Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Montreal, Quebec H2W 1T8; and Department of Medicine, Université de Montréal, Montreal, Quebec, Canada H3C 3J7
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ABSTRACT |
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It has been shown
that short-term (hours) treatment with -adrenergic agonists can
stimulate lung liquid clearance via augmented Na+ transport across alveolar
epithelial cells. This increase in Na+ transport with short-term
-agonist treatment has been explained by activation of the
Na+ channel or
Na+-K+-ATPase
by cAMP. However, because the effect of sustained stimulation (days)
with
-adrenergic agonists on the
Na+ transport mechanism is
unknown, we examined this question in cultured rat alveolar type II
cells.
Na+-K+-ATPase
activity was increased in these cells by
10
4 M terbutaline in an
exposure time-dependent manner over 7 days in culture. This increased
activity was also associated with an elevation in transepithelial
current that was inhibited by amiloride. The enzyme's activity was
also augmented by continuous treatment with dibutyryl-cAMP (DBcAMP) for
5 days. This increase in
Na+-K+-ATPase
activity by 10
4 M
terbutaline was associated with an increased expression of
1-Na+-K+-ATPase
mRNA and protein.
-Adrenergic agonist treatment also enhanced the
expression of the
-subunit of the epithelial
Na+ channel (ENaC). These
increases in gene expression were inhibited by propranolol. Amiloride
also suppressed this long-term effect of terbutaline and DBcAMP on
Na+-K+-ATPase
activity. In conclusion,
-adrenergic agonists enhance the gene
expression of
Na+-K+-ATPase,
which results in an increased quantity and activity of the enzyme. This
heightened expression is also associated with augmented ENaC
expression. Although the cAMP system is involved, the inhibition of
enhanced enzyme activity with amiloride suggests that increased
Na+ entry at the apical surface
plays a role in this process.
adenosinetriphosphatase; epithelial sodium channel; pulmonary edema; gene expression; sodium pump; terbutaline; alveolar epithelium
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INTRODUCTION |
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THE ALVEOLAR EPITHELIUM is considered to be not only an
important barrier to alveolar flooding but also the most likely site of
fluid reabsorption after pulmonary edema (29). This vectorial fluid
transport is dependent in part on active
Na+ transport across the alveolar
epithelium (29). Results obtained from alveolar epithelial cells in
culture suggest that Na+ enters
these cells by an apical Na+
channel (27) and is actively pumped out of the cells by basolateral Na+-K+-ATPase
(31). This active Na+ transport
can be modulated by -adrenergic agonists (6, 23, 40), probably via
the cAMP second messenger system, since cAMP analogs increase lung
liquid clearance in vivo (4, 22, 40) as well as
Na+ transport in alveolar type II
cells (24).
The exact mechanism of activation of
Na+ transport by -adrenergic
agonists is not fully understood. A paradigm can be developed based on
data available in the literature. We know that acute exposure of
alveolar type II cells to
-adrenergic agonists augments short-circuit current over a period of 20-30 min (16). This rise
in short-circuit current is secondary to heightened transepithelial Na+ transport (24), which is
probably caused by activation of the Na+ channel on the apical surface
of cells by protein kinase A (PKA; see Ref. 50). The increased activity
of the Na+ channel could lead to
an elevation in intracellular Na+
concentration, which could in turn stimulate
Na+-K+-ATPase
(38). Activation of
Na+-K+-ATPase
could also be secondary to direct or indirect stimulation of the enzyme
by cAMP (41). This model of enhanced transcellular Na+ transport could explain the
increased Na+ transport and lung
liquid clearance seen in animals (5, 6, 40) and humans (39) after acute
(1-4 h) exposure to
-adrenergic agonists.
However, it has been shown recently that septic animals presenting
enhanced lung liquid clearance also manifest heightened secretion of
endogenous catecholamines (36). In this model, we can expect the
alveolar epithelium to be exposed to an elevated circulating level of
catecholamines for a prolonged period of time. The impact of chronic
stimulation with -adrenergic agonists on the
Na+ transport mechanism in
alveolar epithelial cells is not known.
