Departments of 1 Medicine, 5 Molecular Genetics, and 4 Environmental Health, University of Cincinnati College of Medicine, Cincinnati 45267; 3 Department of Pathology, Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2 Department of Thoracic Medicine, National Heart and Lung Institute, London SW3 6LY, United Kingdom
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ABSTRACT |
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Airway
epithelial cells express 2-adrenergic receptors
(
2-ARs), but their role in regulating airway
responsiveness is unclear. With the Clara cell secretory protein (CCSP)
promoter, we targeted expression of
2-ARs to airway
epithelium of transgenic (CCSP-
2-AR) mice, thereby
mimicking agonist activation of receptors only in these cells. In situ
hybridization confirmed that transgene expression was confined to
airway epithelium, and autoradiography showed that
2-AR
density in CCSP-
2-AR mice was approximately twofold that
of nontransgenic (NTG) mice. Airway responsiveness measured by whole
body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline
(ED200) was greater for CCSP-
2-AR than for
NTG mice (345 ± 34 vs. 157 ± 14 mg/ml; P < 0.01). CCSP-
2-AR mice were also less responsive to ozone
(0.75 ppm for 4 h) because enhanced pause in NTG mice acutely
increased to 77% over baseline (P < 0.05) but
remained unchanged in the CCSP-
2-AR mice. Although both
groups were hyperreactive to methacholine 6 h after ozone
exposure, the ED200 for ozone-exposed CCSP-
2-AR mice was equivalent to that for unexposed NTG
mice. These findings show that epithelial cell
2-ARs
regulate airway responsiveness in vivo and that the bronchodilating
effect of
-agonists results from activation of receptors on both
epithelial and smooth muscle cells.
G protein-coupled receptor; adenosine 3',5'-cyclic monophosphate; adenylyl cyclase; ozone
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INTRODUCTION |
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THE AIRWAY EPITHELIUM represents a critical interface between the bronchial smooth muscle and the external environment. As such, the epithelium can be a significant regulator of airway responsiveness (reviewed in Ref. 58). First, the epithelium forms a mechanical barrier that protects the underlying bronchial structures from exposure to environmental spasmogens (19, 42). Second, the epithelium has enzymatic activity that can degrade a variety of bronchospastic substances (12, 47, 53). Third, mucociliary clearance is regulated by the ciliary activity of the epithelium (10), as is the production and content of the epithelial lining fluid (4). These functions of the epithelium affect airway responsiveness indirectly by limiting the exposure of airway smooth muscle to luminal spasmogens.
In addition to these indirect effects, airway epithelial cells may
regulate bronchial tone directly through the release of substances that
either relax or contract airway smooth muscle. Known epithelium-derived
substances that relax airway smooth muscle include PGE2
(22, 59) and nitric oxide (NO)
(26). Some studies (3, 14) have
also suggested the presence of a nonprostanoid epithelium-derived
factor that inhibits contraction. However, the presence of such a
factor has not been a consistent finding, and its significance remains
speculative (24). Contractile factors produced by the
bronchial epithelium include leukotrienes (23), PGF2 (22), and endothelin-1
(29). Airway epithelial cells produce a number of
different cytokines and adhesion molecules that could affect smooth
muscle responsiveness as well (8).
This notion that the epithelium is an important regulator of airway tone is further supported by clinical observations in asthma. Asthma is a chronic inflammatory disorder of the airways characterized by bronchial hyperresponsiveness to a variety of nonspecific stimuli. The pathogenesis of bronchial hyperresponsiveness in asthma is unclear, but there is evidence that disruption of the bronchial epithelium may be a contributing factor. Indeed, alteration of the epithelium may be present even in mild asthma (27, 28). Furthermore, the amount of epithelial damage can be correlated to the degree of airway hyperreactivity (5).
Results of several studies suggest that these inhibitory functions of
the bronchial epithelium are modulated by 2-adrenergic receptors (
2-ARs). The
2-AR is a G
protein-coupled membrane receptor that acts to raise intracellular cAMP
levels via stimulation of adenylyl cyclase (18). The
receptor is expressed on airway smooth muscle cells where it acts to
relax the muscle and promote bronchodilation (reviewed in Ref. 2).
However, the airway epithelium contains a high density of
2-ARs as well (6, 9,
21, 49). Although neither receptor
autoradiography nor in situ hybridization have localized the
2-ARs to specific epithelial cell types, these receptors
have been shown to regulate ion transport (7,
38), mucus secretion (33), ciliary beat
frequency (10), and Clara cell secretory activity
(36, 37). The regulatory role of the epithelial cell
2-AR is further supported by the
observation that mechanical removal of the epithelium from tracheal
rings decreases the bronchodilator effect of isoproterenol in vitro (14, 16, 51). This finding has
been interpreted to indicate that
-agonists stimulate the epithelial
release of relaxant factors that act directly on smooth muscle.
