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Pulmonary Pharmacology & Therapeutics xxx (2013) 1e6

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Pulmonary Pharmacology & Therapeutics

journal homepage: www.elsevier .com/locate/ypupt

Bronchorelaxation of the human bronchi by CFTR activators

Caroline Norez a, Christophe Jayle b, Frédéric Becq a, Clarisse Vandebrouck a,*

a Institut de Physiologie et Biologie Cellulaires CNRS FRE3511, Université de Poitiers, Poitiers, Franceb Service de chirurgie cardiothoracique, CHU La Milétrie, Poitiers, France

a r t i c l e i n f o

Article history:Received 21 December 2012Received in revised form18 June 2013Accepted 21 June 2013

Keywords:Human airwaySmooth muscleCFTRRelaxationPharmacology

* Corresponding author. 1 rue Georges Bonnet, Bât BFrance. Tel.: þ33 049 45 36 49; fax: þ33 549 45 40 1

E-mail address: clarisse.vandebrouck@univ-poitier

1094-5539/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.pupt.2013.06.008

Please cite this article in press as: Norez C,Therapeutics (2013), http://dx.doi.org/10.101

a b s t r a c t

The airway functions are profoundly affected in many diseases including asthma, COPD and cystic fibrosis(CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR(Cystic Fibrosis transmembrane Conductance Regulator) gene, which normally encodes a multifunctionaland integral membrane cAMP regulated and ATP gated Cl� channel expressed in airway epithelial cells.

Using human lung tissues obtained from patients undergoing surgery for lung cancer, we demon-strated that CFTR participates in bronchorelaxation. Using human bronchial smooth muscle cells(HBSMC), we applied iodide influx assay to analyze the CFTR-dependent ionic transport and immuno-fluorescence technique to localize CFTR proteins. Moreover, the relaxation was studied in isolated humanbronchial segments after pre-contraction with carbachol to determine the implication of CFTR inbronchodilation.

We found in HBSMC that the pharmacology and regulation of CFTR is similar to that of its epithelialcounterpart both for activation (using forskolin/genistein or a benzo[c]quinolizinium derivative) and forinhibition (CFTRinh-172 and GPinh5a). With human bronchial rings, we observed that whatever thecompound used including salbutamol, the activation of muscular CFTR leads to a bronchodilation afterconstriction with carbachol.

Altogether, these observations revealed that CFTR in the human airways is expressed in bronchialsmooth muscle cells and can be pharmacologically manipulated leading to the hypothesis that this ionicchannel could contribute to bronchodilation in human.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Cystic fibrosis (CF), the most common lethal autosomal reces-sive genetic disease, is caused bymutations of the CFTR gene, whichencodes the CF transmembrane Conductance Regulator (CFTR), amultifunctional cAMP-dependent Cl� channel in the apical mem-brane of secretory epithelial cells [1]. In CF, defective function ofCFTR in airway epithelial cells and submucosal glands leads toprogressive airway obstruction that begins early in life. Failure of Cl-

secretion through CFTR or associated ion channels results in thedeshydration of endobronchial secretions. Dessicated secretionsblock the airways and prevent elimination of bacteria [2]. Bronchialhyper-reactivity is also a common feature in CF, occurring in asmany as 40% of affected individuals, which further contributes tothe airway obstruction [3].

Whereas CFTR has been generally regarded as specificallyexpressed in epithelial cells [1], evidence for its expression and/or

36, BP 633, Poitiers F-86022,4.s.fr (C. Vandebrouck).

All rights reserved.

et al., Bronchorelaxation of6/j.pupt.2013.06.008

function as a Cl- conductance has been obtained in cardiac musclecells [4,5], brain [6], endothelia [7,8], in aortic, pulmonary arterial,airway smoothmuscle cells (SMC) and diaphragmmuscle [9e13]. Apossible role for Cl� pathways in the modulation of airway smoothmuscle function and implications for fundamental studies of airwayfunction as well as therapeutic approaches to pulmonary diseasehas been suggested [14]. CFTR is also expressed in rat and micetracheal smooth muscle cells (TSMC) [15]. Exploiting new phar-macological tools (CFTR activators and inhibitors) we alsoprovided evidence for its contribution to the bronchodilation [15] inrat and mice.

