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Published ahead of print on December 9, 2005, doi:10.1165/rcmb.2005-0091OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 410-416, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0091OC

beta2-Adrenoceptor Agonist Modulates Endothelin-1 Receptors in Human Isolated Bronchi

Christophe Faisy, Francisco Pinto, Claire Danel, Emmanuel Naline, Paul-Andre Risse, Ingrid Leroy, Dominique Israel-Biet, Jean-Yves Fagon, Maria-Luz Candenas and Charles Advenier

UPRES EA220, Faculté de Médecine Paris-Ouest and UFR Biomédicale des Saints-Pères, and Service de Réanimation Médicale, Hôpital Européen Georges Pompidou, Paris, France; and Centro de Investigaciones Cientificas Isla de la Cartuja, Instituto de Investigaciones Quimicas, Sevilla, Spain

Correspondence and requests for reprints should be addressed to Christophe Faisy, M.D., Ph.D., UPRES EA220, UFR Biomédicale des Saints-Pères, 45 rue des Saints-Pères, 75006 Paris, France. E-mail: christophe.faisy{at}wanadoo.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Chronic exposure of human isolated bronchi to beta2-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. Our objective was to determine whether ET-1 receptors ETA and ETB are involved in fenoterol enhancement. Twenty-two human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 µM fenoterol (15 h, 21°C). Removing the epithelium after fenoterol incubation limited the maximal contraction (0.10 ± 0.36 g without epithelium versus 1.18 ± 0.22 with, n = 8, P = 0.04). After 15 h incubation, 14 and 8 paired rings were fixed, respectively, for immunolabeling of bronchial ETA and ETB receptors, and to determine the mRNA expression levels using real-time quantitative reverse transcription polymerase chain reaction. ETA and ETB receptor mRNA expressions were 1.27- ± 0.14-fold (not significant) and 2.24- ± 0.28-fold (P < 0.01) higher, respectively, in fenoterol-treated bronchi than in paired controls. Fenoterol incubation significantly increased epithelial ETA and ETB receptor labeling intensity scores (P = 0.001 and P = 0.002, respectively, versus controls), and enhanced the diffuse localization of ETA receptors on the epithelial cells (P = 0.002 versus controls), but did not change the ETB-receptor immunolabeling intensity on airway smooth muscle. We conclude that fenoterol-induced sensitization of human isolated bronchi involves epithelial ETA and ETB receptors, which suggests perturbation of the epithelial regulation of airway smooth muscle contraction in response to ET-1.

Key Words: airway hyperresponsiveness • asthma • ETA and ETB receptors • fenoterol


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Endothelin-1 (ET-1) is a 21–amino acid constrictor peptide first isolated, purified, and sequenced from the conditioned medium of porcine endothelial cell cultures by Yanagisawa and coworkers (1). After its discovery in 1988, ET-1 initially attracted attention in the cardiovascular field, and it is only more recently that the involvement of this peptide and its role in the physiology and pathophysiology of the airways has been envisaged (2, 3). Indeed, ET-1 is synthesized and released by several airway cell types, including predominantly epithelial cells but also endothelial cells, macrophages, and airway smooth muscle cells (2–4). In the human respiratory tract, ET-1 mediates airway smooth muscle contraction, growth, and mucus secretion (2, 5–12). In addition, ET-1 can induce airway inflammation, hyperresponsiveness, and remodeling in animals and humans (3, 11, 13), suggesting that it could be a major component in the pathophysiology of asthma (11, 13–15). In stable chronic obstructive pulmonary disease (COPD), the role of ET-1 remains more speculative because of conflicting findings (2, 13, 16–18). However, sputum and plasma ET-1 levels rise during COPD exacerbations (19).

