Transgenic overexpression of beta 2-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Dennis W. McGraw1, Susan L. Forbes1, Judith C. W. Mak2, David P. Witte3, Patricia E. Carrigan4, George D. Leikauf3, and Stephen B. Liggett1,5

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


    ABSTRACT
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INTRODUCTION
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Airway epithelial cells express beta 2-adrenergic receptors (beta 2-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta 2-ARs to airway epithelium of transgenic (CCSP-beta 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 beta 2-AR density in CCSP-beta 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-beta 2-AR than for NTG mice (345 ± 34 vs. 157 ± 14 mg/ml; P < 0.01). CCSP-beta 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-beta 2-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED200 for ozone-exposed CCSP-beta 2-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta 2-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta -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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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), PGF2alpha (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 beta 2-adrenergic receptors (beta 2-ARs). The beta 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 beta 2-ARs as well (6, 9, 21, 49). Although neither receptor autoradiography nor in situ hybridization have localized the beta 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 beta 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 beta -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 beta 2-AR-regulated, epithelium-derived relaxant factor in some doubt. The physiological and pathophysiological significance of epithelial cell beta 2-ARs therefore remains unclear, primarily because the effects of epithelial cell beta 2-ARs in vivo cannot be easily differentiated from the direct bronchodilator effects of beta 2-ARs on airway smooth muscle.

In the current study, we selectively overexpressed the beta 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 beta 2-AR signaling pathway to be selectively enhanced without directly affecting the density or activity of smooth muscle beta 2-ARs. Because the receptor is not a secreted product, we reasoned that the effects of epithelial cell beta 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)-beta 2-AR mice. Our results showed that overexpression of the beta 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 beta 2-AR to the airway epithelium was achieved with the use of the rat CCSP promoter (50). The CCSP-beta 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 beta 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-beta 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 beta 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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Fig. 1.   A: Clara cell secretory protein (CCSP)-beta 2-adrenergic receptor (AR; CCSP-beta 2-AR) transgene was composed of the rat CCSP promoter, the human beta 2-AR open reading frame (ORF), and the SV40 small T intron and polyadenylation [poly(A)] site. UTR, untranslated region. B: Southern blot analysis of genomic DNA from tail clips showed that the 2 founder mice (lines 714 and 717) used to establish transgenic (TG) lines each had >40 copies of the transgene DNA. NTG, nontransgenic.

Transgene expression and localization. Screening for CCSP-beta 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 beta 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 beta 2-AR ORF (40). Lung RNA (20 µg) and beta 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).

The distribution of transgene expression within the lung was assessed by in situ hybridization as previously reported (57). Tracheae and lungs were rapidly dissected, fixed in 4% paraformaldehyde, cryoprotected with 30% sucrose in PBS, and frozen in optimum cutting temperature compound. Cryostat sections (7 µm) were then mounted on silane-coated slides. An antisense cRNA probe for the human beta 2-AR was prepared as described above for the ribonuclease protection assays except that the probe was labeled with 35S-UTP. A sense cRNA probe was prepared with SP6 polymerase for use as a negative control. Hybridization was performed with 0.5-1.0 × 106 counts/min of labeled probe in a final volume of 30 µl/slide. After overnight incubation at 42°C, the sections were treated with 50 µg/ml of RNase A and 100 U/ml of RNase T1 for 30 min at 37°C and washed to a final stringency with 0.1× saline-sodium citrate at 50°C. Slides were dipped in NTB2 emulsion (Eastman Kodak) diluted 1:1 with 0.6 M ammonium acetate and exposed for 2 wk, after which they were developed with D19 developer (Eastman Kodak) and counterstained with hematoxylin and eosin.

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 beta 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 beta 2-ARs was determined in the presence of 100 µM CGP-20712A, a selective beta 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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ABSTRACT
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CCSP-beta 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-beta 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 beta 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-beta 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 beta 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-beta 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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Fig. 2.   Transgene expression in CCSP-beta 2-AR mice. A: expression of the transgene was screened for by RT-PCR with a sense primer from the CCSP promoter and an antisense primer from the beta 2-AR ORF. PCR products were detected in the presence (+) but not in the absence (-) of RT. B: quantitation of transgene expression was done by ribonuclease protection assays with an antisense riboprobe specific for the human beta 2-AR. Phosphorimage analysis of the gels showed that transgene expression between lines 714 and 717 was not different and was greater than expression in line 721. No signal was present in lung RNA from NTG mice. Nos. at left, no. of bp.