The purpose of this work was to study the effect of sustained exposure
(days) of alveolar type II cells to a -adrenergic agonist on the
Na+ transport mechanism and its
modulation. After determining the impact of long-term
-adrenergic
agonist treatment on
Na+-K+-ATPase
activity and transepithelial current, we tried to dissect the response
by evaluating the role of cAMP and establishing if it is associated
with enhanced expression of
Na+-K+-ATPase
and the epithelial Na+ channel
(ENaC). The possible involvement of
Na+ entry in this response was
then ascertained by studying the influence of amiloride.
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MATERIALS AND METHODS |
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Experimental Protocols
Effect of long-term exposure to terbutaline on
Na+-K+-ATPase
activity and transepithelial current.
To determine whether long-term treatment with -adrenergic agonists
modulates
Na+-K+-ATPase
activity and transepithelial current, we used three different experimental protocols. In the first experimental protocol, alveolar type II cells were exposed continuously to terbutaline
(10
4 M; Sigma, St. Louis,
MO) for 7 days starting from the day of cell isolation. The medium
containing terbutaline was changed every 2 days. Control cells were
treated in an identical fashion except that no terbutaline was present
in the media.
Na+-K+-ATPase
activity was measured by radiometric phosphate release assay after
day 0 (day of cell isolation; control:
n = 9; 60 min exposure to terbutaline:
n = 5), 2 days (control and
terbutaline: n = 6), 5 days (control:
n = 7; terbutaline:
n = 6), and 7 days (control and
terbutaline: n = 6) of treatment.
Modulation of
Na+-K+-ATPase
activity by cAMP.
To evaluate the role of the intracellular cAMP system in long-term
enhancement of
Na+-K+-ATPase
activity by terbutaline, cultured cell monolayers were exposed
continuously to 103 M
dibutyryl-cAMP (DBcAMP; Sigma) for 5 days starting from the time of
cell isolation.
Na+-K+-ATPase
activity was measured (n = 5) after 5 days of continuous treatment. To ascertain whether the long-term effect
of terbutaline on
Na+-K+-ATPase
involves activation of cAMP-dependent PKA, we measured PKA activity in
alveolar type II cells exposed to
10
4 M terbutaline.
Treatment with terbutaline started at the end of the cell isolation
period and lasted for 10 min (n = 4),
60 min (n = 6), or 24 h
(n = 7).
Effect of long-term exposure to terbutaline on
Na+ transporter
mRNA and/or protein expression.
To evaluate if long-term -adrenergic agonist treatment is associated
with changes in expression of the
Na+ channel and
Na+-K+-ATPase,
alveolar type II cells were exposed continuously to
10
4 M terbutaline for 6 days from the cell isolation period. The medium containing terbutaline
was changed every 2 days. On day 2 (n = 8), day
4 (n = 8), and
day 6 (n = 8) in culture, we determined mRNA
expression of the
1-subunit of
Na+-K+-ATPase
and of the
1-subunit of ENaC.
We also established if the changes in expression observed were related
to
-adrenergic agonist stimulation by continuously exposing cell
monolayers to either 10
5 M
propranolol alone (n = 8) or
10
5 M propranolol plus
10
4 M terbutaline
(n = 8) from the cell isolation period
for up to 2 days in culture. The mRNA expression of
Na+-K+-ATPase
and ENaC was determined by Northern blotting at the end of this
continuous 2 days of treatment.
Modulation by amiloride of increased
Na+-K+-ATPase
activity induced by -adrenergic stimulation.
To determine whether the long-term effect of terbutaline or DBcAMP on
Na+-K+-ATPase
activity was secondary to increased
Na+ uptake, cell monolayers were
treated continuously with either 10
4 M amiloride alone,
10
4 M amiloride plus
10
4 M terbutaline, or
10
4 M amiloride and
10
3 M DBcAMP continuously
for 5 days from the cell isolation period. At the end of the 5-day
treatment period,
Na+-K+-ATPase
activity was measured by phosphate release assay.
