Pediatric Pulmonary Division, University of Connecticut School of Medicine, Farmington, Connecticut 06030
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ABSTRACT |
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Corticosteroids enhance -adrenergic
responses by actions at both
-adrenoceptor (
-AR) and post-
-AR
sites. The present study investigated the effects of dexamethasone on
-AR density, high-affinity
-agonist binding, Gs
and Gi
protein expression, and cAMP responses in bovine
tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM
cells increased total
-AR density 1.6- to 1.9-fold as assessed by
the saturation binding of [3H]CGP-12177 and by
displacement of radioligand binding with isoproterenol. Isoproterenol
bound to the
-AR at two sites, a high-affinity site with a density
of 5.9 ± 1.2 fmol/mg protein and a low-affinity site with a density
of 16.9 ± 1.0 fmol/mg protein. Dexamethasone increased both high- and
low-affinity isoproterenol binding sites to 11.1 ± 2.2 and 25.9 ± 2.1 fmol/mg protein, respectively, without influencing agonist binding
affinities. Dexamethasone also selectively increased Gs
protein levels from 0.99 ± 0.14 to 1.46 ± 0.17 µg/mg protein
without affecting Gi
levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP
generation in dexamethasone-treated bTSM cells. These findings provide
new insights into the corticosteroid regulation of
-adrenergic signaling pathways in airway smooth muscle.
-adrenergic receptors; bovine; CGP-12177; corticosteroid; G
proteins; isoproterenol
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INTRODUCTION |
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THE -ADRENERGIC RECEPTOR (
-AR) is
linked to its effector enzyme, adenylyl cyclase, via an intermediary
guanine nucleotide regulatory binding protein termed Gs.
The stimulatory effect of Gs on adenylyl cyclase activity
is antagonized by a corresponding inhibitory G protein (Gi)
that is activated by various other receptor systems. Agonist binding to
Gs-coupled
-ARs results in the activation and release of
the
-subunit of Gs (Gs
) that, in turn,
activates adenylyl cyclase. In contrast, agonist binding to
Gs-uncoupled
-ARs does not result in adenylyl cyclase
activation. To promote agonist-induced signal transduction, the
Gs-coupled
-AR has a much higher agonist binding
affinity state than the uncoupled
-AR. Thus
-adrenergic agonists
interact with the
-AR at two affinity states, and the interaction
between
-AR and Gs proteins is reflected in the amount
of high-affinity agonist-
-AR binding.
There is considerable evidence that asthma is associated with
dysfunction of the -AR-adenylyl cyclase pathway. Some inflammatory mediators have been shown to downregulate
-AR expression (1, 6, 12);
however, more recent studies (10, 12, 14) suggest that the
-adrenergic defect in asthma results from impaired signal transduction, at least in part due to cytokine-induced overexpression of Gi proteins. As a result, the airway relaxant response
to
-adrenergic agonists is attenuated in asthmatic individuals. The
administration of corticosteroids to asthmatic individuals reduces
their chronic airway inflammation and helps restore
-adrenergic
responsiveness. Glucocorticoids directly influence many steps in the
-adrenergic signaling pathway, including supersensitization through
increased
-agonist stimulation of adenylyl cyclase (3) and
upregulation of
-AR gene transcription and receptor expression in
lung tissues and cells (7, 18-20, 22). Glucocorticoids have also
been shown to increase high-affinity
-agonist binding in human
neutrophils (2) and in fetal rat lung tissue (17). Similar studies have not been done in airway smooth muscle, and the direct effects of
corticosteroids on smooth muscle Gs protein expression have not been examined. Therefore, the present study was designed to investigate the effects of dexamethasone (Dex) on
-AR density, high-affinity
-agonist binding, and Gs
protein levels
in airway smooth muscle cells.
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MATERIALS AND METHODS |
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Cell culture and cell membrane preparation. Bovine tracheal
smooth muscle (bTSM) cells were generously provided by Dr. Mark Madison
(Department of Medicine, University of Massachusetts Medical School,
Worcester, MA) (5). The cells were maintained in culture in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.5 µg/ml of amphotericin B. Under these
culture conditions, bTSM cells grew with a population doubling every
1.5-3 days and reached confluence in 6-7 days. Cell
passages 4 through 7 were used for the experiments.