ET-1 acts via two different receptors, ETA and ETB, that belong to the superfamily of seven transmembrane, G protein–coupled receptors. Coupling Gi, Gq, or Gs proteins with ETA or ETB receptors induces different effects in airways (2, 10, 20). ETA and ETB receptors also act via the modulation of ionic transmembrane channels (2, 3, 6, 20, 21). In human airways, ETA and ETB distribution is not well known. Autoradiographic studies showed that the ETB receptor is the predominant subtype in airway smooth muscle (3, 11, 22). ETA and ETB receptors are also present in other structures within the human bronchial wall, such as epithelium, submucosal glands, and blood vessels, but their respective distributions in these cell types remain unknown (2, 3, 13).

beta2-Adrenoceptor agonists are the most potent known airway smooth muscle relaxants and they have been used for several decades to treat asthma and COPD. However, regular use of beta2-adrenoceptor agonists alone may enhance nonspecific airway responsiveness and airway inflammation, and may thereby be deleterious by affecting asthma control (23). That heightened risk was at first identified with fenoterol (23), which is a short-acting beta2-adrenoceptor agonist with high intrinsic efficacy (24). Other studies showed that inhaled beta2-adrenoceptor agonists, and not only fenoterol, could increase airway responsiveness in humans without diminishing their relaxant effects (25–27). The mechanisms involving these untoward effects are unclear, but have been suggested to involve switching beta2-adrenoceptor coupling from the Gs to the Gi protein, beta2-adrenoceptor polymorphism at codons 16 and 27, or the role of the S-isomers of beta2-adrenoceptor agonists on airway hyperresponsiveness (23, 28). We recently demonstrated that prolonged exposure to a pharmacologic dose of beta2-adrenoceptor agonist, especially fenoterol, sensitizes human bronchi to ET-1 and that this phenomenon involves, at least in part, proinflammatory pathways mediated by NF-{kappa}B, mitogen-activated protein kinases, and activation of the transient receptor potential vanilloid-1 (TRPV-1) in sensory nerves present within the airways (29, 30).

The purpose of this work was to study the involvement of ETA and ETB receptors in the fenoterol-induced bronchial sensitization to ET-1. Therefore, we examined the effect of prolonged fenoterol exposure on the expression and distribution of ET-1 receptors in human isolated bronchi.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Tissue Preparations
The study was approved by our local Ethics Committees (Comité de Protection des Personnes se Prêtant à la Recherche Biomédicale, Versailles, France and Consejo Superior de Investigaciones Científicas, Madrid, Spain). Bronchial tissues were surgically removed from 22 patients with lung cancer (15 men and 7 women, 59 ± 10 yr of age); all were ex-smokers or smokers. The latter stopped tobacco use at least 4 wk before surgery. Just after resection, bronchial segments (inner diameter 1–2 mm) were collected as far as possible from the malignant lesion. The absence of tumoral infiltration was retrospectively established in all bronchi. They were placed in oxygenated Krebs–Henseleit solution (vehicle composition in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 11.7 glucose). After removal of adhering lung parenchyma and connective tissues, rings of similar weight and length from the same bronchus were prepared and randomly distributed in paired groups.

Functional Study
A 50/50 mixture of (R)- and (S)-fenoterol isomers (Boehringer-Ingelheim, Ingelheim am Rhein, Germany) was first dissolved in distilled water and then 40 µl of this solution was added to 1 ml of Krebs–Henseleit solution to obtain a final fenoterol concentration of 0.1 µM. After isolation of paired bronchial rings, the control ring was placed in Krebs–Henseleit solution and the pretreated ring was immersed in 0.1 µM fenoterol and both were incubated for 15 h at room temperature (29, 30). Then, functional experiments were conducted by cumulative additions of ET-1 (Sigma, St. Louis, MO) to the rings in organ baths, as previously reported (29, 30). When required, the epithelium was removed after incubation by gently and repeatedly rubbing the luminal surface with a cotton-tipped applicator (31, 32).

