Departments of 1 Medicine and 4 Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati 45267, 3 Department of Zoology, Miami University, Oxford 45056; 5 Department of Pathology, Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2 Cardiovascular Research Institute, University of California, San Francisco, California 94143
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ABSTRACT |
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Clearance
of edema fluid from the alveolar space can be enhanced by endogenous
and exogenous -agonists. To selectively delineate the effects of
alveolar type II (ATII) cell
2-adrenergic receptors (
2-ARs) on alveolar fluid clearance (AFC), we generated
transgenic (TG) mice that overexpressed the human
2-AR
under control of the rat surfactant protein C promoter. In situ
hybridization showed that transgene expression was consistent with the
distribution of ATII cells. TG mice expressed 4.8-fold greater
2-ARs than nontransgenic (NTG) mice (939 ± 113 vs.
194 ± 18 fmol/mg protein; P < 0.001). Basal AFC
in TG mice was ~40% greater than that in untreated NTG mice (15 ± 1.4 vs. 10.9 ± 0.6%; P < 0.005) and
approached that of NTG mice treated with the
-agonist formoterol
(19.8 ± 2.2%; P = not significant).
Adrenalectomy decreased basal AFC in TG mice to 9.7 ± 0.5% but
had no effect on NTG mice (11.5 ± 1.0%).
Na+-K+-ATPase
1-isoform
expression was unchanged, whereas
2-isoform expression
was ~80% greater in the TG mice. These findings show that
2-AR overexpression can be an effective means to
increase AFC in the absence of exogenous agonists and that AFC can be
stimulated by activation of
2-ARs specifically expressed
on ATII cells.
G protein; adenylyl cyclase; -agonist; mouse; pulmonary edema
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INTRODUCTION |
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THE ALVEOLAR EPITHELIUM FORMS a critical barrier against the movement of water into the alveolar space and plays an important role in the active removal of excess alveolar fluid and protein. This reabsorption of excess fluid is driven by the vectorial transport of sodium ions across the alveolar epithelium (reviewed in Ref. 21). Sodium ion uptake probably occurs primarily across alveolar type II (ATII) cells where it enters the cell through amiloride-sensitive and amiloride-insensitive channels on the apical surface (20). Sodium ions are then actively pumped from the basolateral surface of the ATII cell by Na+-K+-ATPase (28). Clearance of alveolar fluid (AFC) results from the passive movement of water that subsequently follows the active transport of sodium ions and perhaps chloride (17).
Endogenous catecholamines and exogenously administered
-agonists can accelerate the removal of excess fluid from the
alveolar space. These effects have been observed in vivo for several
different mammalian species (2, 3, 12) and ex vivo in
human resected lung (34, 35). It has also been shown in
animal models that the enhanced clearance of pulmonary edema fluid
after neurological insult (16), hemorrhage (27,
30), and sepsis (31) is dependent on the release of
endogenous catecholamines. Activation of the
2-adrenergic receptor (
2-AR) on alveolar
epithelial cells may therefore serve a protective function that limits
alveolar flooding and enhances its resolution. This has led to the
proposal that
-agonist therapy could be a possible treatment for
patients with acute pulmonary edema (21, 40).
The effects of -agonists on lung function in intact animals are
potentially confounded by the multiple cell types that express
2-ARs. The receptor is expressed on airway epithelium
and smooth muscle and vascular endothelium and smooth muscle as well as
on one or more cell types that line the alveolus (6, 10).
The cell-specific effects of
2-AR activation on AFC,
particularly those of the vasculature and alveolar epithelium, may
therefore be difficult to distinguish when functional studies are being done. Recently, McGraw et al. (24) and others
(25, 39) have shown that
2-AR
overexpression can stimulate the signaling cascade in vivo by
increasing the pool of spontaneously activated receptor and/or
enhancing the sensitivity to endogenous agonists. Receptor activation
can be limited to a specific cell type via transgenesis by directing
expression with a cell-specific promoter, thereby permitting the in
vivo effects of
2-AR signaling in a given cell to be
differentiated from those of other cell types within the same
environment (24). In the present study, we generated
transgenic mice that selectively overexpressed the
2-AR
in ATII cells. Our goal was to distinguish the effects of
2-AR activation in ATII cells on AFC in intact mice from
those of other cells in the alveolus and to delineate a mechanism by
which this effect might occur. In addition, potential untoward effects
of such overexpression on lung development and function were assessed.