Confluent bTSM cells were placed in low-serum medium (3%) in the
absence (control) and presence of Dex (1 µM). Our preliminary work
with these cells demonstrated that a low concentration of serum was
necessary for them to express -ARs at consistent levels and to
demonstrate reproducible cAMP responses to isoproterenol. Additional
studies with Dex concentrations of 0.01-10 µM showed that
1-10 µM elicited maximal increases in
-AR expression and
isoproterenol-induced generation of cAMP in airway smooth muscle cells
(26).
After 24 h of incubation, the cells were homogenized in iced buffer consisting of 50 mM Tris · HCl (pH 8.3 at 25°C), 1 mM EDTA, and 1 mM dithiothreitol (DTT). Large cell fragments and debris were removed by an initial low-speed centrifugation (1,600 g), and the cell membrane was then pelleted by centrifugation at 35,000 g for 20 min at 4°C. The membrane pellet was resuspended in fresh homogenization buffer and centrifuged again at 35,000 g. The resulting washed membrane pellet was then suspended in binding buffer consisting of 50 mM Tris · HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and 1 mM DTT. Protein concentrations in the final suspensions were determined by the method of Lowry et al. (16) with bovine serum albumin as the standard.
-AR binding studies. The hydrophilic
-AR
ligand
(
)-4- (3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one
([3H]CGP-12177; specific activity 49.0 Ci/mmol)
was chosen for radioligand studies because of its highly specific,
single-site binding and its low nonspecific binding [< 20% of
total binding around the binding affinity (Kd)
concentration]. Initial studies assessed the saturation binding
of [3H]CGP-12177 in control and Dex-treated
bTSM cells to determine the influence of corticosteroids on
[3H]CGP-12177 binding. Triplicate control and
Dex samples containing ~100 µg of membrane protein were incubated
with increasing concentrations of [3H]CGP-12177
(0.007-2.0 nM) for 45 min at 37°C. Specific binding was
determined by the amount of ligand at each concentration displaced by
10 µM propranolol. Incubations were stopped with eight volumes of
iced buffer. Bound [3H]CGP-12177 was extracted
by rapid filtration through fiberglass filters (Whatman GF/C), rinsed
two times with iced buffer containing 50 mM
Tris · HCl, 1 mM EDTA, 1 mM DTT, and 0.1% bovine
serum albumin. The filters were then counted for receptor-bound
radioactivity in a liquid scintillation counter (EcoLite, ICN, Costa
Mesa, CA). Maximum binding (Bmax) and
Kd were determined for control and Dex-treated bTSM
cell membranes with a computerized noniterative curve-fitting program (LIGAND).
To determine the effects of corticosteroids on -adrenergic agonist
binding, competitive binding studies were performed with displacement
of bound [3H]CGP-12177 by the
-adrenergic
agonist isoproterenol. In paired experiments, aliquots containing
~100 µg of control and Dex-treated membrane protein samples in
binding buffer were exposed in triplicate to a constant concentration
of [3H]CGP-12177 (2 nM) and 18 different
concentrations of isoproterenol (0 and 1 × 10
12 to 3 × 10
4
M) for 45 min at 37°C. Nonspecific binding was determined by the
presence of 20 µM propranolol. Bound
[3H]CGP-12177 was extracted and counted as
above. The data from all paired experiments (n = 5 for both
control and Dex-treated bTSM) were analyzed with computerized
noniterative curve fitting for one- and two-site
displacement curves.
Western analysis of Gs
and Gi
protein
expression. Separate studies addressed the direct effects of
corticosteroids on Gs
and Gi
protein
subunit expression as determined by quantitative Western analysis (11,
21). Washed membrane pellets (n = 9 each) were obtained from
paired control and Dex-treated bTSM as in Cell culture and cell
membrane preparation. Five serial dilutions ranging
from 0.15 to 1.0 µg/µl of each control and Dex-treated membrane
protein sample were made. One serial dilution of membrane protein was
loaded to each of five lanes of a SDS-polyacrylamide gel (12%).