After confirming that the beta 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 beta 2-AR riboprobe, we found that transgene expression in the CCSP-beta 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-beta 2-AR (Fig. 3C) or NTG (data not shown) mice when probed with the sense riboprobe.


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Fig. 3.   Distribution of transgene expression in CCSP-beta 2-AR mice. In situ hybridization of frozen lung sections was performed with a 35S-UTP-labeled human beta 2-AR riboprobe. Dark-field photographs of sections counterstained with hematoxylin and eosin are shown. A: section of a bronchus from a CCSP-beta 2-AR TG mouse hybridized with the antisense riboprobe. L, lumen. Specific hybridization (arrow) was present over the airway epithelium (E) but not over the underlying cartilage (C) or smooth muscle (M). B: no specific signal was observed in NTG lung sections hybridized with the same probe. C: similarly, no signal was present when CCSP-beta 2-AR lung sections were hybridized with a beta 2-AR sense probe.

A previous study (6) has demonstrated that >90% of all beta 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 beta -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 beta 2-AR density of membranes prepared from CCSP-beta 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 beta 2-AR density, CGP-20712A was included to inhibit 125ICYP binding to the beta 1-AR. Compared with NTG mice, CCSP-beta 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-beta 2-AR mice (Fig. 4, C and D, respectively), confirming that binding was due to the beta 2-AR.


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Fig. 4.   Localization of beta 2-AR expression by receptor autoradiography. Frozen lung sections from NTG (A-C) and CCSP-beta 2-AR (D-F) mice were incubated with [125I]iodocyanopindolol (ICYP) in the presence of 100 µM CGP-20712A. Dark-field examination showed that grain density over the airway epithelium (Ep) of CCSP-beta 2-AR mice (D) was greater than that of NTG mice (A). However, no differences were observed for smooth muscle, bronchial vessels (Bv), or alveoli (A). Bronchi (Br) and surrounding lung structures are shown in bright-field photomicrographs of sections counterstained with hematoxylin (B and E). Addition of 200 µM (-)-isoproterenol inhibited 125ICYP binding to lung sections from both NTG (C) and CCSP-beta 2-AR (F) mice.

Long-term observation of CCSP-beta 2-AR TG mice (up to 20 mo) showed that epithelial cell overexpression of the beta 2-AR was not associated with increased morbidity or mortality. CCSP-beta 2-AR mice develop and grow normally compared with their NTG littermates. The gross structural anatomy of the lungs of CCSP-beta 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-beta 2-AR mice.


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Fig. 5.   Histological analysis of CCSP-beta 2-AR mice. The histology of NTG mice (A-C) was compared with that of TG mice (D-F). Staining with hematoxylin and eosin (A and D), Masson's trichrome (B and E), and Alcian blue (C and F) showed no differences between NTG and CCSP-beta 2-AR mice.

To examine the potential effects of targeted epithelial cell beta 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-beta 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-beta 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-beta 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 beta 2-AR in bronchial epithelial cells afforded protection against cholinergic-mediated bronchoconstriction in vivo. To confirm that this effect was the result of beta 2-AR activation, NTG and CCSP-beta 2-AR mice were treated with the beta -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-beta 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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Fig. 6.   In vivo assessment of airway responsiveness to methacholine. Methacholine-induced bronchoconstriction was assessed in conscious NTG and CCSP-beta 2-AR mice with the use of whole body plethysmography to measure enhanced pause (Penh). ED200, dose required to increase Penh to 200% of baseline. The methacholine dose-response curve of the CCSP-beta 2-AR mice was shifted significantly to the right compared with that of the NTG mice. Values are means ± SE of measurements from 10 mice/group. P < 0.01 for ED200 values.

The results of the aforementioned experiments indicated that the CCSP-beta 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-beta 2-AR mice to beta -agonists was also different from that of NTG mice. For these experiments, NTG and CCSP-beta 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-beta 2-AR mice. In CCSP-beta 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 beta 2-AR was as effective as inhaled albuterol that acts on both epithelial cell and smooth muscle cell receptors.


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Fig. 7.   Effect of beta -agonists on airway responsiveness in NTG and CCSP-beta 2-AR mice. Mice were pretreated with aerosolized albuterol (1 mg/ml) for 20 min before methacholine challenge. Data are means ± SE from 10 mice/group. Compared with baseline, albuterol increased the methacholine ED200 in NTG mice to 388 ± 68 mg/ml (P < 0.01). In CCSP-beta 2-AR, the methacholine ED200 increased to 455 ± 62 mg/ml, but the difference was not significant. The postalbuterol methacholine ED200 values for NTG and CCSP-beta 2-AR mice were not significantly different.