RNA Isolation and Real-Time RT–PCR
After preparation, paired bronchial rings from eight different patients were immersed in RNAlater (Sigma) for 24 h without previous incubation (H0, n = 8) or were incubated with 0.1 µM fenoterol or vehicle for 6 h (H6, n = 4) or for 15 h (H15, n = 8) before immersion in RNAlater (24 h). Bronchi were then stored at –80°C until use. Total RNA was isolated and treated with RNase-free, FPLC pure DNase I (Amersham Biosciences, Essex, UK) in DNase buffer (40 mM Tris–HCl, pH 7.5, 6 mM MgCl2) containing 10 units of RNasin (Promega Corp., Madison, WI) to eliminate contaminating genomic DNA, then quantified spectrophotometrically at 260 nm. DNase-treated total RNA (2 µg) was reverse transcribed using a first-strand cDNA synthesis kit (Amersham Biosciences).

An end-point PCR assay was used to analyze the presence and to establish the identity of the amplified products by DNA sequence analysis, as previously described (33, 34). Real-time PCR was used to quantify the expression of the genes encoding the ETA receptor (EDNRA) and the ETB receptor (EDNRB) using the iCycler iQ real-time detection system (Bio-Rad Laboratories, Hercules, CA). The specific oligonucleotide primers used for PCR amplification were designed with Primer 3 software (35) and synthesized and purified by Sigma Genosys (Cambridge, UK). The sequences of the primer pairs were: EDNRA encoding the ETA receptor, forward 5'-GCTCTTTGCTGGTTCCC TCTT-3' and reverse 5'-GGTCATCAGACTTTTGGACTGG-3', to amplify a 243 base-pair (bp) PCR product; EDNRB encoding the ETB receptor, forward 5'-TCTTTTGCCTGGTCCTTGTCT-3' and reverse 5'-GCAGTTTTTGAATCTTTTGCTCAC-3', to amplify a 215-bp PCR product; and beta-actin (internal control), forward 5'-TCCCTGGAGAA GAGCTACGA-3' and reverse 5'-ATCTGCTGGAAGGTGGA CAG-3', yielding a 362-bp PCR product. In addition to beta-actin, we analyzed the expression of two additional housekeeping genes: PPP1CB, which encodes Homo sapiens protein phosphatase 1 catalytic subunit beta-isoform (33); and POLR2A, encoding H. sapiens polymerase (RNA) II (DNA directed) polypeptide A (36). Sequences of forward and reverse primers for PPP1CB were: 5'-AACCATGAGTGTGC TAGCATCA-3' and 5'-CACCAGCATTGTCAAACTCGCC-3', designed to amplify a PCR product of 472 bp. Sequences of forward and reverse primers for POLR2A were: 5'-ACATCACTCGCCTCTTC TACTCC-3' and 5'-GTCTTGTCTCGGGCATCGT-3', giving a PCR product of 268 bp.

The PCR reaction mixture contained 0.2 µM primers, 1.5 U of JumpStart Taq DNA polymerase (Sigma), the buffer supplied, 2.5 mM MgCl2, 200 µM dNTP and cDNA in 25 µl. The PCR buffer also contained SYBR green I (1:75,000 dilution of the 10,000 x stock solution; Molecular Probes, Leiden, The Netherlands) and fluorescein (diluted 1:100,000) used as a reference dye to normalize any fluorescein signal fluctuation in the reactions. Control samples without the reverse-transcription step and no added RNA were also included in each plate to detect any possible contamination. After a hot start (3 min at 94°C), the parameters used for PCR amplification were: 10 s at 94°C, 20 s at 60°C, and 30 s at 72°C for 45 cycles, and fluorescence was measured after amplification step.