Recombinant rat standard Gs
protein (47 kDa, 0.04 µg;
Santa Cruz Biotechnology, Santa Cruz, CA) and standard
Gi
protein (42 kDa, 0.0107 µg; Santa Cruz
Biotechnology) were loaded in separate lanes on each gel as positive
controls and for density comparison of each protein sample band.
Prestained high-range molecular-mass standards (Life
Technologies) were also loaded in one lane as a transfer marker. The
paired control and Dex-treated gels were run in parallel, and protein
separation was performed on the Mini-PROTEAN II cell (Bio-Rad,
Hercules, CA) at 200 V for 30-40 min. Separated proteins were
transferred electrophoretically (200 V, 400 mA, 1 h) to nitrocellulose
membranes (0.2 µm; Schleicher & Schuell) in transfer buffer
containing 25 mM Tris base, 192 mM glycine and 20% (vol/vol) methanol
(pH 8.2)
Thereafter, the nitrocellulose blots were incubated at room temperature
for 1 h with blocking solution [0.2 M NaCl, 50 mM Tris · HCl, 0.2% (vol/vol) Tween, and 5% nonfat
milk]. The blots were primed with rabbit polyclonal antibody
raised to the Gs subunit (1:1,000 dilution; Santa Cruz
Biotechnology) for 1 h and then incubated with peroxidase-conjugated
goat anti-rabbit IgG (1:10,000 dilution; Calbiochem, La Jolla, CA) for
1 h. In-between each incubation step, the blots were washed six times
(5 min each time) with buffer containing 0.2 M NaCl, 50 mM
Tris · HCl, and 0.2% (vol/vol) Tween. The blots were
then exposed to enhanced chemiluminescence substrate (Pierce, Rockford
IL) for 5 min and were immediately visualized on X-ray film (Kodak
XAR-5). The visualized Gs
sample and standard bands were
quantified with a densitometer.
The nitrocellulose membranes were then reprobed with a polyclonal
antibody raised to the Gi subunit. Each nitrocellulose membrane was incubated for 30 min at 60°C in stripping buffer containing 2% (wt/vol) SDS, 62.5 mM
Tris · HCl (pH 6.8), and 100 mM
-mercaptoethanol
and then rinsed three times with water. The membranes were reblotted
with the same protocol and solutions as for anti-Gs
antibody above except that the primary antibody was changed to a
polyclonal antibody recognizing all three subtypes of Gi
(dilution 1:1,000; Santa Cruz Biotechnology). The sample and standard
Gi
bands were quantified with a densitometer. To ensure
that there was no sequence effect to the blots, three of the nine
paired blots were stained with anti-Gi
first, then
stripped and restained with anti-Gs
. The resulting blots
were no different than the other six done in the opposite sequence.
The density units from each protein sample were compared with the
density units of the known standard Gs or
Gi
proteins. Thus the approximate amounts of
Gs
and Gi
proteins per membrane protein
sample (in ng/µg) were obtained. Plots of the amounts of
Gs
or Gi
protein as a function of the
corresponding amount of membrane protein were linear in all cases, and
their slopes represent the average amount of Gs
or
Gi
protein (in ng) per microgram of membrane protein
from the control or Dex-treated bTSM cells.
cAMP assay. To assess the physiological relevance of our
observed changes in -adrenoceptor and Gs
densities,
additional studies determined the influence of Dex on basal and maximal
-agonist-stimulated levels of cAMP in the bTSM cells. Cells grown to
confluence in T-25 plates were placed in low-serum medium for 24 h in
the absence and presence of 1 µM Dex. After a 30-min incubation with
the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 10 µM), duplicate bTSM samples were exposed for 2 min to varying
concentrations of isoproterenol (0 and 10
8 to
10
4 M) (27). The cells were quickly lysed in 6%
perchloric acid and scraped from the flasks. Cellular cAMP levels were
determined by displacement of [3H]cAMP from a
commercial binding protein (Amersham International, Arlington Heights,
IL). The cAMP values are expressed as a percentage of baseline,
unstimulated levels in both control and Dex-treated cells.