Methods
Cell isolation. Alveolar epithelial type II cells were isolated from male Sprague-Dawley rats weighing 175-200 g by enzymatic tissue digestion with elastase (Worthington Biochemical, Freehold, NJ) and purified by a differential adherence technique in rat IgG-coated bacteriological plastic plates (21). The cells were suspended in minimum essential medium (GIBCO, Burlington, Ontario, Canada) containing 10% fetal bovine serum (GIBCO), 0.08 mg/l gentamicin, 0.2% NaHCO3, 0.01 M HEPES, and 2 mM L-glutamine and then plated at a density of 4 × 105 cells/cm2 in plastic cell culture flasks (25 cm2) kept in a humidified 5% CO2-air incubator at 37°C.Na+-K+-ATPase
activity.
Na+-K+-ATPase
activity in alveolar type II cells was assessed as ouabain-inhibitable
ATPase hydrolysis under maximal velocity conditions by radiometric
monitoring as described previously (41). Exposure to the agents tested
was terminated on day 2,
day 5, and day
7 in culture by washing the cell monolayers with
ice-cold phosphate-buffered saline. The cells were scraped to obtain a cell pellet and centrifuged at 3,500 rpm at 4°C for 15 min. The cell pellet was resuspended in 250 µl of ice-cold homogenate solution (250 mM sucrose, 10 mM Tris, and 1 mM EGTA, pH 7.4) and then treated with a 15-s burst of sonication at 60 W on ice using an ultrasound cell
processor (Broun-Sonic, Allentown, PA). Whole cell homogenates containing 100-200 µg of protein were preincubated in a final volume of 1 ml of assay buffer (100 mM NaCl, 10 mM KCl, 5 mM
MgCl2, 4 mM ATP, 1 mM EGTA, 5 mM
sodium azide, and 50 mM Tris, pH 7.4) in the presence or absence of 1 mM ouabain for 30 min at 37°C. ATPase hydrolysis was then started
by adding ATP solution containing 5-10 µCi of
[-32P]ATP (ICN
Biochemicals, Montréal, Québec, Canada) as a tracer and
terminated every 3 min by placing aliquots of the assay mixture into an
ice-cold stop solution (1 M perchloric acid and 0.35 M NaH2PO4).
The release of Pi was detected as
Cerencov radiation after extraction of
Pi by activated charcoal
absorption.
Na+-K+-ATPase
activity was calculated as the difference between the slopes of
regression lines adapted to Pi
release in the presence or absence of ouabain. Data were standardized
to cellular protein content using the method described by Bradford
(10).
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RESULTS |
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Chronic stimulation with the -adrenergic agonist did not influence
monolayer formation. Alveolar type II cells in both the presence and
absence of 10
4 M
terbutaline spread to form confluent cell monolayers by
day 3 in culture. There were no
significant differences in cellular protein content on
day 2 and day
5 of culture for cells treated with terbutaline
compared with nontreated cell monolayers.
Na+-K+-ATPase
activity in cultured rat alveolar type II cells was augmented by
continuous exposure to the -adrenergic agonist. It increased in an
exposure time-dependent manner over 7 days in culture, when the cells
were kept in the presence of
10
4 M terbutaline (Fig.
1). This increase in
Na+-K+-ATPase
activity was also paralleled by a rise in transepithelial current
measured between day 4 and
day 6 of continuous treatment with
terbutaline (Fig. 2). At least 48 h of
treatment with the
-adrenergic agonist were necessary to stimulate
Na+-K+-ATPase
activity at day 5 in culture (Fig.
3).
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To examine the possible role of the cAMP system in this response, we
first determined if a permeable cAMP analog could also modulate
Na+-K+-ATPase
activity. After 5 days of continuous treatment with DBcAMP (103 M),
Na+-K+-ATPase
activity rose to 2.5 times the control value (Table
1). Furthermore, the percentage of
activated PKA increased to two times the control value when the cells
were treated continuously with
10
4 M terbutaline for 24 h
after cell isolation (Fig. 4). This rise in
PKA activity was not seen after 10 or 60 min of terbutaline treatment
of freshly isolated alveolar type II cells.
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We then determined if this sustained treatment with terbutaline is
associated with increased expression of the two systems involved in
Na+ transport in epithelial cells,
the -subunit of ENaC and the
1-subunit of
Na+-K+-ATPase.