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METHODS |
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Transgenic mice.
Expression of the human 2-AR was directed to ATII cells
with the promoter from the rat surfactant protein (SP) C gene
(14). To construct the SP-C-
2-AR transgene,
a 3.7-kb HindIII-HindIII fragment from the rat
SP-C promoter (a gift from Dr. J. Whitsett, Children's Hospital
Medical Center, Cincinnati, OH), a 1.5-kb HindIII-PshAI fragment encoding the human
2-AR open reading frame (ORF), and a 0.85-kb
XhoI-BamHI fragment encoding the SV40 small t
intron and polyadenylation signal were ligated together in the vector
pUC18. The orientation of each fragment was confirmed by sequence
analysis and restriction enzyme digestion. The 6.05-kb transgene was
released from the vector by NotI digestion, gel purified,
and dialyzed against 5 mM Tris · HCl (pH 7.4) and 0.1 mM EDTA.
The purified DNA was then injected into fertilized eggs of FVB/N mice,
and the eggs were implanted into pseudopregnant females with methods
previously described (39). Founder mice were identified by
Southern blot analysis of genomic DNA prepared from tail clips.
Independent lines of heterozygous SP-C-
2-AR mice were
maintained by mating the transgenic mice with nontransgenic FVB/N mice.
Subsequent screening for the heterozygous progeny was by PCR analysis
of the genomic DNA with a forward primer from the
2-AR
ORF (5'-GGAGCAGAGTGGATATCACG-3') and a reverse primer from the SV40
polyadenylation region (5'-GTCACACCACAGAAGTAAGG-3'). Heterozygous mice
from generations 2 to 5 between the ages of 8 and
16 wk were used for all studies.
Transgene expression and localization.
To assess transgene expression among the independent transgenic lines,
RNase protection assays (RPAs) were performed with a
32P-labeled antisense riboprobe corresponding to the distal
500 bp of the human 2-AR ORF as previously reported
(23). McGraw et al. (24) have previously
shown that this probe does not recognize the endogenous mouse
2-AR transcript. Lung RNA was prepared from lungs with
TRI Reagent (Molecular Research Center, Cincinnati, OH). For the RPA,
RNA (20 µg) and the
2-AR riboprobe were hybridized overnight, digested with RNase, and subjected to denatured PAGE analysis. A radiolabeled antisense riboprobe for mouse
-actin was
included in each reaction to serve as an internal positive control and
to account for differences in gel loading. The gels were visualized
with a phosphorimager (Molecular Dynamics) and analyzed with the
ImageQuant software package (Molecular Dynamics).
Receptor density and adenylyl cyclase. Lung membranes were prepared from individual mice by homogenizing the entire lung or trachea in 10 ml of hypotonic lysis buffer (5 mM Tris, pH 7.4, and 2 mM EDTA) containing the protease inhibitors leupeptin, aprotinin, benzamidine, and soybean trypsin inhibitor (10 µg/ml each). The homogenates were centrifuged at 40,000 g for 10 min at 4°C. The supernatant was removed, and the pellets containing the crude membrane particulates were suspended in assay buffer (75 mM Tris, pH 7.4, 12.5 mM MgCl2, and 2 mM EDTA). Receptor expression was determined by radioligand binding with [125I]iodocyanopindolol as previously described (23).
Adenylyl cyclase activity in these membrane preparations was assessed with a column chromatography method as previously reported (22).AFC studies. The mice were killed by an overdose of pentobarbital sodium (200 mg/kg ip), and a 20-gauge trimmed angiocath plastic needle (Becton Dickinson) was inserted into the trachea. The lungs were then inflated with 7 cmH2O continuous airway pressure with 100% oxygen throughout the experiment. An infrared lamp placed 30 cm above the body was cycled on and off to maintain body temperature at 37°C. Body temperature was monitored by placing a temperature probe (Yellow Springs Instruments) into the abdominal cavity via a 0.5-cm incision. In some experiments, bilateral adrenalectomies were performed 8-12 h before the AFC studies.