Reagents. DMEM and nonessential amino acid solution were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum, penicillin-streptomycin solution, and amphotericin B were obtained from Gemini Bio-Products (Calabasas, CA). [3H]CGP-12177 and the cAMP assay kit were purchased from Amersham Life Science (Cleveland, OH). Isoproterenol hydrochloride, dithiothreitol, EDTA, dexamethasone 21-acetate, Trizma hydrochloride, and Trizma base were obtained from Sigma (St. Louis, MO). Acrylamide, SDS, N,N'-methylene-bis-acrylamide, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate were obtained from Bio-Rad. Tween solution was obtained from Fisher Scientific (Pittsburgh, PA). Methanol was obtained from J. T. Baker (Phillipsburg, NJ), and glycine was from ICN (Aurora, OH). IBMX was purchased from Research Biochemicals International (Natick, MA).
Statistical analysis. All data are expressed as means ± SE.
Slopes of the G-membrane protein relationships were obtained by
least-squares analysis. Statistical comparisons between control and
Dex-treated results were performed by two-tailed, unpaired t-tests and by repeated-measures analysis of variance for
isoproterenol-cAMP dose-response relationships (StatView 4.5, SAS
Institute, Cary, NC). Cellular sensitivity to isoproterenol was
characterized by the log concentration of isoproterenol associated with
half-maximal responses (i.e., the pC2 values). P
values < 0.05 were considered to be significant.
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RESULTS |
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-AR expression. The LIGAND analysis of
-AR
saturation binding with [3H]CGP-12177
demonstrated a single binding site in all control and Dex-treated bTSM
membrane samples (n = 4 each). This finding was also reflected
in linear Scatchard plots (correlation coefficients
0.791 to
0.984) and Hill coefficients near unity (range 0.847-1.03). Dex exposure did not affect the Kd of
[3H]CGP-12177 for the
-AR, with a
Kd value of 0.164 ± 0.067 nM for Dex-treated bTSM
cells vs. 0.213 ± 0.126 nM for control cells (P = 0.50). In
contrast, maximal
-AR binding was significantly increased with Dex,
with Bmax equaling 30.2 ± 6.2 fmol/mg protein in
Dex-treated cells and 15.8 ± 1.5 fmol/mg protein in control cells
(P = 0.018). These findings suggested that Dex exposure increased total
-AR numbers without changing the
Kd for a
-adrenergic-antagonist ligand.
-Agonist binding. In comparison to the ligand
saturation binding experiments, LIGAND analyses of the isoproterenol
displacement studies demonstrated two-site binding with biphasic curves
and nonlinear Scatchard relationships. In each control and Dex
experiment (n = 5 each), two-site binding resulted in a
significantly better fit than one-site binding. Addition of the
nonhydrolyzable GTP analog guanosine
5'-(
,
-imino)-triphosphate resulted in loss of high-affinity
binding sites and a shift of the inhibition curves to the right (data
not shown). The individual control and Dex displacement binding curves
were combined in order for LIGAND to determine the average values for
the high- and low-affinity agonist binding parameters (Fig.
1). The average high Kd
was ~1,000 times higher than the average low Kd.
Neither Kd value was changed by Dex treatment
(Table 1). In contrast, Dex increased total isoproterenol binding (total Bmax) by 62% over control
values (compared with the 91% increase obtained in the
[3H]CGP-12177 saturation experiments).
Low-affinity binding sites were increased by 53% above control levels,
and high-affinity sites were increased by 88%. The ratio of
high-affinity to total binding (i.e., high Bmax/total
Bmax) was not affected by Dex exposure. The increase in
high-affinity
-agonist binding suggested that corticosteroid
treatment either increased Gs protein expression or
enhanced
-AR-Gs coupling in bTSM (or both).
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Gs and
Gi
protein expression.