In the present study, we found that CFTR is expressed andfunctional in human bronchial smooth muscle cells (HBSMC) andproposed its implication in bronchodilation.

2. Materials and methods

2.1. Isolation and organ culture of human bronchi

Our institutional researchethicboard approved the protocol of ourstudy. Bronchial ringsof 5e12mminexternaldiameterwereobtained

the human bronchi by CFTR activators, Pulmonary Pharmacology &

C. Norez et al. / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e62

from normal lung tissue at the periphery of lesions removed atoperative lobectomydue to lungcarcinoma.Specimenswereobtainedfrom 26 patients (18 males 58� 8 years; 8 females 55� 7 years).

The preparation was rinsed in culture medium (DMEMeHEPESsupplemented with 1% penicillinestreptomycin, 1% Na pyruvate, 1%non-essential amino acids). Smooth muscle part of the bronchi wasdissected under sterile conditions in culturemedium andwas cut inseveral pieces (1e2mm2). Smoothmuscle pieces were placed at thebottom of individual wells of 6-well culture plates containing cul-ture medium enriched with 10% fetal calf serum (FCS). Organ cul-ture plates were placed in a humidified incubator at 37 �C under 5%CO2 in air. The medium was changed every 48 h. After one week,confluent cells were rinsed twice with Hanks’ balanced salt solu-tion and then passaged with trypsin-EDTA. Isolated cells were thenseeded in a 24-well culture plate for functional study of chloridechannel activity. Cells were left in culture medium for 48 h beforethey were growth arrested using serum-free culture mediumsupplemented with 1% insulinetransferrineselenium (ITS) (aspreviously described) [10].

2.2. Immunofluorescence study

Cells grown on glass coverslips were washed 3 times in Tris-buffered saline (TBS) and after fixation, non-specific binding siteswere blocked with TBS containing 0.5% BSA and 0.05% saponin for1 h. Cells were incubated with an anti-CFTR C-terminal monoclonalantibody (1:100, Ig2a, mouse anti-human, R&D Systems) for 2 h atroom temperature. After 3 washes, cells were incubated with theFluoProbes 488 (1:400, Interchim, Montluçon, France) secondaryantibody. In the control, the primary antibody was omitted. Nucleiwere stained in blue with TO-PRO-3 iodide (Molecular Probes,Eugene, OR) for 15 min at room temperature (1:1000 in TBS).Fluorescencewas examinedwith a spectral confocal station FV 1000installed on an inverted microscope IX-81 (Olympus, Tokyo, Japan).

2.3. Contraction measurement on isolated bronchial rings

The bronchial tissue was cut into rings of 3 mm length. Bron-chial rings were placed into Krebs solution containing (in mM): 120NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 15 NaHCO3, 11.1 D-glucose, pH 7.4. Human bronchial rings were mounted between afixed clamp at the base of a water-jacketed 5 ml organ bath con-tained an oxygenated (95% O2 and 5% CO2) Krebs solution and anIT1-25 isometric force transducer (Emka Technologies, Paris,France) (14, 15). All experiments were performed at 37 �C. A basaltension of 1 g was applied in all experiments. During 1 h, tissueswere rinsed three times in Krebs solution and the basal tone wasalways monitored and adjusted to the range 400e1000 mg [16].1 mM Carbachol (denoted CCh) were used to evoke the sustainedcontractile response. Once the sustained tension was established,the tissues were allowed to equilibrate before cumulative additionof agonist to the bath. Cumulative concentrationeresponse re-lationships for the relaxant effect of MPB compound were deter-mined in bronchial rings following stable contraction. The relaxanteffect of CFTR agonists was expressed as percentage contraction ofthe agonist-constricted human bronchial rings. IC50 was calculatedas the drug concentration inducing a half-maximal dilatation (orinhibition of contraction). Data are presented as mean � S.E.M. of nexperiments. In a separate set of experiments, the epithelial layer ofbronchial segments was gently removed by a cotton swab.