However, this effect has not been consistently observed
(1, 3), leaving the concept of a
physiologically relevant
2-AR-regulated,
epithelium-derived relaxant factor in some doubt. The physiological and
pathophysiological significance of epithelial cell
2-ARs
therefore remains unclear, primarily because the effects of epithelial
cell
2-ARs in vivo cannot be easily differentiated from
the direct bronchodilator effects of
2-ARs on airway
smooth muscle.
In the current study, we selectively overexpressed the
2-AR in the airway epithelium of transgenic (TG) mice
and examined the role that these receptors play in the regulation of
airway tone. The use of such a targeted strategy permitted the
epithelial
2-AR signaling pathway to be selectively
enhanced without directly affecting the density or activity of smooth
muscle
2-ARs. Because the receptor is not a secreted
product, we reasoned that the effects of epithelial cell
2-AR activation could be distinguished in vivo from
those of other cells by comparing airway responsiveness of
nontransgenic (NTG) mice to that of TG Clara cell secretory protein
(CCSP)-
2-AR mice. Our results showed that overexpression of the
2-AR in the airway epithelium of TG mice reduced
airway responsiveness, thereby supporting a role for this population of
receptors in the regulation of airway tone.
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METHODS |
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TG mice.
Targeted expression of the human 2-AR to the airway
epithelium was achieved with the use of the rat CCSP promoter
(50). The CCSP-
2-AR transgene was composed
of a 2.4-kb Hind III-Hind III fragment from the
rat CCSP promoter, a 1.5-kb Hind III-PshA I
fragment from the human
2-AR [1.2 kb of open reading
frame (ORF) and 0.3 kb of 3'-untranslated region (UTR)], and a 0.85-kb Xho I-BamH I fragment encoding the SV40 small T
intron and polyadenylation [poly(A)] signal. The fragments were
ligated together in the vector pUC18, with the orientation of each
fragment confirmed by sequence analysis and restriction enzyme
digestion. A 4.75-kb Not I fragment (Fig.
1) was then isolated for injection. The
DNA was gel purified and dialyzed against 5 mM Tris · HCl (pH
7.4) and 0.1 mM EDTA. Transgene DNA was injected into fertilized eggs
of FVB/N mice and implanted into pseudopregnant females as previously
described (54). Three founder mice were identified by
Southern blot analysis of genomic DNA prepared from tail clips.
Independent lines of heterozygous CCSP-
2-AR mice were
maintained by mating TG mice with NTG 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 poly(A)
region (5'-GTCACACCACAGAAGTAAGG-3'). Heterozygous mice from
generations 2 to 4 between the ages of 10 and 14 wk were used for all studies.
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Transgene expression and localization.
Screening for CCSP-2-AR transgene expression in the lung
was initially done by RT-PCR. Total RNA was extracted from freshly isolated lungs with Tri-Reagent (Molecular Research Center, Cincinnati, OH). An aliquot of RNA was reverse transcribed with random hexamers with murine leukemia virus reverse transcriptase (PerkinElmer) as previously described (13). Transgene cDNA was amplified
with 150 nM each of a sense primer from the CCSP promoter
(5'-CATCAGCCCACATCTACAGACAGC-3') and an antisense primer from the
2-AR ORF (5'-GACCAGCACATTGCCAAACAC-3'). The PCR was
started at 95°C for 120 s, followed by amplification for 35 cycles at 95°C for 60 s and 60°C for 60 s, followed by a
final extension at 72°C for 7 min. The PCR products were detected in
agarose gels stained with ethidium bromide. To quantitatively assess
transgene expression among the independent TG lines, ribonuclease protection assays were performed as previously reported
(40) with a 32P-labeled antisense riboprobe
corresponding to the distal 500 bp of the human
2-AR ORF
(40). Lung RNA (20 µg) and
2-AR riboprobe were hybridized overnight, digested with RNase, and subjected to
denatured PAGE analysis. The gels were visualized with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed with the ImageQuant software package (Molecular Dynamics).
Receptor density and function. 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). 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 (ICYP) as described (40). Adenylyl cyclase activity in these membrane preparations was assessed by a column chromatography method as previously reported (39).
Receptor localization.