AFC was measured as previously reported (2, 9, 12, 19). The lungs were instilled with Ringer lactate containing 5% bovine serum albumin and 0.1 µCi of 131I-albumin (Merck-Frosst, Montreal, PQ) as an alveolar protein tracer. Osmolarity of the instillate was adjusted to 340 mosM, which we have shown to be isosmolar with mouse plasma (9). In some experiments, different concentrations of theWestern blot analysis.
The expression of Na+-K+-ATPase
1- and
2-isoforms in microsomal membrane
preparations was assessed by Western blot analysis as previously
reported (11). For each sample, whole lungs from four mice
were homogenized in 7 ml of buffer (250 mM sucrose, 30 mM histidine, pH
7.2, and 2 mM EDTA) with two 30-s bursts of a Polytron homogenizer.
Microsomes were prepared from these homogenates as previously reported
except that NaI treatment was omitted (13). For
Western blotting, the samples were incubated for 30 min at 37°C in 50 mM Tris (pH 6.9), 5% SDS, 1%
-mercaptoethanol, and 10% glycerol.
Proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After
electrophoresis, the gels were transferred overnight to polyvinylidene
difluoride membranes. The blots were blocked for 1 h at room
temperature in 5 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 that
contained 1% blocking reagent (Boehringer Mannheim). The blots were
then incubated for 1 h at room temperature in 5 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 containing either the
Na+-K+-ATPase
1-isoform-specific
monoclonal antibody
6f (University of Iowa Developmental Hybridoma
Bank, Iowa City) or the
2-isoform-specific monoclonal
antibody McB2 (provided by Dr. Kathleen Sweadner, Massachusetts General
Hospital, Charlestown, MA). Afterward, the blots were washed
and then incubated with a peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch). Immunoreactivity was
visualized with an enhanced chemiluminescence system (Amersham Life
Sciences) and Kodak BioMax MR X-ray film. Exposure times were
individually optimized to provide the maximal signal intensity for each
isoform. The resulting signals were quantified by densitometry with
ImageQuant software (Molecular Dynamics). Two microsomal preparations,
each composed of four pooled lungs, were analyzed for each group. Each
blot contained multiple protein concentrations (30, 60, and 120 µg)
to ensure linearity of the signal.
Statistical analysis. Data are reported as means ± SE. Statistical comparisons between the nontransgenic and transgenic groups were performed with a two-tailed Student's t-test. Differences were considered significant at P < 0.05.
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RESULTS |
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Transgenic mice.
Southern blot analysis was used to screen for SP-C-2-AR
founder mice. Figure 1A shows
that the transgene was detected in 5 of 20 mice screened. To generate
transgenic lines, founder mice (designated 2.2,
3.1, 3.2, 4.3, and 5.2)
were mated with nontransgenic FVB/N mice. Transgenic progeny were
detected in all lines except those from founder 3.2. For
each transgenic line established, the transgene was inherited in
~50% of the progeny with an equal distribution between males and
females. Growth, development, and survival of mice from each transgenic
line were not different from those of nontransgenic littermates.
Histological examination of the lungs as well as of other organs from
SP-C-
2-AR mice was unremarkable.
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Transgene expression and localization.
To confirm that the SP-C-2-AR transgene was being
expressed in each transgenic line, total cellular RNA was subjected to RPAs. RNA was hybridized with an antisense riboprobe corresponding to
the distal 500 bp of the human
2-AR. McGraw et al.
(23) have shown that this probe is specific for the human
receptor and does not detect the mouse
2-AR transcript.
As shown in Fig. 1B, significant levels of the human
2-AR transcript were expressed only in the transgenic
lines established from founders 2.2 and 4.3.
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2-AR expression and adenylyl cyclase activity.
Having confirmed that the SP-C-
2-AR transgene was
expressed in the lung and that the pattern of mRNA expression was
consistent with the location of ATII cells, we next quantified
2-AR density by radioligand binding with
[125I]iodocyanopindolol. For these experiments, binding
was performed on membranes prepared from whole lung homogenates and is
expressed as femtomoles of receptor per milligram of membrane protein.