We next examined whether the acute response to ozone was altered in CCSP-beta 2-AR mice. For these experiments, NTG and CCSP-beta 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-beta 2-AR mice showed no increase in Penh during the same time period.


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Fig. 8.   In vivo assessment of airway responsiveness to ozone. NTG and CCSP-beta 2-AR mice were exposed to ozone as described in METHODS, and Penh was measured by whole body plethysmography at the indicated time points after exposure. Data for each time point are means ± SE of measurements from 10 mice. Compared with baseline, Penh increased acutely in NTG mice (5 min) and remained elevated up to 1 h (* P < 0.05). In contrast, no increase in Penh occurred in the CCSP-beta 2-AR mice.

To assess whether the extent of ozone-induced hyperresponsiveness was also affected in CCSP-beta 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-beta 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-beta 2-AR mice. The postexposure ED200 for methacholine for the CCSP-beta 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-beta 2-AR mice.


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Fig. 9.   Airway reactivity after ozone. Methacholine challenge was performed in NTG and CCSP-beta 2-AR mice 6 and 24 h after ozone exposure, and an ED200 was calculated for each mouse. Data are means ± SE of 10 mice/group. The methacholine ED200 measured 6 h after ozone decreased from 157 ± 14 to 56 ± 12 mg/ml (P < 0.01) in NTG mice and from 345 ± 34 to 143 ± 35 (P < 0.05) in CCSP-beta 2-AR mice. The ED200 for ozone-treated CCSP-beta 2-AR was not significantly different from that of nonexposed NTG mice. At 24 h postexposure, hyperresponsiveness to methacholine had resolved in both groups.

The results of the whole body plethysmography studies showed that CCSP-beta 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 beta -agonists could be assessed. PGE2 levels in the BAL fluid from NTG (0.56 ± 0.15 ng/ml) and CCSP-beta 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-beta 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-beta 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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Fig. 10.   PGE2 and nitric oxide (NO) content in bronchoalveolar (BAL) fluid from NTG and CCSP-beta 2-AR mice. A: PGE2 content was measured with an enzyme immunoassay. Data are means ± SE of duplicate measurements from 4 different mice/group. PGE2 levels in BAL fluid from NTG and CCSP-beta 2-AR mice were not different. B: total nitrite content was measured by the Griess reaction after enzymatic conversion of nitrates (see RESULTS). Data are means ± SE of duplicate measurements from 4 different mice/group. Nitrite content in BAL fluid from NTG and CCSP-beta 2-AR mice was not different.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The beta 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 beta 2-AR signal transduction pathway. However, the physiological and potential pharmacological significance of epithelial cell beta 2-ARs have remained questionable, largely because of the inability to differentiate their effects in complex in vivo models from those of beta 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 beta 2-AR specifically in epithelial cells, but not in smooth muscle cells, in the airways of TG mice. Increasing the expression of beta 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 beta 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 beta 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 beta 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 beta 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 beta 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-beta 2-AR mice were significantly less reactive than their NTG littermates. Remarkably, the degree of protection afforded to the CCSP-beta 2-AR mice was the same as that achieved by inhalation of the beta -agonist albuterol in NTG mice. Although there was a trend toward increased protection in CCSP-beta 2-AR mice treated with albuterol, suggesting that epithelial beta 2-ARs act in an additive fashion with smooth muscle beta 2-ARs to bronchodilate, the difference did not reach significance. These data suggest, then, that epithelial cell beta 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 beta 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-beta 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 beta -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-beta 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 beta 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-beta 2-AR mice. The relative protective effect of beta 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-beta 2-AR mice was not different from that for unexposed NTG mice. CCSP-beta 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 beta 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-beta 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 beta 2-AR activation and thus limit the response to an agent such as methacholine. However, the observation that CCSP-beta 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 beta 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-beta 2-AR mice, we measured the content of PGE2 and NO in BAL fluid from NTG and CCSP-beta 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 beta 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 beta 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. beta -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-beta 2-AR TG mice, suggesting that this scenario is unlikely.

Regardless of the mechanism, our findings clearly show that overexpression of the beta 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 beta 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-beta 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 beta 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 beta 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 beta 2-ARs present on these cells. Furthermore, the effect of epithelial cell beta 2-AR activation is distinct from the effects of beta 2-AR activation in other lung cell types. The inhibition of bronchoconstriction in TG mice overexpressing the beta 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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aizawa, H, Miyazaki N, Shigematsu N, and Tomooka M. A possible role of airway epithelium in modulating hyperresponsiveness. Br J Pharmacol 93: 139-145, 1988[Abstract].