The distribution of 2-AR expression within the lung was
assessed by receptor autoradiography (35). The tracheae of
anesthetized mice were cannulated, and the lungs were inflated with
tissue embedding fluid diluted 1:4 with PBS. The tracheae and lungs
were removed as a block, snap-frozen in isopentane cooled with liquid nitrogen, and stored at
80°C until further use. The tissues were subsequently sectioned (10 µm) on a cryostat at
30°C and thaw mounted onto slides coated with gelatin. Slides were stored at
80°C
and showed no loss of binding capacity under these conditions. For
autoradiography, the slides were allowed to warm to room temperature, after which they were washed for 15 min in incubation buffer (25 mM
Tris · HCl, pH 7.4, 154 mM NaCl2, 1.1 mM ascorbic
acid, and 0.25% polypep). Binding reactions were then carried out at
37°C for 120 min in incubation buffer containing 25 pM
125ICYP. Specific binding of
2-ARs was
determined in the presence of 100 µM CGP-20712A, a selective
1-AR antagonist. Nonspecific binding was determined by
incubation of adjacent tissue sections with 125ICYP in the
presence of 200 µM (
)-isoproterenol. After the incubation period,
the slides were washed twice for 15 min in ice-cold 25 mM
Tris · HCl (pH 7.4), rinsed in cold distilled water to remove buffer salts, and dried rapidly under a stream of cold air. The slides
were postfixed in paraformaldehyde vapor at 60°C for 30 min and then
dipped directly in Ilford K-5 emulsion. After 3 days at 4°C, the
emulsion was developed and fixed. Sections were stained with
hematoxylin and examined under a Zeiss microscope equipped with dark-
and bright-field illumination. Grain counts, assessed as optical
density, were measured with the Image Analysis System (Seescan,
Cambridge, UK) with a constant magnification (×200) and corrected for
background and nonspecific binding. The results represent the means of
nine separate fields (300 µm2/field) taken from three
different mice, each within the NTG and TG groups (27 fields
total/group).
Pulmonary function. Airway responsiveness (i.e., bronchoconstriction) was assessed noninvasively in conscious, unrestrained mice with a whole body plethysmograph (Buxco Electronics, Troy, NY) as previously described (31). With this system, the volume changes that occur during a normal respiratory cycle are recorded as the pressure difference between an animal-containing chamber and a reference chamber. The resulting signal is used to calculate respiratory frequency, minute volume, tidal volume, and enhanced pause (Penh). Penh was used as the measure of bronchoconstriction and was calculated from the formula: Penh = pause × (peak expiratory pressure/peak inspiratory pressure), where pause is the ratio of time required to exhale the last 30% of tidal volume relative to the total time of expiration (20). Mice were placed in the plethysmograph and the chamber was equilibrated for 10 min. They were exposed to aerosolized PBS (to establish baseline) followed by incremental doses of methacholine (1-640 mg/ml). Each dose of methacholine was aerosolized for 2 min, and respiratory measurements were recorded for 2 min afterward. During the recording period, an average of each variable was derived from every 30 breaths (or 30 s, whichever occurred first). The maximum Penh value after each dose was used to measure the extent of bronchoconstriction. On a separate day, the mice were submitted to the same protocol except that they were first treated with aerosolized albuterol (1 mg/ml) for 20 min. The concentration-response data for each mouse were fit to a curve by an iterative least squares technique, and the dose of methacholine required to increase Penh to 200% of baseline (ED200) was derived.
Ozone exposure. Mice were exposed for 4 h to filtered room air containing 0.75 ppm ozone in a 0.32-m3 stainless steel inhalation chamber that was capable of complete air exchange every 2 min. Zhao et al. (60) have previously shown that this level of exposure does not result in inflammation or structural lesions. Ozone was generated from 100% extra-dry oxygen (Matheson, Columbus, OH) with a model V1-0 ultraviolet ozonator (OREC, Phoenix, AZ), and its concentration was measured continuously with an ultraviolet photometric ozone analyzer (model 1008-PC, Dasibi Environmental, Glendale, CA). This instrument has an internal calibration system and was routinely checked and calibrated against a US Environmental Protection Agency transfer standard. The status of the mice and the concentration of ozone were checked hourly throughout the exposure period. Penh was measured by whole body plethysmography for 2-min intervals at 5, 15, 30, 45, 60, 90, and 120 min after ozone exposure. Methacholine challenge testing was performed 6 and 24 h postozone exposure with the protocol described in Pulmonary function. In studies carried out to characterize the increase in Penh that we observed in NTG mice after ozone exposure, mice were pretreated with either aerosolized albuterol or atropine. Albuterol (1 mg/ml) markedly reduced the increase in Penh induced by ozone (2.47 ± 0.41 without albuterol, 1.26 ± 0.07 with albuterol; n = 4 animals; P < 0.03). These results confirmed that bronchospasm accounted for most, although not all, of the increase in Penh that occurred acutely after exposure to ozone. In contrast, atropine (0.6 mg/ml) had little or no effect (<20% reduction, which was not significant) on the ozone response of NTG mice, consistent with a minimal role for cholinergic mechanisms in mediating the acute ozone-induced bronchospasm.