As shown in Fig. 3A,
2-AR density in membranes from nontransgenic mice was
194 ± 18 fmol/mg (n = 5). Receptor density in the
transgenic lines with undetectable transcripts was not different from
that in the nontransgenic lines (data not shown). In contrast,
2-AR density in mice from line 2.2, which had
the highest level of transcript expression, was nearly fivefold greater
than that in the nontransgenic mice (939 ± 113 fmol/mg;
P < 0.001; n = 5). Transgenic mice
from line 4.3, which expressed lower levels of transcript,
displayed a smaller but still significant increase in
2-AR density compared with the nontransgenic group
(349 ± 41 fmol/mg protein; P < 0.01;
n = 4). Subsequent studies were carried out with the
higher-expressing line 2.2.
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AFC studies.
To assess the in vivo effects of 2-AR overexpression and
activation in ATII cells, we measured AFC rates in nontransgenic and
SP-C-
2-AR mice using an in situ lung preparation
(9). As in a prior study by Ma et al. (19),
AFC was calculated from the increase in the concentration of an
impermeant volume indicator (131I-albumin) that occurred
over 15 min after instillation into the airspace. Figure
4A shows that the basal AFC
rate in SP-C-
2-AR mice was 38% greater than that in the
nontransgenic mice (10.9 ± 0.6 and 15.0 ± 1.4%;
P < 0.005). To assess whether increased AFC in the
transgenic mice was the result of an increase in the pool of
spontaneously activated receptors or an increased sensitivity to
endogenous catecholamines, mice in both groups were adrenalectomized. Adrenalectomy had no effect on basal AFC in nontransgenic mice (11.6 ± 0.9%). In contrast, basal AFC in the
SP-C-
2-AR mice was significantly reduced by
adrenalectomy (to 9.7 ± 0.2%), which was no different from AFC
in nontransgenic mice. Enhancement of AFC in SP-C-
2-AR
mice was therefore ameliorated by elimination of endogenous
catecholamines.
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Na+-K+-ATPase
1- and
2-isoform expression.
Previous studies (4, 26, 38) have suggested that
-agonists may increase Na+ transport, and thus AFC, by
increasing the activity of Na+-K+-ATPase. This
effect may result from increased production or translocation of
Na+-K+-ATPase subunits into the basolateral
membrane, in particular the
1-subunit isoform
(4). This finding, together with the observations that
1) the
-subunit contains the catalytic region of
Na+-K+-ATPase (18) and
2) the
1-isoform is also the most abundant
-isoform in the lung (29), prompted us to examine
whether expression of the
1-isoform was altered in the
lungs of SP-C-
2-AR mice. Using Western blot analysis
with a monoclonal antibody specific for the
1-isoform,
we found no difference in
1-isoform content in
microsomal membranes prepared from the lungs of nontransgenic and
SP-C-
2-AR mice (Fig. 5).
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DISCUSSION |
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-Agonists have been shown to stimulate AFC in a number of
different mammalian species, including studies of excised human lungs
(2, 3, 12, 34, 35). It has also been shown that endogenous
catecholamines increase AFC in animal models of pulmonary edema caused
by neurological insult (16), hemorrhagic shock (30), and sepsis (31). These
catecholamine-mediated increases in AFC were blocked by the
-antagonist propranolol, indicating that the
-AR signaling system
may have an important protective function that serves to prevent or
limit alveolar flooding and enhance its resolution. Activation of the
2-AR signaling cascade by agonists or other means could
therefore be a potential treatment for pulmonary edema as previously
suggested (40).
Recent studies have demonstrated that receptor overexpression can
be a useful method of activating 2-AR signaling pathways in a cell type-specific manner. For example, McGraw et al.