At the end of each PCR run, the data were automatically analyzed by the system and an amplification plot was generated for each DNA sample. From each of these plots, the iCycler software calculated the threshold cycle (CT), defined as the fractional cycle number at which fluorescence reaches 10x the standard deviation of the baseline. Quantitative real-time PCR values are expressed as the fold change of the target-gene expression relative to beta-actin mRNA in each sample using the following formula: fold change Formula, where {Delta}CT = CTtarget gene – CTbeta-actin and –{Delta}{Delta}CT = {Delta}CTtest sample – {Delta}CTcontrol. Real-time PCR data from a human bronchus incubated in vehicle for 15 h was arbitrarily chosen as the control and its mRNA/beta-actin mRNA was taken as 1; this sample was included in all PCR experiments to correct for possible interassay variations. Three serial dilutions of the cDNA template were prepared from each tissue and each dilution was amplified in triplicate. The experimental approach was further validated by the observation that the differences between the CT for the target gene and beta-actin remained essentially constant for each starting DNA amount.

Immunohistochemical Study
After 15 h of incubation at room temperature, 14 paired rings (control and pretreated) from 14 different patients were fixed for 24 h in 10% formalin and then embedded in paraffin. Immunolabeling used the immunoperoxidase method. Serial sections, 5 µm thick, were mounted on Superfrost Plus slides (Fisher Scientific, Fairlawn, NJ). After paraffin removal and tissue-section rehydration in graded ethanols, endogenous peroxidase activity was blocked by 100% H2O2 once for 5 min and nonspecific antibody binding sites were blocked with 20% normal swine serum. Sheep anti-human ETA or ETB monoclonal antibody (Alexis Biochemicals, San Diego, CA), diluted 1:500, was incubated with sections for 45 min at room temperature. Specific ETA or ETB antibody binding was subsequently detected with biotinylated mouse anti-sheep antibody (1:100; Vector Laboratories, Burlingame, CA) and streptavidin–peroxidase complex with nickel chloride for enhancement (Vector). Specificity of the antibodies was confirmed by co-incubating the primary antibody with specific immunizing blocking peptide (Tebu-Bio, Le Perray en Yvelines, France) in four human bronchi (data not shown). Mayer's hematoxylin was used as the counterstain. The sections were examined by light microscopy by two investigators blinded to the treatment group. Three localization patterns of labeling in the epithelial cells, airway smooth muscle, and submucosal glands were defined: apical, diffuse, and diffuse pattern with apical enhancement. Intracellular localization of labeling was expressed in percentage of bronchi (n = 14). The intensity of immunolabel was graded 0–4 semiquantitatively: 0, no staining; 1, focal or weak diffuse staining; 2, diffuse mild staining; 3, diffuse moderate staining; and 4, diffuse intense staining (37). Serial sections were also stained with periodic acid Schiff to identify mucus secretion by surface epithelial cells and submucosal glands, with specific monoclonal mouse anti-human actin (smooth muscle) antibody labeling (Dako, Glostrup, Sweden).

Data and Statistical Analysis
Values are expressed as means ± SEM. Each fenoterol-treated ring had its own paired control. Contractile responses are expressed in g calculated as follows: contraction (g) = total measured tension – basal tone. Emax, expressed in g, represents the maximal contraction induced by 0.1 µM ET-1 (5). {Delta}Emax represents the difference between Emax values obtained for the fenoterol-pretreated and its paired control bronchial rings. Potency (–log EC50) could not be calculated because a maximal contractile plateau in response to ET-1 was not achieved with the highest peptide concentration used. Fenoterol sensitization of human bronchi is characterized by a {Delta}Emax of ET-1 > 0 after 15 h (29). {Delta}mRNA represents the fold change in mRNA levels between tissues pretreated with fenoterol for 6 or 15 h and its paired control tissues, calculated for each individual pair. The results were analyzed using a two-tailed Student's t test, for paired or unpaired samples, and {chi}2 test for comparison of categorical variables (StatView 5.0; SAS Institute, Cary, NC). A P value < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Functional Study
Incubation of the 22 bronchi from 22 patients with 0.1 µM fenoterol (15 h, 21°C) significantly enhanced the ET-1-induced contraction (Figure 1A). Emax was 2.84 ± 0.27 x g in the presence of fenoterol versus 1.87 ± 0.20 x g in the paired control rings, P < 0.0001 ({Delta}Emax = 0.97 ± 0.14 x g). Removing the epithelium significantly limited the effect of fenoterol incubation on {Delta}Emax in eight paired bronchi: {Delta}Emax = 1.18 ± 0.22 x g with epithelium versus 0.10 ± 0.36 x g in absence of epithelium (P < 0.05, Figure 1B).