Bronchoalveolar lavage studies.
To perform bronchoalveolar lavage (BAL), mice were killed by a lethal
injection of pentobarbital sodium and exsanguinated via transection of
the abdominal aorta. The tracheae were then exposed and cannulated with
a polyethylene catheter. BAL was performed by instilling 1 ml of PBS
warmed to 37°C. The lavage fluid was allowed to dwell for 5 min,
after which it was withdrawn by gentle aspiration. The typical fluid
return was ~0.8 ml, with no difference observed between the TG and
NTG mice. The BAL fluid was centrifuged at 1,000 g for 10 min at 4°C to remove cells, and the resulting supernatant was frozen
and stored at 80°C until further use. Levels of PGE2 in
BAL fluid were measured by a colorimetric enzyme immunoassay from a
commercial kit (Amersham Pharmacia Biotech, Piscataway, NJ). Duplicate
samples (50 µl) from each BAL specimen were assayed directly in a
96-well plate. Optical density was read at 450 nm. The PGE2
content of each sample, measured in triplicate, was determined from a
reference curve of known PGE2 standards. NO content in BAL
fluid was determined by a spectrophotometric method based on the Griess
reaction to measure nitrite (55). To enzymatically reduce
BAL fluid nitrate to nitrite, samples were first incubated with 80 U/l
of nitrate reductase, 1 µM NADPH, 0.5 mM glucose 6-phosphate, and 160 U/l of glucose-6-phosphate dehydrogenase for 3 h at 25°C. The
sample (50 µl) was mixed with 50 µl of 1% sulfanilamide in 5%
H3PO4 and 50 µl of 0.1%
N-(1-napthyl)ethylenediamine. After incubation for 10 min,
absorbance was read at 550 nm. The nitrite content of BAL samples was
subsequently calculated from a reference curve that was generated from
the absorbance values of sodium nitrite standards. The nitrite
concentration for each sample was determined from the mean of duplicate
measurements. To standardize BAL recovery, the urea content of BAL
fluid was determined with a colorimetric assay (Sigma, St. Louis, MO).
The manufacturer's protocol was followed except that sample size was increased to 50 µl, and the total reaction volume was reduced to 1 ml. These conditions provided for a linear calibration curve. Aliquots
from each BAL sample were measured in duplicate.
Statistical analysis. Data are reported as means ± SE. Statistical comparisons between NTG and TG groups were performed with a two-tailed Student's t-test. For the whole body plethysmography studies, the effect of a given treatment within the NTG or TG group was assessed by paired analysis when appropriate. Differences were considered significant at the P < 0.05 probability level.
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RESULTS |
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CCSP-2-AR founder mice were identified by Southern
blot analysis of genomic DNA with a cDNA probe corresponding to the
SV40 poly(A) fragment of the transgene. The transgene was detected in 3 of 36 mice initially screened. These founder mice, designated lines 714, 717, and 721, were mated with NTG
FVB/N mice to generate heterozygous offspring. Genotypic analysis of
the progeny showed that the transgene was inherited in ~50% of the
progeny, with equal distribution between males and females.
Quantitative Southern blotting showed that heterozygous mice from
lines 714 and 717 contained ~40 copies of the
transgene (Fig. 1), whereas line 721 contained <5 copies
(data not shown).
Expression of the CCSP-2-AR transgene was initially
confirmed by RT-PCR. Total RNA prepared from whole lung homogenates was reverse transcribed and subjected to PCR with a forward primer from the
CCSP promoter and a reverse primer from the
2-AR ORF. As
shown in Fig. 2A, only
mice with positive genomic screens were found to express the transgene
mRNA. No PCR products were detected in the absence of reverse
transcriptase. CCSP-
2-AR transgene expression was
quantitated by ribonuclease protection assays (Fig. 2B). For
these studies, total cellular RNA prepared from whole lung homogenates
was hybridized with an antisense riboprobe corresponding to the distal
500 bp of the human
2-AR ORF. Because this region has
only ~70% homology with the mouse gene, the riboprobe does not
protect the endogenous mouse transcript from RNase digestion. Therefore, the full-length protected fragment (Fig. 2B,
arrow) was only detected in mice expressing the
CCSP-
2-AR transgene. With this technique, we found that
transgene expression in the lungs of mice from lines 714 and
717 was equivalent and much greater than that of line
721. The two higher expressing lines were subsequently propagated
for further studies.