(24) found that the contractile response to methacholine
was markedly inhibited in transgenic mice that overexpressed the
2-AR in airway smooth muscle. In fact, these transgenic
mice were less reactive to methacholine than nontransgenic mice treated
with a
-agonist. Agonist-independent enhancement of cardiac function
has also been observed in transgenic mice that overexpressed the
2-AR in cardiomyocytes (25, 39). These
studies show that the activation of receptor signaling by
overexpression can effectively mimic agonist-mediated activation. This
overexpression strategy can further be used to distinguish the
physiological effects of
2-AR activation among different
cell types within a complex tissue or organ by using cell-specific
promoters to target expression. In addition, because desensitization
does not appear to be appreciable in these overexpressing mice, genetic
transfer may be superior to continuous high-dose agonist where
tachyphylaxis could limit effectiveness.
A primary objective of this study was therefore to determine whether
AFC could be persistently upregulated by overexpression of the
2-AR in ATII cells. Using the rat SP-C promoter to
direct expression to ATII cells, we generated transgenic mice that
expressed the
2-AR approximately fourfold over that of
their nontransgenic littermates. Physiological assessment of these
SP-C-
2-AR mice showed that their basal AFC rate was
~40% greater than that of the nontransgenic mice and was equivalent
to AFC rates in nontransgenic mice treated with the
-agonist
formoterol. The SP-C-
2-AR mice also displayed a trend
toward a further increase in AFC when they were treated with
formoterol, but the difference did not reach significance.
Nevertheless, these results show that overexpression of the
2-AR is as effective as short-term
-agonist treatment with regard to enhancing AFC in vivo.
The cell types responsible for 2-AR-mediated increases
in AFC have not been clearly elucidated in prior work, primarily
because agonists could not be delivered in a cell-specific manner. A
key role for the ATII cell has been supported by histological evidence that suggests that the ATII cell has the metabolic and anatomic potential to support the active transepithelial fluxes required for
driving fluid transport. Studies of isolated ATII cells have also
demonstrated the presence of basolateral
Na+-K+-ATPase, apical Na+ channels,
and functional
2-ARs (reviewed in Ref. 20).
However, ATI cells may also have
2-ARs on their surface
(5), and there is evidence to suggest that these cells
have Na+-K+-ATPase activity as well
(33). Activation of
2-ARs on other cells
lining the alveolus, such as vascular smooth muscle and possibly
vascular endothelium (6), could potentially alter vascular
capacity and additionally contribute to the effects of
-agonists on
AFC (10). By selectively targeting expression (and thus
receptor activation) to the ATII cell, we were able to eliminate the
potential confounding effects of
2-AR activation in
other cell types. With this strategy, we found that the increase in AFC
due to transgenic activation of
2-AR signaling in ATII cells alone was nearly the same as that of nontransgenic mice treated
with formoterol, an agonist that presumably acted on all cell types
lining the alveolus. The absence of any additional increase in AFC by
activation of receptors on these other cell types suggests that the
effects of
-agonists on AFC are mediated predominantly through their
effects on ATII cells. Still, because the ATII cell is the progenitor
of the ATI cell, it is possible that overexpression of the
2-AR is being maintained after differentiation. However,
previous work (36) has shown that the half-life of receptor turnover in vivo is ~18 h, and in situ hybridization studies with many transgenes directed by the SP-C promoter,
including those of the present work, showed that transgene expression
is limited to ATII cells (14). It is therefore unlikely
that persistent expression of the
2-AR transgene in
ATI cells occurred in the absence of ongoing transcriptional activity.
Different modeling techniques indicate that the increase in basal
(i.e., agonist-independent) receptor signaling that occurs with
2-AR overexpression may be due to a larger pool of
spontaneously active receptors (24, 25). However, in an in
vivo setting, the physiological effect of
2-AR
overexpression could be the result of enhanced sensitivity to
endogenous agonists (i.e., epinephrine). In measurements of isolated
lung membrane adenylyl cyclase activity, we observed a leftward shift
in the dose-response curve for agonist-stimulated adenylyl cyclase
activity in the transgenic mice but no increase in maximal agonist
activity. An increase in tachyphylaxis or desensitization is unlikely
to explain this finding because overexpression tends to attenuate
desensitization by increasing the number of receptors available for
interacting with agonists. Moreover, an increase in sensitivity without
a change in the maximal response is predicted by the current models in
the case of receptor overexpression, to the point where some other
component becomes the limiting factor in the maximal achievable
response (i.e., the spare receptor phenomenon) (1). Our
results thus support the concept that spare
2-ARs are
present in the peripheral lung.