2.   Barnes, PJ. Beta-adrenergic receptors and their regulation. Am J Respir Crit Care Med 152: 838-860, 1995[ISI][Medline].

3.   Barnes, PJ, Cuss FM, and Palmer JB. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br J Pharmacol 86: 685-691, 1985[Abstract].

4.   Basbaum, CB, and Finkbeiner WE. Mucous-producing cells of the airways. In: Lung Cell Biology, edited by Massaro D.. New York: Dekker, 1989, p. 37-79.

5.   Beasley, R, Roche WR, Roberts JA, and Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 139: 806-817, 1989[ISI][Medline].

6.   Carstairs, JR, Nimmo AJ, and Barnes PJ. Autoradiographic visualization of beta -adrenoreceptor subtypes in human lung. Am Rev Respir Dis 132: 541-547, 1985[ISI][Medline].

7.   Corrales, RJ, Coleman DL, Jacoby DB, Leikauf GD, Hahn HL, Nadel JA, and Widdicombe JH. Ion transport across cat and ferret tracheal epithelia. J Appl Physiol 61: 1065-1070, 1986[Abstract/Free Full Text].

8.   Davies, RJ, and Devalia JL. Asthma. Epithelial cells. Br Med Bull 48: 85-96, 1992[Abstract].

9.   Davis, PB, Silski CL, Kercsmar CM, and Infeld M. beta -Adrenergic receptors on human tracheal epithelial cells in primary culture. Am J Physiol Cell Physiol 258: C71-C76, 1990[Abstract/Free Full Text].

10.   Devalia, JL, Sapsford RJ, Rusznak C, Toumbis MJ, and Davies RJ. The effects of salmeterol and salbutamol on ciliary beat frequency of cultured human bronchial epithelial cells, in vitro. Pulm Pharmacol 5: 257-263, 1992[ISI][Medline].

11.   Dupuy, PM, Shore SA, Drazen JM, Frostell C, Hill WA, and Zapol WM. Bronchodilator action of inhaled nitric oxide in guinea pigs. J Clin Invest 90: 421-428, 1992[ISI][Medline].

12.   Dusser, DJ, Nadel JA, Sekizawa K, Graf PD, and Borson DB. Neutral endopeptidase and angiotensin converting enzyme inhibitors potentiate kinin-induced contraction of ferret trachea. J Pharmacol Exp Ther 244: 531-536, 1988[Abstract].

13.   Eason, MG, and Liggett SB. Human alpha 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of alpha 2C10, alpha 2C4, and alpha 2C2 mRNA in multiple tissues. Mol Pharmacol 44: 70-75, 1993[Abstract].

14.   Flavahan, NA, Aarhus LL, Rimele TJ, and Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 58: 834-838, 1985[Abstract/Free Full Text].

15.   Franconi, GM, Rubinstein I, Levine EH, Ikeda S, and Nadel JA. Mechanical removal of airway epithelium disrupts mast cells and releases granules. Am J Physiol Lung Cell Mol Physiol 259: L372-L377, 1990[Abstract/Free Full Text].

16.   Goldie, RG, Papadimitriou JM, Paterson JW, Rigby PJ, Self HM, and Spina D. Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists. Br J Pharmacol 87: 5-14, 1986[Abstract].

17.   Green, SA, Cole G, Jacinto M, Innis M, and Liggett SB. A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J Biol Chem 268: 23116-23121, 1993[Abstract/Free Full Text].

18.   Green, SA, and Liggett SB. Molecular basis of G-protein-coupled receptor signaling. In: The Genetics of Asthma, edited by Liggett SB, and Meyers D.. New York: Dekker, 1996, p. 67-90.

19.   Gumbiner, B. Structure, biochemistry, and assembly of epithelial tight junctions. Am J Physiol Cell Physiol 253: C749-C758, 1987[Abstract/Free Full Text].

20.   Hamelmann, E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, and Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 156: 766-775, 1997[Abstract/Free Full Text].

21.   Henry, PJ, Rigby PJ, and Goldie RG. Distribution of beta 1- and beta 2-adrenoceptors in mouse trachea and lung: a quantitative autoradiographic study. Br J Pharmacol 99: 136-144, 1990[Abstract].

22.   Holtzman, MJ. Arachidonic acid metabolism in airway epithelial cells. Annu Rev Physiol 54: 303-329, 1992[ISI][Medline].