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After confirming that the 2-AR transgene could be
detected in whole lung RNA, we next used in situ hybridization to
assess its distribution within the lung. With the human-specific
2-AR riboprobe, we found that transgene expression in
the CCSP-
2-AR mice was limited to the airway epithelium
(Fig. 3A). No specific binding
was observed in lung sections from NTG mice (Fig. 3B). Similarly, no specific binding occurred in either
CCSP-
2-AR (Fig. 3C) or NTG (data not shown)
mice when probed with the sense riboprobe.
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A previous study (6) has demonstrated that >90% of all
2-ARs in the lung are localized to the alveolar wall.
Because the human TG receptor cannot be differentiated from the
endogenous mouse receptor with
-AR radioligands, we anticipated that
receptor overexpression in one population of cells (i.e., those of the bronchial epithelium) might go undetected in receptor binding studies
with membranes prepared from whole lung homogenates. Indeed, our
initial radioligand assays with 125ICYP showed no
difference in
2-AR density of membranes prepared from
CCSP-
2-AR lungs or tracheae versus those prepared from
NTG mice. Analysis of adenylyl cyclase activities in these membranes also showed no difference. We therefore used receptor autoradiography with 125ICYP to establish that the receptor protein was
overexpressed in the airway epithelium. To specifically assess
2-AR density, CGP-20712A was included to
inhibit 125ICYP binding to the
1-AR.
Compared with NTG mice, CCSP-
2-AR mice had increased
grain density over the bronchial epithelium (Fig.
4, A and D). Grain
counts over the airway epithelium of the TG mice were approximately
twofold greater than those of the NTG mice (1.07 ± 0.07 vs.
0.59 ± 0.04 optical density, respectively, P < 0.05). No difference in grain density was observed
for other lung structures (bronchial smooth muscle, alveoli, and
vessels), indicating that receptor overexpression was confined to the
airway epithelium. (
)-Isoproterenol inhibited 125ICYP
binding in both NTG and CCSP-
2-AR mice (Fig. 4,
C and D, respectively), confirming that binding
was due to the
2-AR.
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Long-term observation of CCSP-2-AR TG mice (up to 20 mo)
showed that epithelial cell overexpression of the
2-AR
was not associated with increased morbidity or mortality.
CCSP-
2-AR mice develop and grow normally compared with
their NTG littermates. The gross structural anatomy of the lungs of
CCSP-
2-AR mice was unremarkable. Examination of
hematoxylin and eosin-stained lung sections showed no histological
abnormalities (Fig. 5, A and
D). Additional staining with Masson's trichrome showed no
evidence of increased collagen deposition (Fig. 5, B and
E), and staining with Alcian blue showed no differences in
the number of mucus-secreting cells (Fig. 5, C and
F). Remodeling of the lung, at least as detected by these
techniques, did not occur in CCSP-
2-AR mice.
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To examine the potential effects of targeted epithelial cell
2-AR overexpression on airway responsiveness, we
measured methacholine-induced bronchoconstriction in vivo so that the
integrity and function of the epithelium could be maintained. For these
experiments, we used whole body plethysmography to measure
Penh, a derived parameter that has been shown to correlate
highly with airway resistance as measured by invasive methods
(20). Conscious, unrestrained NTG and
CCSP-
2-AR mice were exposed to incremental doses of
aerosolized methacholine for 2 min followed by recording of respiratory
parameters for 2 min. As shown in Fig. 6,
the CCSP-
2-AR mice required higher doses of methacholine
to induce a change in Penh compared with the NTG mice. The
ED200 for methacholine in CCSP-
2-AR mice was
approximately twice that of NTG mice (345 ± 34 vs. 157 ± 14 mg/ml, respectively; P < 0.01; n = 10). These results therefore indicated that overexpression of the
2-AR in bronchial epithelial cells afforded protection
against cholinergic-mediated bronchoconstriction in vivo. To confirm
that this effect was the result of
2-AR activation, NTG
and CCSP-
2-AR mice were treated with the
-antagonist
propranolol (0.5 g/l in the drinking water) for 6 days. Afterward, the
mice were exposed to 160 mg/ml (the ED200 for NTG mice) of
aerosolized methacholine. The Penh value for the 160 mg/ml
concentration before propranolol treatment was then compared with that
after treatment. For NTG mice, the Penh value before
propranolol treatment (1.55 ± 0.12; n = 10) was
not different from that after treatment (1.49 ± 0.35;
n = 9). In contrast, the Penh of untreated
CCSP-
2-AR mice increased significantly after treatment
with propranolol (0.99 ± 0.10 vs. 1.72 ± 0.33; P < 0.03; n = 10 and 7, respectively).