To further confirm that heightened agonist sensitivity was the
mechanism underlying the in vivo gain of function in the
SP-C-2-AR mice, we performed adrenalectomies to minimize
the effects of endogenous catecholamines (5). Mice were
studied within 12 h of surgery to minimize the
non-catecholamine-mediated effects of adrenalectomy on AFC, such as
those resulting from the loss of corticosteroid production. Although
adrenalectomy had no effect on the basal AFC rate in nontransgenic
mice, AFC rates in the SP-C-
2-AR mice were significantly
reduced compared with those in the nontransgenic mice. The finding of
an effect only in the
2-AR overexpressors is further
consistent with catecholamine depletion as the primary mechanism of
adrenalectomy in this study. Taken together, these observations
indicate that the increased AFC rate in SP-C-
2-AR mice
was indeed the result of heightened sensitivity to endogenous catecholamines.
Having demonstrated that AFC could indeed be increased by
2-AR overexpression in ATII cells, we began to explore
the cellular mechanisms by which it occurred. Experiments performed on
cultured ATII cells have shown that
-agonists increase
Na+-K+-ATPase activity.
Na+-K+-ATPase is an integral membrane protein
localized to the basolateral membrane surface of ATII cells that
transports three sodium ions out of the cell and two potassium ions
into the cell for each molecule of ATP consumed (reviewed in Ref.
17). It is a heteromeric enzyme composed of a 97-kDa
-subunit and a 53-kDa glycosylated
-subunit. The
-subunit
contains both the cation and nucleotide binding sites and is the
catalytic component of the enzyme. The
-subunit is thought to
regulate heterodimer assembly and stability, trafficking to the
basolateral surface, and possibly the control of potassium ion
kinetics. The mechanism by which
2-ARs regulate Na+-K+-ATPase function is not entirely clear.
Previous work by Bertorello et al. (4) showed that
isoproterenol increased the amount of the
1-isoform, the
predominant Na+-K+-ATPase
-isoform in the
lung, in the basolateral membranes of cultured ATII cells. Because of
the rapid onset of the effect, their interpretation was that
-agonists increased Na+-K+-ATPase activity
by recruiting
1-subunits from an intracellular compartment to the basolateral surface. We therefore measured
1-subunit expression in lung microsomes with Western
blot analysis. Our results showed that
1-subunit
expression in SP-C-
2-AR mice was not different from that
in the nontransgenic mice. This finding is analogous to that of Suzuki
et al. (38), who found that the increase in
Na+-K+-ATPase activity caused by terbutaline
was not associated with changes in
1-subunit expression.
Our findings are, however, different from those of the former
investigators (4). This difference could be
partly due to our use of microsomal fractions derived from whole lungs
rather than isolated ATII cells, which may have caused us to
underestimate the changes that occurred specifically in these cells.
Despite these differences, recent data bring into question whether
increases in 1-subunit expression could account for
2-AR-mediated increases in AFC in vivo. Using
adenoviral-mediated gene transfer, Factor et al. (7) found
that in vivo delivery of the
1-subunit to rat lungs did
not result in an increase in AFC rates. In contrast, delivery of the
1-subunit increased both
Na+-K+-ATPase activity and AFC rates. Because
the
1-subunit does not possess catalytic activity, this
effect may have resulted from increased formation and recruitment of
active heterodimers. However, the absence of an effect by
1-subunit overexpression could indicate that the
increase in AFC due to
1-subunit overexpression resulted from interactions with other
-isoforms, although this possibility was not specifically addressed in their study (7). To
date, four different Na+-K+ ATPase
-isoforms
have been identified, with each having a unique tissue distribution
(18). Transcripts for the
2-isoform have been detected in RNA prepared from whole lung homogenates, but the
abundance is less than that of the
1-isoform
(29). Nevertheless, even this small amount of
2-isoform could have potential importance with regard to
physiological function. The ratio of
1- to
2-isoform expression in the heart is similar to that in
the lung (29), yet James et al. (11) showed
that the
2-isoform had a distinct role in regulating
heart contractility in vivo by using knockout mice that expressed
reduced levels of this isoform.