Figure 1
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  		Figure 1. Concentration–response curves for endothelin-1 (ET-1) in 22 human bronchi after 15 h of incubation at room temperature with 0.1 µM fenoterol (circles) or Krebs–Henseleit solution for paired control (squares) (A). Effect of the epithelium removal (B) on the fenoterol-induced enhancement of the maximal contraction in response to 0.1 µM ET-1 (n = 8). Filled squares, control with epithelium; filled circles, fenoterol (0.1 µM) with epithelium; open squares, control without epithelium; open circles, fenoterol (0.1 µM) without epithelium. {Delta}Emax: difference between maximal contractions of fenoterol-pretreated bronchi and paired control bronchi in response to 0.1 µM ET-1. Values are means ± SEM. *P < 0.05, {Delta}Emax with epithelium versus {Delta}Emax without; ***P < 0.0001, fenoterol versus paired control.

 
RT-PCR
By using end-point PCR and agarose-gel electrophoresis, we were able to detect the presence of single bands of the size predicted for the ETA-receptor (243 bp), the ETB-receptor (215 bp), PPP1CB (472 bp), POLR2A (268 bp), and beta-actin (362 bp), which appeared in all bronchi assayed (n = 32 bronchial rings from eight different patients). The identity of all cDNA fragments was confirmed by DNA sequence analysis (data not shown).

Real-time quantitative RT-PCR showed that, relative to beta-actin, PPP1CB and POLR2A mRNA amounts were not altered by the incubation time and/or fenoterol treatment. Moreover, PPP1CB and POLR2A mRNA levels in fenoterol-treated tissues were virtually identical to those observed in its paired control strips, giving {Delta}mRNA values close to 1 (Table 1).


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  		TABLE 1. {Delta}mRNA FOR ETA- AND ETB-RECEPTORS IN HUMAN BRONCHI AFTER INCUBATION WITH 0.1 µM FENOTEROL OR KREBS-HENSELEIT (PAIRED CONTROLS) FOR 6 (H6) OR 15 (H15) h

 
ETA-receptor mRNA expression did not differ significantly between H0 and H6 h (n = 4) and between H0 and H15 h in control bronchi (n = 8, Figure 2A). Compared with paired control tissues, ETA-receptor mRNA rose slightly after ring exposure to fenoterol for 6 or 15 h (Figure 2A, Table 1). ETA-receptor mRNA was 1.28- ± 0.08-fold higher in tissues treated with fenoterol for 6 h (P < 0.05, n = 4, Table 1). With the exception of one of the tissue pairs assayed, ETA-receptor mRNA expression was higher in tissues treated with fenoterol for 15 h than in its paired controls (n = 8, Table 1). However, the differences between the mRNA levels did not reach statistical significance.


Figure 2
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  		Figure 2. Real-time quantitative RT–PCR analysis of the expression of ETA (A) and ETB-receptor mRNA (B) in human bronchi at 0 h (H0, n = 8) and after incubation with 0.1 µM fenoterol (filled bars) or Krebs–Henseleit solution (controls; open bars) for 6 (H6, n = 4 tissue pairs) or 15 h (H15, n = 8 tissue pairs). Values are mean ± SEM units relative to beta-actin mRNA expression. *P < 0.05, **P < 0.01, fenoterol versus paired control.