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The results of the aforementioned experiments indicated that the
CCSP-2-AR TG mice were hyporesponsive to methacholine
and that this effect was specifically mediated rather than an artifact of transgene insertion in the genome. Our next goal was to assess whether the in vivo response of CCSP-
2-AR mice to
-agonists was also different from that of NTG mice. For these
experiments, NTG and CCSP-
2-AR mice were pretreated with
aerosolized albuterol (1 mg/ml) for 20 min before methacholine
challenge. Figure 7 shows that albuterol
caused a rightward shift in the methacholine dose response of NTG mice,
increasing the ED200 from 157 ± 14 to 388 ± 68 mg/ml (P < 0.01; n = 10). Of note, the
ED200 value for the albuterol-treated NTG mice was not
statistically different from that of the untreated
CCSP-
2-AR mice. In CCSP-
2-AR mice,
pretreatment with albuterol also raised the ED200 for
methacholine (345 ± 34 to 455 ± 62 mg/ml; n = 10), but this difference was small and did not reach significance.
Thus in these mice, epithelial cell overexpression of the
2-AR was as effective as inhaled albuterol that acts on
both epithelial cell and smooth muscle cell receptors.
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We next examined whether the acute response to ozone was altered in
CCSP-2-AR mice. For these experiments, NTG and
CCSP-
2-AR mice were exposed to 0.75 ppm ozone for 4 h. Penh was measured at frequent intervals during the
initial 2-h postexposure period. As shown in Fig.
8, Penh immediately increased
to 77% over baseline in NTG mice and then remained significantly
elevated for 1 h after exposure (P < 0.05). In
contrast, CCSP-
2-AR mice showed no increase in
Penh during the same time period.
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To assess whether the extent of ozone-induced hyperresponsiveness was
also affected in CCSP-2-AR mice, methacholine challenge testing was performed 6 and 24 h after ozone exposure.
Ozone-induced airway hyperresponsiveness was observed for both NTG and
CCSP-
2-AR mice (Fig. 9).
Compared with the preozone values, the ED200 for methacholine 6 h after ozone exposure decreased from 157 ± 14 to 56 ± 12 mg/ml (P < 0.01) in NTG mice and
from 345 ± 34 to 143 ± 35 mg/ml (P < 0.05)
in CCSP-
2-AR mice. The postexposure ED200 for methacholine for the CCSP-
2-AR mice remained
significantly greater than that of the exposed NTG mice
(P < 0.05) and, in fact, was not different from that
of unexposed NTG mice. However, this may have been due to the
differences in baseline Penh rather than differences in
hyperreactivity per se. Ozone-induced hyperresponsiveness was transient
in both groups of mice (Fig. 9). By 24 h postexposure, the
ED200 for methacholine increased to 133 ± 26 mg/ml in
the NTG mice and to 255 ± 45 mg/ml in the
CCSP-
2-AR mice.
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The results of the whole body plethysmography studies showed that
CCSP-2-AR mice were less responsive to methacholine and ozone than were NTG mice. To address the possibility that these effects
were due to the release of bronchodilator substances by the bronchial
epithelial cells, we measured the concentration of two candidates,
PGE2 and NO, in BAL fluid. For these measurements, mice
were lavaged with 1 ml of PBS that was allowed to dwell for 5 min. In
another group of mice, 10
6 M isoproterenol was added to
the lavage fluid so that the response to
-agonists could be
assessed. PGE2 levels in the BAL fluid from NTG (0.56 ± 0.15 ng/ml) and CCSP-
2-AR (0.66 ± 0.07 ng/ml) mice were not different (Fig.
10A). Addition of 1 µM isoproterenol to the lavage fluid had no effect on
PGE2 levels in BAL fluid from either group even though cAMP
content in the BAL fluid was approximately four times greater than that
in the untreated samples (data not shown). Likewise, basal levels of
nitrite in BAL fluid, as measured by the Griess reaction, in NTG
(1.05 ± 0.16 µM) and CCSP-
2-AR (1.35 ± 0.11 µM) mice were not different (Fig. 10B). Exposure to
isoproterenol had no effect on nitrite content in the BAL fluid from
either CCSP-
2-AR or NTG mice. Normalization of
PGE2 and NO measurements to either urea or protein had no
impact on the results.