23.   Holtzman, MJ, Hansbrough JR, Rosen GD, and Turk J. Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells. Biochim Biophys Acta 963: 401-413, 1988[ISI][Medline].

24.   Jacoby, DB. Role of the respiratory epithelium in asthma. Res Immunol 148: 48-58, 1997[ISI][Medline].

25.   Jacoby, DB, Ueki IF, Widdicombe JH, Loegering DA, Gleich GJ, and Nadel JA. Effect of human eosinophil major basic protein on ion transport in dog tracheal epithelium. Am Rev Respir Dis 137: 13-16, 1988[ISI][Medline].

26.   Kobzik, L, Bredt DS, Lowenstein CJ, Drazen J, Gaston B, Sugarbaker D, and Stamler JS. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol 9: 371-377, 1993[ISI][Medline].

27.   Laitinen, LA, Heino M, Laitinen A, Kava T, and Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131: 599-606, 1985[ISI][Medline].

28.   Laitinen, LA, Laitinen A, and Haahtela T. Airway mucosal inflammation even in patients with newly diagnosed asthma. Am Rev Respir Dis 147: 697-704, 1993[ISI][Medline].

29.   Laporte, J, D'Orleans-Juste P, and Sirois P. Guinea pig Clara cells secrete endothelin 1 through a phosphoramidon-sensitive pathway. Am J Respir Cell Mol Biol 14: 356-362, 1996[Abstract].

30.   Laporte, J, Hallee A, Maghni K, Robidoux C, Borgeat P, and Sirois P. Metabolism of arachidonic acid by guinea pig Clara cells. Prostaglandins 41: 263-281, 1991[Medline].

31.   Lee, JJ, McGarry MP, Farmer SC, Denzler KL, Larson KA, Carrigan PE, Brenneise IE, Horton MA, Haczku A, Gelfand EW, Leikauf GD, and Lee NA. Interleukin-5 expression in the lung epithelium of TG mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 185: 2143-2156, 1997[Abstract/Free Full Text].

32.   Leikauf, GD, Simpson LG, Santrock J, Zhao Q, Abbinante-Nissen J, Zhou S, and Driscoll KE. Airway epithelial cell responses to ozone injury. Environ Health Perspect 103: 91-95, 1995[ISI][Medline].

33.   Leikauf, GD, Ueki IF, and Nadel JA. Autonomic regulation of viscoelasticity of cat tracheal gland secretions. J Appl Physiol 56: 426-430, 1984[Abstract/Free Full Text].

34.   Leikauf, GD, Ueki IF, Nadel JA, and Widdicombe JH. Bradykinin stimulates Cl secretion and prostaglandin E2 release by canine tracheal epithelium. Am J Physiol Renal Fluid Electrolyte Physiol 248: F48-F55, 1985[Abstract/Free Full Text].

35.   Mak, JC, Nishikawa M, Shirasaki H, Miyayasu K, and Barnes PJ. Protective effects of a glucocorticoid on downregulation of pulmonary beta 2-adrenergic receptors in vivo. J Clin Invest 96: 99-106, 1995[ISI][Medline].

36.   Massaro, GD, and Davis LD. Demonstration of beta-adrenergic receptors in rat bronchiolar epithelial cells employing 9-amino-acridyl propranolol fluorescent microscopy. J Histochem Cytochem 32: 122-123, 1984[Abstract].

37.   Massaro, GD, Fischman CM, Chiang MJ, Amado C, and Massaro D. Regulation of secretion in Clara cells: studies using the isolated perfused rat lung. J Clin Invest 67: 345-351, 1981[ISI][Medline].

38.   McCann, JD, and Welsh MJ. Regulation of Cl- and K+ channels in airway epithelium. Annu Rev Physiol 52: 115-135, 1990[ISI][Medline].

39.   McGraw, DW, Donnelly ET, Eason MG, Green SA, and Liggett SB. Role of beta ARK in long-term agonist-promoted desensitization of the beta 2-adrenergic receptor. Cell Signal 10: 197-204, 1998[ISI][Medline].

40.   McGraw, DW, Forbes SL, Kramer LA, and Liggett SB. Polymorphisms of the 5' leader cistron of the human beta 2-adrenergic receptor regulate receptor expression. J Clin Invest 102: 1927-1932, 1998[Abstract/Free Full Text].

41.   Milano, CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA, and Lefkowitz RJ. Enhanced myocardial function in TG mice overexpressing the beta 2-adrenergic receptor. Science 264: 582-586, 1994[ISI][Medline].