 
ETB receptor mRNA expression in control human bronchi at H0 did not differ significantly from H6 (n = 4), but was significantly lower after incubation for 15 h in Krebs–Henseleit solution (n = 8; Figure 2B). When each pair of tissues was individually compared, ETB receptor mRNA values were higher in tissues exposed to fenoterol for 15 h than in paired control tissues (Table 1, Figure 2B).

Similar {Delta}mRNA values for ETA and ETB receptors were found when PPP1CB or POLR2A, instead of beta-actin, was used as the internal standard (data not shown).

Immunohistochemical Study
ETA receptor. In 14 control bronchi from 14 patients, immunolabeling of ETA receptors showed them to be located in the bronchial epithelium (apical pattern: 71%; diffuse with apical enhancement: 7%; diffuse: 22%) and in the submucosal glands (diffuse pattern). Variable, weak labeling of the vascular endothelium and absence of labeling in airway smooth muscle cells were also observed. Prolonged exposure to 0.1 µM fenoterol (15 h, 21°C) significantly increased the epithelial ETA receptor labeling intensity score (P = 0.01, n = 14; Figures 3A and 4B). Furthermore, fenoterol incubation significantly modified the localization of the epithelial ETA receptor labeling pattern (apical: 50%; diffuse with apical enhancement: 21%; diffuse: 29%; P = 0.002 versus paired controls). Fenoterol exposure also increased, but not significantly, the ETA-receptor labeling intensity score in submucosal glands (P = 0.08, n = 14; Figure 3B).


Figure 3
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  		Figure 3. Epithelial (A) and submucosal (B) ETA-receptor labeling intensity score in 0.1 µM fenoterol-pretreated bronchi (filled bars) and in paired controls (open bars). Values are means ± SEM (n = 14). *P < 0.05, fenoterol versus paired control.

 
ETB receptor. In control bronchi, ETB receptors were immunolocalized in the bronchial epithelium (apical pattern: 50%; diffuse with apical enhancement: 50%; diffuse: 0%), in airway smooth muscle (diffuse), and in the submucosal glands (diffuse). Variable, weak labeling of the vascular endothelium was also seen. Prolonged exposure to 0.1 µM fenoterol (15 h, 21°C) significantly increased the epithelial ETB receptor labeling intensity score (P = 0.01, n = 14; Figures 4D and 5A). Fenoterol incubation did not affect the epithelial ETB receptor localization (apical pattern: 50%; diffuse with apical enhancement: 50%; diffuse: 0%; comparable to paired controls). The smooth muscle ETB receptor labeling intensity score was similar in control and fenoterol-sensitized bronchial rings (1.50 ± 0.17 versus 1.50 ± 0.20, respectively, P > 0.99, n = 14). Fenoterol incubation also had not effect on ETB receptor labeling in submucosal glands (P = 0.72, n = 14; Figure 5B).


Figure 4
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  		Figure 4. Epithelial ETA (A, B) or ETB (C, D) receptor immunolabeling of untreated control bronchi (A, C) and in paired fenoterol-pretreated bronchi (B, D). Fenoterol enhances ETA and ETB receptor labeling in epithelial cells (arrows). Hematoxylin counterstain (x400).

 

Figure 5
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  		Figure 5. Epithelial (A) and submucosal (B) ETB receptor labeling intensity score in 0.1 µM fenoterol-pretreated bronchi (filled bars) and in paired controls (open bars). Values are means ± SEM (n = 14). *P < 0.05, fenoterol versus paired control.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we showed that prolonged exposure of human isolated bronchial rings to fenoterol, a beta2-adrenoceptor agonist, induced epithelium-dependent hyperresponsiveness to ET-1 with increased expression of the ETA and ETB receptor mRNAs and enhanced intensity of epithelial ETA and ETB receptor labeling. We also observed a fenoterol-induced change of ETA receptor localization in the epithelium.