-ENaC mRNA expression in cultured rat alveolar type II cells was
increased by continuous exposure to terbutaline
(10
4 M; Figs.
5 and
6A). Terbutaline
treatment also elevated the expression of the
1-subunit of
Na+-K+-ATPase
mRNA (Figs. 5 and 6B). The increased
expression of
-ENaC and
1-Na+-K+-ATPase
was inhibited by continuous cotreatment with terbutaline and
propranolol for 2 days, starting on the day of cell isolation (Figs. 5
and 7).
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This augmented activity and gene expression of
1-Na+-K+-ATPase
were also associated with heightened expression of
1-Na+-K+-ATPase
protein. The
1-subunit of
Na+-K+-ATPase
detected at 96 kDa increased in a time-dependent manner over 7 days in
culture when the cells were exposed continuously to
10
4 M terbutaline (Fig.
8).
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To determine the possible role of Na+ entry in modulation of this response, we studied the effect of amiloride. When amiloride was added to the medium with terbutaline or DBcAMP, the rise in Na+-K+-ATPase activity seen after 5 days of continuous treatment was inhibited (Table 1). There was also significant inhibition of terbutaline-increased transepithelial current with amiloride (Fig. 2).
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DISCUSSION |
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This study provides novel evidence that sustained -adrenergic
agonist stimulation can lead to enhanced
Na+-K+-ATPase
activity associated with increased expression of
Na+-K+-ATPase
and of the Na+ channel in alveolar
epithelial cells.
We first tried to determine the effect of sustained stimulation with
terbutaline on
Na+-K+-ATPase
activity. The continuous presence of terbutaline for up to 7 days
produces a gradual increase in
Na+-K+-ATPase
activity (Fig. 1) and transepithelial current (Fig. 2). This response
is mediated by cAMP, since it could be reproduced by long-term exposure
to the membrane-permeable cAMP analog DBcAMP (Table 1). The role of the
cAMP system is also suggested by the augmented activity of PKA, which
we recorded 24 h after initiating treatment with the -adrenergic
agonist (Fig. 4).
We thought it was important to evaluate the effect of long-term
exposure to the -adrenergic agonist on the
Na+ transport mechanism, even if
short-term modulation of the Na+
channel (50) and
Na+-K+-ATPase
(41) is relatively well established, because long-term treatment could
elicit not only changes in activity but also in the quantity of these
molecules. This is especially possible since the
-adrenergic agonist
has been shown to modify the expression of other genes (26, 45). We
then determined if the increased activity of
Na+-K+-ATPase
is associated with heightened gene expression of systems involved in
transepithelial Na+ transport. We
demonstrated that sustained stimulation with the
-adrenergic agonist
augmented the gene expression of ENaC, the Na+ channel that is responsible
for apical entry of Na+ into the
cell (Figs. 5 and 6). Expression of the
Na+-K+-ATPase
gene, the mechanism for Na+
extrusion on the basolateral side of the cell, was also enhanced by
long-term
-adrenergic stimulation (Figs. 5 and 6). This response was
mediated by
-receptors since it was inhibited by propranolol (Fig.
7).
Because the increase in gene expression was only transient, we also
wanted to determine if it was associated with a greater level of
expression of the protein. We only measured changes in 1-Na+-K+-ATPase
protein since we could not identify a functional ENaC antibody to work
in Western blotting of proteins obtained from cell lysates. We used a
monoclonal antibody against the
1-subunit of
Na+-K+-ATPase
that is actively involved in the transport mechanism (38, 42). The
increased expression of
1-Na+-K+-ATPase
protein (Fig. 8) suggests that the augmented activity measured on
days 5 and
7 is partially related to an elevation in
1-Na+-K+-ATPase
protein. However, the sustained increment of protein expression (Fig.