Given these observations, we considered the possibility that
2-AR activation in ATII cells could regulate AFC by
modulating Na+-K+-ATPase
2-subunit expression. Western blots of microsomal
fractions prepared from whole lungs showed that
2-isoform expression in the SP-C-
2-AR
mice was ~75% greater than that in nontransgenic mice. Given that
the
2-AR is not a secreted product and that in situ
hybridization showed a pattern of transgene expression limited to ATII
cells, the increase in
2-isoform content that we
observed was most likely the result of changes in ATII cell expression.
In light of the findings by James et al. (11)
demonstrating the physiological relevance of low-level
2-subunit expression in the heart and the previously
cited work by Factor et al. (7) showing that increased
1-subunit expression does not increase AFC, our findings
suggest the possibility that an additional mechanism of
2-AR-mediated increase in AFC may be due to regulation
of other Na+-K+-ATPase
-subunit isoforms.
However, activation of the
2-AR is necessary to elicit
this increase in AFC because loss of circulating epinephrine from
adrenalectomy ablated the effect. Of note, although an increase in the
expression of the Na+-K+-ATPase
2-subunit may play a role in
2-AR-mediated increases in AFC, we have not directly
assessed this. Studies to differentiate
2-subunit
function by oubain sensitivity and direct overexpression of the
2-subunit via transgenesis are necessary to confirm this link. The possibility that other components of ion transport, such as
the apical Na+ channel or Cl
channel, are
additionally upregulated in these mice must also be considered.
Of note, our data indicate that the increased basal AFC due to
2-AR overexpression in ATII cells likely results from
enhanced signaling to endogenous catecholamines. This is supported by
the leftward shift in agonist dose-response curves that was observed in
the in vitro assays of adenylyl cyclase activity. We must also consider, though, that second messenger pathways other than cAMP may
underlie
2-AR-mediated effects on AFC. Indeed, Suzuki et al. (38) observed that acute treatment of cultured ATII
cells with
-agonists produced increases in
Na+-K+-ATPase that could not entirely be
explained by changes in cAMP content. These findings, though, may have
been related to the short duration of
2-AR activation
because a later study (26) suggested a more
prominent role for cAMP-dependent processes when
-agonist exposure
was prolonged (i.e., 5-7 days). However, it is of interest to note
that tumor necrosis factor-
, which may actually lower cAMP, has been
shown to increase AFC (32). Furthermore, a number of
recent investigations have demonstrated that the
2-AR can couple to Ca2+-activated K+ channels
(15), Cl
channels (8), and the
Na+/H+ exchange regulator (37)
independently of cAMP. Such coupling could explain the upregulation of
basal AFC observed in the absence of increased adenylyl cyclase
activity and cAMP levels.
In summary, we have used a cell-specific promoter to overexpress the
2-AR in ATII cells of transgenic mice, thereby
permitting us to selectively delineate the effects of the ATII cell
2-AR from those of other cells in an in vivo setting.
Our findings demonstrate that overexpression of the
2-AR
in ATII cells can increase AFC as effectively as
-agonist treatment.
Transgenic activation of
2-AR signaling was associated
with upregulation of the Na+-K+-ATPase
2-isoform, suggesting, but not conclusively proving, a
potential role for this isoform in regulating AFC. Last, these findings
support the notion that activation of
2-AR signaling could be used as a therapeutic option to enhance the resolution of
pulmonary edema.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Caroline Halfter Spahn Trust Genetic Research Fund and National Heart, Lung, and Blood Institute Grants HL-03986, HL-41496, HL-55184, and HL-51856.
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FOOTNOTES |
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* Dennis W. McGraw, Norimasa Fukuda, and Paul F. James contributed equally to this work.
Address for reprint requests and other correspondence: S. B. Liggett, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0564 (E-mail: stephen.liggett{at}uc.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.
Received 10 October 2000; accepted in final form 1 June 2001.
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