Paired Western analyses of Gs
protein expression in
control and Dex-treated membrane protein samples (n = 9)
demonstrated prominent bands at 45 kDa (Fig.
2A). The densities
of the five bands in the five protein sample lanes in each blot were
analyzed relative to the density of a known amount of Gs
protein standard (0.04 µg). Plots of the amount of Gs
protein present versus the amount of membrane protein sample loaded on
the gel showed linear relationships (Fig. 2B), the
slopes of which represented the average amount of Gs
present (in ng/µg or µg/mg membrane protein). Comparison of paired control and Dex-treated samples showed that the Dex-treated lines had
consistently steeper slopes than the control lines. Statistical comparison between control and Dex-treated Gs
protein
levels showed significantly increased Gs
expression in
Dex-treated bTSM (1.46 ± 0.17 vs. 0.99 ± 0.14 µg/mg protein in
control bTSM; P = 0.048).
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A single prominent band was obtained at 40 kDa in each blot for
Gi control and Dex-treated protein samples (Fig.
3A). However, the slopes of the
relationship between Gi
protein levels and the amount of
membrane protein samples were not different between control and
Dex-treated cells (Fig. 3B), suggesting that Dex treatment did
not affect the amount of Gi
in these cells. Indeed,
average Gi
levels were not changed with Dex treatment
(0.421 ± 0.096 µg/mg protein) compared with levels in paired
control cells (0.408 ± 0.072 µg/mg protein; P = 0.82). Thus
Dex selectively increased Gs
protein levels without
increasing Gi
protein levels in cultured bTSM cells.
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cAMP responses. Treatment of control bTSM cells
(n = 4 samples) with increasing doses of
isoproterenol resulted in dose-dependent increases in cellular cAMP
levels to a maximal stimulated level that was 309 ± 41% of the
unstimulated baseline. Dex treatment potentiated the cAMP response
(P = 0.046 vs. control value by ANOVA) and increased the
maximal response to 541 ± 77% of baseline (P = 0.021 vs. control value). Unstimulated baseline levels of cAMP were not
affected by Dex exposure (P = 0.30). There was also no
difference in bTSM sensitivity to isoproterenol in Dex-treated (pC2 6.23 ± 0.28 log M) and control
cells (5.57 ± 0.24
log M; P = 0.10).
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DISCUSSION |
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This study investigated the effects of corticosteroids on -AR
density,
-agonist binding, and Gs
and
Gi
protein levels in bTSM cells. Pretreatment of bTSM
cells for 24 h with 1 µM Dex increased total
-AR binding 1.9-fold
over the control value in the saturation binding studies and 1.6-fold
over the control value in the agonist displacement studies. These
findings are consistent with observations in various other tissues and
cell types and have been attributed to corticosteroid time- and
concentration-dependent increases in
2-AR mRNA levels
from enhanced gene transcription (18, 28). The increased
-AR binding
was accompanied by a 1.8-fold increase in maximal
-agonist-induced
cAMP generation in the cells. The parallel increases in
-AR
expression and
-agonist-mediated cAMP generation have also been seen
in rabbit tracheal smooth muscle after in vivo Dex administration (25).
Thus there appears to be a direct relationship between
-AR density
and maximal
-agonist cAMP responsiveness in airway smooth muscle.
The isoproterenol displacement studies revealed that both high- and
low-affinity -agonist binding was enhanced by corticosteroid exposure due to increases in
-agonist binding sites and not to changes in agonist Kd values. Isoproterenol bound
to
-ARs at two affinities in bTSM cells, with a proportion of
high-to-low binding of ~1:2 and a difference in
Kd values of ~1,000-fold. Both high- and
low-affinity binding sites were increased by Dex treatment. The
fraction of high-affinity
-agonist binding was not affected by Dex
in the bTSM cells, although cortisone treatment can enhance the
percentage of high-affinity
-agonist binding in human neutrophil
membranes (2). Of interest, methylprednisolone has been shown to
restore airway
-adrenergic responsiveness in the Basenji-Greyhound
dog model of asthma (24). At least one mechanism accounting for the
impairment of
-AR signaling in this model is a decrease in
high-affinity
-agonist binding sites in the lungs of these animals
(4). Our studies suggest that corticosteroids could improve
-adrenergic responsiveness in the Basenji-Greyhound dogs by
increasing high-affinity
-agonist binding sites in their airways.