8) in the presence of transient changes in mRNA (Fig. 6) probably
indicates that other cellular mechanisms besides the rise in gene
transcription are involved in this response. This increased expression
of
Na+-K+-ATPase
protein probably also explains the persistent elevation in
Na+-K+-ATPase
activity on day 5, even when
terbutaline treatment was stopped on day
2 (Fig. 3). It would have been interesting to evaluate if there was also an increase in
-ENaC protein. However, technical difficulties prevented us from performing this experiment. Other laboratories have been able to measure changes in expression of a
Na+ channel protein in alveolar
type II cells of hyperoxic rats (49). The antibodies used by these
investigators were not directed against purified ENaC but were raised
against an Na+ channel protein
purified from the bovine kidney papilla. This polyclonal antibody
mainly recognizes a protein of 135 kDa in membrane vesicles of alveolar
type II cells (28). However, because it recognizes a protein that is of
a different molecular weight than
-ENaC (11), it could not be used
to determine if changes in
-ENaC mRNA are associated with an
increase in
-ENaC protein. Furthermore, we did not determine if
stimulation by the
-adrenergic agonist was associated with an
increase of other ENaC subunits (
and
) or with an elevation in
the
-subunit of
Na+-K+-ATPase.
We decided to focus on the
-subunits of ENaC and
Na+-K+-ATPase
because they represent the most important subunits for the activity of
these proteins (12, 42).
Because the increase in mRNA of ENaC and
Na+-K+-ATPase
as well as the changes in
Na+-K+-ATPase
protein were relatively modest, their physiological significance needed
to be determined. To address this issue, we measured transepithelial current across monolayers treated continuously for 4-6 days with terbutaline. These treated cells showed a significant increase in
transepithelial current that could be inhibited by the continuous presence of amiloride. Our results suggest that this modest increase in
genes and protein expression had a significant physiological impact on
the monolayer. However, it is also interesting to note that changes in
transepithelial current,
Na+-K+-ATPase
activity, and protein level were persistent 5-6 days after the
initiation of treatment (Figs. 1, 2, and 8) at a time when the mRNA
levels of ENaC and
Na+-K+-ATPase
had returned to the value of nontreated cells (Fig. 6). These results
indicate that modulation by cAMP is not limited to a transcriptional
effect but could also involve some posttranslational modifications such
as changes in protein synthesis or the protein degradation rate. Hence,
although the data suggest that chronic stimulation with the
-adrenergic agonist leads to enhanced transport capacity of the
epithelium, further studies are needed to establish the mechanism
involved in this process.
Our results are quite novel, since no previous publications have
reported a role of -adrenergic agonists in modulating the gene
expression of proteins involved in
Na+ transport. However, chronic
stimulation with
-adrenergic agonists has been shown to enhance the
expression of other genes. Chronic exposure to
1- or
2-adrenergic agonists
stimulates the expression of the
3-receptor gene as well as
protein expression (45).
-Adrenergic stimulation also upregulates
the activity and protein expression of phosphodiesterases (26).
Furthermore,
-adrenergic stimulation has been demonstrated to
enhance myocardial actin gene expression (7). Thus, although
-adrenergic agonists have not been reported to modulate the
expression of Na+ transport
protein in the lung, they alter the gene expression of other proteins
by different mechanisms (7, 26, 45).
Because -adrenergic agonists have been shown to enhance the
expression of different genes (7, 26, 45), it is possible that what we
observed was a nonspecific effect of terbutaline. Although this
question was not specifically addressed in these experiments, more
recent preliminary work done by our group has revealed that, unlike the
heart muscle, continuous treatment with cAMP does not increase the
expression of
-actin in alveolar type II cells (20). However, it is
possible that continuous exposure to the
-adrenergic agonist leads
to the enhanced expression of other genes in alveolar type II cells.
This would not be surprising, since continuous treatment of type II
cells with the
-adrenergic agonist is probably similar to the
stimulation that could be observed in a stress situation. In fact,
chronic elevation of catecholamines probably triggers the expression of
multiple genes. Hence, the increased expression of
Na+ transport protein probably
represents only one system that responds to this stimulus.
To investigate the mechanism that could be involved in this response,
we decided to evaluate first the potential role of
Na+ entry. It has been
demonstrated that -adrenergic treatment leads to activation of the
Na+ channel (50). This would
result in increased Na+ uptake
(24) and would elevate intracellular
Na+ concentration, which would
stimulate
Na+-K+-ATPase
(38). Thus inhibition of Na+ entry
by amiloride could then suppress the rise in
Na+-K+-ATPase
activity measured on day 5. Amiloride
did indeed inhibit the increased activity of the enzyme (Table 1).