The present data showed that ETA and ETB receptor mRNAs are locally produced in the human bronchi. This prompted us to analyze the localization of both receptor proteins by immunohistochemistry. To the best of our knowledge, this is the first description of the distributions of ETA and ETB receptor in human isolated bronchi obtained from ex-smokers. Indeed, autoradiography with [125I]–ET-1 and immunohistochemical studies showed that the localization and the function of ET-1 receptors in airways are highly species-dependent, thereby limiting the significance of the results obtained with animals (2, 3, 5, 38). While it seems that ETB receptors predominate in airway smooth muscle, the distributions of ETB and ETA receptors in human bronchial epithelium and submucosal glands remain unknown. Our observations revealed weak or mild ETA and ETB labeling in epithelial cells, especially in the apical region. In submucosal glands, ETA and ETB receptor labeling varied, with the latter only being present in airway smooth muscle.

Goldie and coworkers found low-level specific [I125]–ET-1 binding to ETB and ETA receptors in the bronchial epithelia of three nonasthmatic and three asthmatic human bronchi (22). Those authors also established that ETB is the predominant receptor in airway smooth muscle, and further established the presence of ETA binding sites in bronchial smooth muscle detected using a quantitative autoradiographic technique. Our results differ from theirs, but the different methods of tissue preparation and ET-1-receptor revelation, and the model of airway inflammation, could explain these discrepancies. Pertinently, the subjects with asthma studied by Goldie and colleagues were on beta2-adrenoceptor agonist therapy and their bronchi developed significantly stronger maximal contractile responses to K+ compared with the subjects without asthma. Moreover, the ETB/ETA receptor ratio was 88/12 in subjects without asthma versus 82/18 in those with asthma. However, Goldie and coworkers found no significant difference between the maximum specific [I125]–ET-1 binding capacities of asthmatic and nonasthmatic airway smooth muscle. The small population enrolled in that study, and the use of prednisone on the part of one of the subjects with asthma, might explain this lack of difference.

In humans and animals, epithelial ETA receptors are involved in nitric oxide (NO) and prostaglandin E2 (PGE2) production via activation of NO synthase and cyclooxygenase (COX), leading to relaxation of the underlying airway smooth muscle (3, 5, 9, 38–41). Conversely, epithelial ETA receptor stimulation may involve bronchoconstriction via lipoxygenase and COX activation (9, 38, 42). In human isolated bronchi, we previously showed that the modulation of ET-1–induced contraction implicated epithelial ETA receptor activation and NO release (40). We also found that L-nitroarginine methylester (L-NAME), an NO synthase inhibitor, failed to enhance the maximal contraction in response to ET-1 after fenoterol sensitization, suggesting that chronic exposure to fenoterol perturbs the epithelial regulation of ET-1–induced airway smooth muscle contraction (29). We showed here that fenoterol incubation upregulates ETA receptor mRNA expression, perhaps via a positive transcriptional mechanism. Indeed, cyclic adenosine monophosphate (cAMP) was shown to upregulate ETA receptor mRNA and increase contraction in response to ET-1 in rat aortic smooth muscle cells (43). In addition, prolonged stimulation of cAMP production could be responsible for an enzymatic inflammatory process (constitutive phospholipase A2 [cPLA2] and COX2) mediated by NF-{kappa}B activation (29, 44). Our results are in accordance with those findings, since fenoterol activates cAMP by stimulating adenylate cyclase. It is also known that neuropeptides upregulate ETA receptor mRNA expression (2). We recently established that exposure to fenoterol induced neurosensitization of human bronchi involving substance P, neurokinin A, and calcitonin gene–related peptide (30). Taken together, these data suggest that prolonged exposure to a beta2-adrenoceptor agonist provokes upregulations of the epithelial ETA-receptor, cPLA2, and COX2 expression, leading to impaired regulation of ET1-mediated contraction by the epithelial ETA receptor. Our results showing that epithelium removal limited the maximal contraction of fenoterol-sensitized bronchi in response to ET-1 support this hypothesis. Indeed, Naline and colleagues showed that the removal of epithelium increased the ET-1–mediated contraction of human isolated bronchi not exposed to fenoterol by abolishing NO release activated by the epithelial ETA receptor stimulation (40).