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DISCUSSION |
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The 2-AR is present on many different cell types
within the lung, including airway epithelial cells. In vitro studies
(14, 16, 51) have demonstrated
that the airway epithelium can modulate bronchial tone and that this
interaction may be at least partly regulated by the
2-AR
signal transduction pathway. However, the physiological and potential
pharmacological significance of epithelial cell
2-ARs
have remained questionable, largely because of the inability to
differentiate their effects in complex in vivo models from those of
2-ARs on other cell types such as airway smooth muscle.
In the current study, we have attempted to address this issue by
overexpressing the
2-AR specifically in epithelial
cells, but not in smooth muscle cells, in the airways of TG mice.
Increasing the expression of
2-ARs has been shown in
both cell (17) and TG (41, 54)
models to increase basal signaling in a manner identical to that evoked
by agonists. In transfected Chinese hamster fibroblasts, Green et al.
(17) observed a direct correlation between
receptor density and basal adenylyl cyclase activity. Furthermore,
a mutant
2-AR that exhibited decreased
agonist-stimulated adenylyl cyclase activation had proportionately
decreased basal levels of activation as well (17).
Similarly, Turki et al. (54) and others (41)
have shown that TG mice overexpressing the
2-AR in the
heart have increased basal adenylyl cyclase activities relative to
their NTG littermates. Various modeling techniques have shown that the
increased basal signaling that occurs with
2-AR
overexpression is due to spontaneous conversion of receptors to the
active (R*) conformation (46). Thus by selectively
targeting expression to a specific cell type, one has the capability to distinguish the effect(s) of
2-AR activation in cell
types that are anatomically in close proximity and otherwise not
amenable to selective activation by agonists.
With the use of this strategy, our results revealed that
2-AR activation in Clara cells had a striking effect on
bronchomotor tone as assessed in vivo by whole body plethysmography. In
response to the inhaled bronchoconstrictor methacholine,
CCSP-
2-AR mice were significantly less reactive than
their NTG littermates. Remarkably, the degree of protection afforded to
the CCSP-
2-AR mice was the same as that achieved by
inhalation of the
-agonist albuterol in NTG mice. Although there was
a trend toward increased protection in CCSP-
2-AR mice
treated with albuterol, suggesting that epithelial
2-ARs
act in an additive fashion with smooth muscle
2-ARs to bronchodilate, the difference did not reach significance. These data
suggest, then, that epithelial cell
2-ARs can have a
substantial impact on airway smooth muscle responsiveness and are thus
indicative of a response not previously quantifiable.
The protective effect of 2-AR overexpression was not
limited to methacholine because the acute response to ozone was also reduced. Unlike methacholine, ozone does not pass beyond the epithelial layer (45) and therefore does not act directly on airway
smooth muscle. Instead, the effects of ozone are due to ozonation and oxidation of proteins and lipids in the epithelium and epithelial lining fluid that either act directly as bronchoconstrictors or lead to
the production of other bronchospastic substances (reviewed in Ref.
32). Ozone caused an acute increase in Penh of NTG mice that persisted up to 1 h after exposure. In contrast,
Penh was unchanged in the CCSP-
2-AR mice
over the same time period. Because Penh parallels measures
of airway resistance, the acute response in ozone-exposed NTG mice
could conceivably be due to edema from inflammation, mucosal damage, or
frank mucus plugging. However, several lines of evidence indicate that
bronchospasm was the primary component. First, spontaneous resolution
was rapid (<2 h). Second, the response was markedly attenuated by the
-agonist bronchodilator albuterol. Third, mice exposed to these
concentrations of ozone showed no evidence of cellular inflammation,
edema, or plugging for up to 4 h after exposure (60).
Furthermore, it would not be expected that edema and mucus plugging
would be readily reversed by a smooth muscle relaxant. Taken together,
these findings indicate that smooth muscle contraction is the major
contributing factor that accounts for the postozone increase in
Penh. The protection afforded to CCSP-
2-AR
mice was therefore effective against two different bronchoconstrictors
(i.e., methacholine and ozone). These bronchospastic agents appear to
act through different mechanisms because the anticholinergic agent
atropine had little effect on the Penh response after ozone.
Epithelial cell 2-AR overexpression did not, however,
prevent the development of ozone-induced airway hyperresponsiveness. The ED200 for methacholine measured 6 h after ozone
was 36% of basal level in NTG mice and 41% of basal level in
CCSP-
2-AR mice. The relative protective effect of
2-AR overexpression was maintained, though, because the
ED200 for the ozone-exposed TG mice was still significantly
less than that for the treated NTG mice. In fact, the ED200
of ozone-exposed CCSP-
2-AR mice was not different from that for unexposed NTG mice. CCSP-
2-AR mice were
therefore protected in the sense that their response to methacholine
after ozone was submaximal.