Because a significant portion of the elevation in activity seen on
day 5 is secondary to increased
Na+-K+-ATPase
protein expression, this suppression by amiloride indicates that
Na+ itself could be an important
modulator of
Na+-K+-ATPase
gene expression. Although it has been known for many years that chronic
elevation of intracellular Na+
leads to an increased number of pumps (8), recent data obtained by
Yamamoto et al. (47) suggest that this increase in the number of
Na+-K+-ATPase
could be related to stimulation of gene expression by a
Na+-responsive element present in
the
Na+-K+-ATPase
promoter. Hence it is possible that long-term treatment with
-adrenergic agonists increases
Na+ entry into cells, which could
result in higher intracellular Na+
concentration and stimulate
Na+-K+-ATPase
gene expression and activity. This hypothesis is, in fact, supported by
the study of Rokaw et al. (37), who showed that chronic stimulation of
Na+ entry leads to elevated
Na+-K+-ATPase
activity that persists after stimulation is stopped. The increased
activity they observed was also associated with a greater quantity of
Na+-K+-ATPase
on the cell surface. Thus these results confirm that modulation of
Na+ entry in cells can cause
changes in
Na+-K+-ATPase
expression. However, we have to be careful when interpreting experiments with amiloride. It has been suggested in the past that
amiloride could directly inhibit DNA and protein synthesis (25, 46).
This conclusion was reached in experiments performed on peripheral
mononuclear cells (46) and 3T3 cells (25). However, the authors did not
consider the possibility that changes in intracellular ion
concentration could have led to their observations. This is particularly important in mononuclear cells, since the presence of ENaC
has been demonstrated (1). Hence, it is possible that, in this cellular
model, amiloride inhibits Na+
entry into cells and that its effect is not nonspecific but related to
the suppression of Na+ entry.
Recent work suggests that the effect of amiloride is probably not one
of nonspecific toxicity. Yamamoto et al. (47), in experiments where
they induced increased
Na+-K+-ATPase
expression with ouabain, were unable to demonstrate inhibition of this
response with amiloride. If amiloride had a nonspecific toxic effect,
its presence should have inhibited the increase in gene expression seen
with ouabain. The study by Rokaw et al. (37) also supports the concept
that the effect of amiloride is not toxic. They reported that chronic
treatment of A6 cells with amiloride leads to a decrease in
short-circuit current and a reduced number of
Na+-K+-ATPase
pumps on the cell surface. A similar response was observed when apical
Na+ was replaced by
tetramethylammonium, a nonpermeable cation. Interestingly, when
amiloride was washed out, the authors noted a gradual recovery of
short-circuit current over 100 min. Again, such a recovery would not be
possible if amiloride had a toxic action. Thus, although we cannot
exclude the possibility that the effect of amiloride may be related to
nonspecific toxicity on protein synthesis, it is quite unlikely. In
this series of experiments, we have, however, not determined if
amiloride had an impact on
-ENaC expression. Further studies are
needed to establish if there is also a modulation of ENaC expression by
Na+.
However, the effect of the -adrenergic agonist could also be
explained by other mechanisms. One of these possible mechanisms is that
the increased activity of PKA, measured after 24 h of stimulation,
leads to phosphorylation of cAMP-responsive element binding protein
(9). This protein could then bind to the cAMP-responsive element (CRE)
site on the gene promoter, which then causes activation of the gene
transcript (9). Such a mechanism is possible for the
Na+-K+-ATPase
pump since the presence of a CRE has been demonstrated in the promoter
region (2). Although we do not know if there is a CRE in the promoter
of
-ENaC, a putative CRE has been found in the 5'-flanking
region of
-ENaC (44). Other cAMP-independent pathways could also be
involved in this response. For example, in the heart, chronic
stimulation with
-adrenergic agonists leads to an increase in actin
mRNA that is mediated by G protein-coupled calcium channel activation
(7). Although we would like to propose that the observed effect of the
-adrenergic agonist is mediated by changes in
Na+ entry into cells, we cannot
exclude the possibility that other regulatory mechanisms, such as CRE
and calcium, are involved in this process.