The role of the epithelial ETB receptor in human bronchus remains unclear. As for the ETA receptor, ETB receptor mRNA expression is upregulated by cAMP and neuropeptides (2, 43). Recently, Blouquit and coworkers showed that ET-1 stimulates transepithelial Cl– secretion via ETB receptors located in the apical membrane, and that this receptor stimulation probably involves the activation of the cAMP pathway (45). In agreement with that study, we found a predominant apical expression of the epithelial ETB receptor in our control human isolated bronchi. Moreover, fenoterol incubation induced a significant increase in both ETB receptor mRNA expression and the intensity of epithelial ETB receptor labeling. Our results and those obtained by Blouquit and colleagues (45) suggest that the epithelial ETB receptor is involved in the fenoterol-induced dysregulation of the ET-1–mediated contraction of human bronchi, perhaps via the activation of the proinflammatory enzymes cPLA2 and COX2 mediated by the cAMP pathway.

Our findings also demonstrated that the ETB receptor is the only ET-1 receptor present on bronchial smooth muscle. Moreover, functional studies conducted by our group and others plead for the involvement of the smooth muscle ETA receptor in human bronchoconstriction (40, 46). Our immunohistochemical labeling technique may lack sensitivity to detect a very small population of ETA receptors in airway smooth muscle. Indeed, semiquantitative grading system is a method less accurate than quantitative image analysis for immunolabeling, and its use could decrease the sensitivity to identify ETA receptors. Fenoterol incubation significantly enhanced ETB receptor mRNA expression, but did not change the intensity of smooth muscle ETB receptor labeling. This observation provides evidence that fenoterol sensitization of human bronchi involves upregulation of epithelial ET-1 receptors. Notably, we also found that ETB receptor mRNA expression was lower after 15 h of incubation in control but not in fenoterol-sensitized bronchi. This finding suggests that fenoterol prevents the decrease in mRNA expression either by increasing ETB receptor transcription and/or by decreasing its mRNA degradation. Further studies are needed to clarify this role and the absence of smooth muscle ETB receptor involvement in fenoterol sensitization. Downregulation of smooth muscle ETB receptors by proinflammatory enzymes could be an alternative approach to explore this phenomenon (47).

Fenoterol-sensitizing effects are not great, making the processes involved in these phenomena difficult to highlight. Moreover, interpreting changes in ETA and ETB receptor mRNA expression in whole bronchi to changes in protein expression over a specific region requires caution although our results showed ETA and ETB receptor immunolabeling localized in few cellular structures. While we recently inhibited the fenoterol sensitization with propranalol, a beta1- and beta2-adrenoceptor antagonist (30), additional studies are needed to define the respective role of these adrenoceptors in the mechanisms of human bronchi sensitization, and selective beta2-adrenoceptor antagonists such as ICI 118,551 could be an alternative approach to precise this point (48).

In summary, our results showed that prolonged exposure of human bronchi to fenoterol induced hyperresponsiveness to ET-1, involving epithelial ETA and ETB receptors. They suggest that fenoterol sensitization perturbs the epithelial regulation of the ET-1–mediated bronchoconstriction.


   Acknowledgments
 
The authors thank Professor Gilles Chatellier (Department of Medical Informatics and Biostatistics, Hôpital Européen Georges Pompidou, Paris, France) for statistical advice.


   Footnotes
 
This work has been supported, in part, by a grant from Ministerio de Educación y Ciencia (SAF2002–04080-C02–01).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0091OC on December 9, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 2, 2005

Accepted in final form October 6, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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J.-M. Tadie, P. Henno, I. Leroy, C. Danel, E. Naline, C. Faisy, M. Riquet, M. Levy, D. Israel-Biet, and C. Delclaux
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