The increase in high-affinity
-agonist binding in the bTSM cells
implied either that Dex enhanced Gs expression in the cell
membrane or that the corticosteroid somehow promoted the interaction
between
-ARs and the Gs proteins.
The mechanism of increased high-affinity binding was evaluated further
by quantitative Western analysis of Gs protein levels. These Western analyses demonstrated that Dex selectively increased Gs
protein levels in bTSM cells without affecting
Gi
levels. Similar observations have been reported in
the rat cerebral cortex, wherein corticosterone increased
Gs
levels 1.4-fold without influencing Gi
levels (23). The unchanged Gi
levels represent an
important control for any potential loading inequalities between the
samples and nonspecific steroid effects. It may be noted that the
molecular masses of the recombinant rat Gs
and
Gi
protein standards were slightly higher than those of
the bovine Gs
and Gi
proteins, likely
reflecting different posttranslational modifications of the recombinant
-protein standards. Corticosteroids can activate the transcription
of many genes by binding as a ligand-receptor complex to certain DNA
target sequences, termed glucocorticoid response elements, in the
promoter region of steroid-sensitive genes. Dex could increase
Gs
protein expression through this mechanism if the
Gs
protein gene contains a glucocorticoid response element. To address this possibility, we reviewed the reported nucleotide sequence of the human Gs
gene (13) and found
no classic sequences for glucocorticoid response elements in the 790 bases upstream from the initiation codon. Corticosteroids may act,
therefore, through trans-activating factors or other indirect
pathways to enhance Gs
protein expression in bTSM cells. Moreover, corticosteroid effects on Gs
protein
expression may vary between cell types, with positive effects reported
in the cerebral cortex (23), osteosarcoma cells (21), and
pheochromocytoma cells (15) but negative effects seen in aortic
endothelial membranes (9).
Agonist-induced GTPase stimulation is closely reflected in the affinity
state of the receptor for the agonist (8). Thus although we did not
measure GTPase activity directly, the finding that Dex increased
high-affinity -agonist binding demonstrates that the steroid
augments the function as well as the expression of Gs
.
The enhancements were similar, with a 1.5-fold increase in
Gs
protein expression and a 1.9-fold increase in
Gs
function (i.e., high-affinity
-agonist binding
sites). These increases were reflected in the potentiated maximal cAMP
response to isoproterenol in Dex-treated bTSM cells (175% of control
value). Although these observations do not rule out any potential
promoting effects of corticosteroids on
-AR-Gs protein
coupling, they suggest that the increased Gs
at least
contributes to this steroid response.
In summary, our study showed that Dex potentiates -agonist cAMP
responses and high-affinity binding in airway smooth muscle through
parallel increases in both
-AR levels and Gs
protein expression. This is the first direct demonstration that corticosteroids can influence Gs
protein levels in airway smooth muscle.
The effect on Gs
is selective in that Gi
protein levels were unaffected. Collectively, these findings provide
new insights into the regulation of
-adrenergic signaling pathways
by corticosteroids and may partly explain the mechanisms of
corticosteroid upregulation of
2-adrenergic
responsiveness in asthmatic patients. Further studies are
needed to fully understand the mechanisms underlying the potentiating effect of corticosteroids on the
-adrenergic signaling pathway in
airway smooth muscle cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mitchell Kresch and his research technician Constance Christian for technical advice with the Western blot studies.
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FOOTNOTES |
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This research was sponsored by a Career Investigator Award cofunded by the American Lung Association and the American Lung Association of Connecticut (to C. M. Schramm).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. M. Schramm, Pediatric Pulmonary Division, Connecticut Children's Medical Center, 282 Washington St., Hartford, CT 06106 (E-mail:cschram{at}ccmckids.org).
Received 14 June 1999; accepted in final form 11 February 2000.
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