Although these results suggest that continuous stimulation with
-adrenergic agonists could increase
Na+-K+-ATPase
activity, it is well known that this causes desensitization and
downregulation of
-receptor function (3). We therefore wanted to
determine the minimal period of exposure to a
- adrenergic agonist
that would be necessary to stimulate
Na+-K+-ATPase
activity. Alveolar type II cells were exposed to terbutaline for 1, 2, or 5 days, and
Na+-K+-ATPase
activity was measured on day 5 in
culture. Two days of treatment with the
-adrenergic agonist were
sufficient to increase Na+-K+-ATPase
activity at day 5 in culture (Fig. 3).
This observation coupled with results demonstrating that PKA activity
is augmented at 24 h suggests that the response to the
-adrenergic
agonist occurs between days 1 and
2. The data indicate that a period of time is necessary for
-adrenergic receptors to regain their
function, since we could not demonstrate PKA activation at 10 and 60 min after cell isolation. The response observed is probably not due to
continuous receptor stimulation for 5 days but to transient stimulation
between days 1 and
2. Although
-receptors were
probably desensitized in our system, their transient stimulation may
have occurred before desensitization.
Overall, the data presented in this paper as well as the data that we
have published previously (41) suggest that there are at least two
different mechanisms by which -adrenergic agonists can modulate lung
liquid clearance. When cells or lung tissues are exposed for a short
period of time to
-adrenergic agonists (1-4 h), there is an
acute stimulation of the Na+
channel (50) and of
Na+-K+-ATPase
(41) that is related to an increase in intracellular cAMP
concentration. These modifications are not, however, associated with
any changes in the expression of the two proteins (41, 50). This
mechanism probably explains the results from experiments in which lung
liquid clearance was stimulated in animals exposed to terbutaline for a
few hours (6, 40). When cells or lung tissues are exposed for a more
prolonged period of time (days) to
-adrenergic agonists, not only is
the activity of the Na+ channel or
Na+-K+-ATPase
augmented but the expression of these genes is also increased. Furthermore, it would seem that intracellular
Na+ concentration could play a
modulatory role in this response. These results could possibly explain
the enhanced Na+ transport and
liquid clearance (30, 32, 36, 48) as well as the augmented
Na+ transport protein expression
(13, 33, 49) seen in hyperoxic or thiourea- or sepsis-induced lung
injury. This paradigm is interesting, but it is important to remember
that, in these models of lung injury, the stress response could lead to
changes in factors other than catecholamines. These other factors could
also be involved in the regulation of expression of these genes. For
example, hyperoxia itself is considered to be a potential stimulant of
Na+-K+-ATPase
expression (14). Corticosteroids, which could be released in response
to stress, have been shown to be important stimulants of
Na+ channel expression (15, 43) as
well as
Na+-K+-ATPase
in certain tissues (35) but not in the fetal lung (43). All of these
data suggest that modulation of
Na+ transport in vivo probably
depends on multiple mechanisms and that a better understanding of these
mechanisms could lead to new treatment strategies for pulmonary edema.
In summary, the present study demonstrates that sustained treatment of
cultured rat alveolar type II cells with a -adrenergic agonist
enhances the gene expression of
Na+-K+-ATPase,
which results in an increase in the quantity and activity of the
enzyme. This enhanced expression is associated with an increased
expression of
-ENaC. Although the cAMP system is involved in this
response, the inhibition of increased enzyme activity with amiloride
suggests that augmented Na+ entry
at the apical surface plays a role in the process.
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ACKNOWLEDGEMENTS |
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Y. Minakata and S. Suzuki contributed equally to this paper.
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FOOTNOTES |
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This work was supported in part by the Medical Research Council of Canada (Grant MT-1203) and by the Canadian Cystic Fibrosis Foundation.
S. Suzuki is the recipient of a fellowship from the Canadian Lung Association, and Y. Berthiaume is a Chercheur-Boursier Clinicien from Fonds de la Recherche en Santé du Québec.
Address for reprint requests: Y. Berthiaume, Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Campus Hôtel-Dieu, 3850 St. Urbain, Montréal, Québec, Canada H2W 1T8.
Received 18 July 1997; accepted in final form 3 April 1998.
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