. In this preparation, in the presence of a chloride gradient, terbutaline was found to increase Isc, but
Isc was not increased by terbutaline when a
sodium gradient was imposed across the apical membrane. However, the
authors were unable to show an increase in amiloride-sensitive
Isc with terbutaline and did not conduct any
single channel studies.
Regulation of sodium-permeant channels by -adrenergic agonists
varies, depending on the type of channel being studied.
We have found that the effect of cAMP on cation channels varies with
the type of channel. Any treatment that increases intracellular cAMP
appears to increase the Po of 20-pS NSC with
little or no change in the density of channels on the apical surface of
epithelial cells. Similar regulation of NSC activity by
-agonists
has also been reported by other investigators. Yue et al.
(44) found that 10 µM terbutaline or PKA and ATP
increased Po and the mean open time without
affecting single channel conductance of the 27-pS NSC in ATII cells.
Marunaka et al. (27) and Tohda and Marunaka
(41) also demonstrated that terbutaline increases
Po of an amiloride-sensitive, calcium-activated,
chloride-inhibitable NSC in fetal rat alveolar epithelium by increasing
the mean open time without any significant change in the mean closed
time or in the single channel conductance. Senyk et al.
(39) and Berdiev et al. (3) showed that
addition of the catalytic subunit of PKA plus ATP to the presumed
cytoplasmic side of the lipid bilayers significantly increased
Po of NSC reconstituted into planar lipid bilayers. Virtually all reports of the effects of agents that increase
intracellular cAMP report an increase in the Po
of NSC. Occasionally, there have been reports that the number of
channels also increases (24, 44). The problem with these
observations is that, even if there were apparent changes in the number
of channels, these may be an experimental artifact, since counting current levels tends to underestimate the number. This is a particular problem if the Po is low (since channel openings
from multiple channels are unlikely to overlap). If the
Po increases significantly, then the probability
that multiple channels will open simultaneously increases
substantially, thus making it appear that the patch now has more
channels than before treatment. As one of us has established in
published work (22, 26), there are relatively straightforward statistical tests that can establish the confidence that one can place in estimates of the number of channels. We have used
them in this work, but such tests have not been commonly used
previously in the lung cell single channel literature, making any
conclusion that both the number of channels and
Po change unclear.
Molecular mechanisms underlying regulation of sodium channels by
-agonists.
As a first step toward elucidation of the mechanism of action of
-adrenergic agents, we looked at the effect of cAMP on sodium channels, since
-agonists are known activators of adenylyl
cyclase. Commercially available analogs of cAMP had a similar effect on NSC as terbutaline. Furthermore, forskolin, a direct stimulator of
adenylyl cyclase, increased the Po of NSC by
sevenfold. One of the ways by which cAMP produces its effects is by
stimulating PKA. H-89, a commercially available PKA inhibitor, blocked
the effect of terbutaline, implying a cAMP- and PKA-dependent
mechanism of action. H-89 also blocked the effect of forskolin. In our
studies, propranolol (a
-antagonist) abolished the terbutaline
effect, implying that the effect was mediated via activation of
-adrenergic receptors. The presence of both
1 and
2 receptors has been reported in vivo by
autoradiographic techniques (7). Taken together, these
findings point to a regulatory role for
-adrenergic agents in
regulation of lung sodium and water transport.
|
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Jain, Dept. of Pediatrics, Emory Univ. School of Medicine, 2040 Ridgewood Dr. NE, Atlanta, GA 30322 (E-mail: ljain{at}emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00356.2001
Received 11 November 2001; accepted in final form 26 November 2001.
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