42.   Munakata, M, Huang I, Mitzner W, and Menkes H. Protective role of epithelium in the guinea pig airway. J Appl Physiol 66: 1547-1552, 1989[Abstract/Free Full Text].

43.   Plopper, CG. Comparative morphologic features of bronchiolar epithelial cells. The Clara cell. Am Rev Respir Dis 128: S37-S41, 1983[ISI][Medline].

44.   Plopper, CG, Mariassy AT, and Hill LH. Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung: I. A comparison of rabbit, guinea pig, rat, hamster, and mouse. Exp Lung Res 1: 139-154, 1980[ISI][Medline].

45.   Pryor, WA, Squadrito GL, and Friedman M. A new mechanism for the toxicity of ozone. Toxicol Lett 82-83: 287-293, 1995.

46.   Samama, P, Cotecchia S, Costa T, and Lefkowitz RJ. A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem 268: 4625-4636, 1993[Abstract/Free Full Text].

47.   Sekizawa, K, Nakazawa H, Ohrui T, Yamauchi K, Ohkawara Y, Maeyama K, Watanabe T, Sasaki H, and Takishima T. Histamine N-methyltransferase modulates histamine- and antigen-induced bronchoconstriction in guinea pigs in vivo. Am Rev Respir Dis 147: 92-96, 1993[ISI][Medline].

48.   Shaul, PW, North AJ, Wu LC, Wells LB, Brannon TS, Lau KS, Michel T, Margraf LR, and Star RA. Endothelial nitric oxide synthase is expressed in cultured human bronciolar epithelium. J Clin Invest 94: 2231-2236, 1994[ISI][Medline].

49.   Spina, D, Rigby PJ, Paterson JW, and Goldie RG. Autoradiogrphic localization of beta-adrenoceptors in asthmatic human lung. Am Rev Respir Dis 140: 1410-1415, 1989[ISI][Medline].

50.   Stripp, BR, Sawaya PL, Luse DS, Wikenheiser KA, Wert SE, Huffman JA, Lattier DL, Singh G, Katyal SL, and Whitsett JA. cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 267: 14703-14712, 1992[Abstract/Free Full Text].

51.   Stuart-Smith, K, and Vanhoutte PM. Heterogeneity in the effects of epithelium removal in the canine bronchial tree. J Appl Physiol 63: 2510-2515, 1987[Abstract/Free Full Text].

52.   Sweatman, WJ, and Collier HO. Effects of prostaglandins on human bronchial muscle. Nature 217: 69, 1968[ISI][Medline].

53.   Taisne, C, Norel X, Walch L, Labat C, Verriest C, Mazmanian GM, and Brink C. Cholinesterase activity in pig airways and epithelial cells. Fundam Clin Pharmacol 11: 201-205, 1997[ISI][Medline].

54.   Turki, J, Lorenz JN, Green SA, Donnelly ET, Jacinto M, and Liggett SB. Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in TG mice. Proc Natl Acad Sci USA 93: 10483-10488, 1996[Abstract/Free Full Text].

55.   Verdon, CP, Burton BA, and Prior RL. Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. Anal Biochem 224: 502-508, 1995[ISI][Medline].

56.   Vornanen, M. Adrenergic responses in different sections of rat airways. Acta Physiol Scand 114: 587-591, 1982[ISI][Medline].

57.   Wang, J, Niu W, Nikiforov Y, Naito S, Chernausek S, Witte D, LeRoith D, Strauch A, and Fagin JA. Targeted overexpression of IGF-I evokes distinct patterns of organ remodeling in smooth muscle cell tissue beds of TG mice. J Clin Invest 100: 1425-1439, 1997[Abstract/Free Full Text].

58.   Widdicombe, JH. Physiology of airway epithelia. In: The Airway Epithelium, edited by Farmer SG, and Hay DWP. New York: Dekker, 1991, p. 41-64.

59.   Widdicombe, JH, Ueki IF, Emery D, Margolskee D, Yergey J, and Nadel JA. Release of cyclooxygenase products from primary cultures of tracheal epithelia of dog and human. Am J Physiol Lung Cell Mol Physiol 257: L361-L365, 1989[Abstract/Free Full Text].

60.   Zhao, Q, Simpson LG, Driscoll KE, and Leikauf GD. Chemokine regulation of ozone-induced neutrophil and monocyte inflammation. Am J Physiol Lung Cell Mol Physiol 274: L39-L46, 1998[Abstract/Free Full Text].


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