As mentioned earlier, airway epithelial cells could regulate airway
responsiveness through one or more of several different mechanisms. Overexpression of the 2-AR in
airway epithelial cells could have caused chronic remodeling of the
airways. However, histological analysis showed no structural
alterations in the airways of the CCSP-
2-AR mice, and
the protection afforded to the TG mice was inhibited by short-term
treatment with propranolol. Clara cells also have enzymatic activity
(e.g., acetylcholinesterase) that could potentially be upregulated by
2-AR activation and thus limit the response to an agent
such as methacholine. However, the observation that
CCSP-
2-AR mice were also protected against ozone-induced
bronchospasm (which was noncholinergic) suggests the presence of a
generalized pathway rather than one specific for a given agent.
The bronchial epithelium is the source of a number of substances that
may directly affect airway smooth muscle tone. We therefore considered
the possibility that activation of epithelial cell 2-ARs
stimulates the release of a relaxant factor. The two candidates we
chose to consider were PGE2 and NO because both are known
to relax airway smooth muscle (11, 52) and
both are produced by Clara cells (30, 48). To
investigate whether either of these substances could account for the
inhibition of bronchoconstriction in the CCSP-
2-AR mice,
we measured the content of PGE2 and NO in BAL fluid from
NTG and CCSP-
2-AR mice. Evaluation of this fluid showed
no differences in the levels of PGE2 or NO, suggesting that
neither agent was contributing to the protective effect of
2-AR overexpression. However, epithelial release of
these mediators may be primarily directed toward the serosal surface
(25, 34) and therefore underestimated in BAL
fluid. Thus we cannot conclusively exclude PGE2, NO, or
other factors as mediators of the epithelial cell
2-AR-mediated relaxation at this time. It is also
possible that overexpression altered the production and release of
Clara cell secretory products that in some way modify airway compliance or reactivity.
-Agonists have previously been shown to increase the
secretory activity of Clara cells (37). An increase in
such a component of the epithelial lining fluid could thus potentially act to limit the effect of methacholine and ozone by stabilization of
the airways, making them more resistant to closure. Baseline lung
function and histology, however, were not different in the CCSP-
2-AR TG mice, suggesting that this scenario is unlikely.
Regardless of the mechanism, our findings clearly show that
overexpression of the 2-AR in epithelial cells modulates
airway responsiveness to contractile agents. Previous ex vivo studies that used airway ring preparations have been inconsistent in
demonstrating a modulatory role for the epithelial cell
2-ARs. This may be due to the fact that the
bronchoactive substances have direct access to airway smooth muscle via
the exposed serosal surface, thus potentially bypassing protective
effects of the epithelium. Furthermore, mechanical removal of the
epithelium may introduce artificial confounding variables caused by
mast cell degranulation (15). Because our goal was to
assess the physiological relevance of this pathway on airway
responsiveness in vivo, we assessed bronchoconstriction with whole body
plethysmography to measure Penh. Penh is a
derived measure of bronchoconstriction that has been shown to strongly
correlate with airway resistance as measured by traditional invasive
methods (20). Whole body plethysmography also permits
assessment of airways distal to the trachea (20). This is
particularly relevant with regard to the CCSP-
2-AR mouse because Clara cells comprise up to 50% of the epithelial cells lining
the terminal bronchioles of murine airways (43,
44). Furthermore, at least two studies (51,
56) have demonstrated that the effect of epithelial cell
2-ARs varied among different airway generations, with
their effect being most pronounced in the smaller airways.
In summary, we have found that TG mice overexpressing
2-ARs in airway epithelial cells exhibit decreased
responsiveness to methacholine and ozone. Although the mechanism by
which this protection is afforded is unclear, these findings show that
the bronchial epithelium is capable of modulating airway tone and that
this interaction is at least partly regulated by
2-ARs
present on these cells. Furthermore, the effect of epithelial cell
2-AR activation is distinct from the effects of
2-AR activation in other lung cell types. The inhibition
of bronchoconstriction in TG mice overexpressing the
2-AR in the airway epithelium suggests the intriguing
possibility that delivery of this gene in vivo could be used in the
management of bronchospastic lung disease.
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ACKNOWLEDGEMENTS |
---|
This study was funded by National Heart, Lung, and Blood Institute Grants HL-45967, HL-41496, and HL-54829.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: S. B. Liggett, University of Cincinnati College of Medicine, 231 Bethesda Ave., 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. §1734 solely to indicate this fact.
Received 24 September 1999; accepted in final form 20 March 2000.
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