2.4. Tissue measurement of CFTR activity

CFTR Cl� channel activity was assayed on bronchial samples bymeasuring the influx of iodide (125I). At the beginning of each

Please cite this article in press as: Norez C, et al., Bronchorelaxation ofTherapeutics (2013), http://dx.doi.org/10.1016/j.pupt.2013.06.008

experiment, tissues were washed twice with Krebs buffer. Initialradioactivity (kinitial) of Krebs plus Na125I (1 mCi Na125I/l, NEN,Boston, MA) was determined using a Packard Cobra�II gammacounter (Perkin Elmer life Sciences, Courtaboeuf, France). The influxof extracellular 125I wasmeasured during 3 firstmin in Krebs-Na125Ialone (kbasal) to establish the baseline influx then, during 3 min inthe Krebs-Na125I containing the appropriate cocktail of drug (kdrug).The activation of CFTR channels was calculated as a ratio:(kdrug � kbasal)/kinitial. The effect of CFTR inhibitors was expressed asa percentage of maximal activation.

2.5. Single cell measurement of CFTR activity

CFTR function was assessed by single-cell fluorescence imaging,using the potential-sensitive probe, bis-(1,3-diethylthiobarbituricacid)trimethine oxonol (DiSBAC2(3); Molecular Probes, Eugene,OR), as previously reported [17,18].

2.6. Statistics

Results are expressed as means � SEM of n observations. Sets ofdata were compared with a Student’s t test. Differences wereconsidered statistically significant when P < 0.05. ns: non signifi-cant difference, *P < 0.05, **P < 0.01, ***P < 0.001. All statisticaltests were performed using GraphPad Prism version 4.0 for Win-dows (Graphpad Software, San Diego, CA). n corresponds to thenumber of experiments and N corresponds to the number ofpatient.

2.7. Drugs and chemical reagents

The CFTR activator benzo[c]quinolizinium compound 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride (MPB-104) wasprepared as described previously [19]. Chemicals used for prepar-ing the MPB-104 provide by Sigma. The CFTR inhibitor 20-deoxy-adenosine-methylglyoxal adducts (GPinh5a) was provided by Jean-Luc Decout [20]. Carbamylcholine, monoclonal antibody anti-smooth muscle actin, Na pyruvate, non-essential amino acidswere purchased from Sigma (Saint Quentin Fallavier, France).DMEMeHEPES, Fetal Calf Serum (FCS), PenicillineStreptomycin,trypsin-EDTA and Hanks’ balanced salt solution were purchasedfrom Gibco (Invitrogen Corporation, Cergy Pontoise, France).CFTRinh-172 [21] was purchased from Calbiochem (USA). All drugswere prepared in dimethyl sulfoxide (DMSO) except carbachol andGPinh5a that were prepared as stock solution in distilled water.

3. Results

3.1. CFTR expression in organ culture of HBSMC

Immunostaining with monoclonal anti-a-smooth muscle actinantibody was positive for all cells demonstrating the presence of anhomogenous population of smooth muscle cells (data not shown).To study the presence of CFTR in HBSMC, its localization was per-formed by indirect immunofluorescence confocal microscopy usinganti-CFTR C-terminal monoclonal antibody. Fig. 1A shows diffuseexpression of CFTR throughout the cytoplasm of organ culture ofHBSMC. No staining was detected when the primary antibody wasomitted (data not shown).

3.2. Functionality of CFTR protein in HBSMC

In the next series of experiments we studied the transportfunction of CFTR in HBSMC by using a cocktail composed by theadenylate cyclase activator forskolin (Fsk) and the isoflavone

the human bronchi by CFTR activators, Pulmonary Pharmacology &

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Fig. 1. Presence and functional evaluation of CFTR by DisBAC2(3) assay in HBSMC. A.Immunofluorescence study of CFTR in HBSMC. Scale bar is 10 mm. B. Example of typicaltime course obtained with HBSMC stimulated by a mixture of 10 mM Forskolin (Fsk)plus 30 mM Genistein (Gst) used to activate CFTR. CFTRinh-172 (10 mM) is used to inhibitCFTR. C. Histograms report the mean of the relative fluorescence collected from twoseparate experiments (N ¼ 2) with a total of cells from 14 to 25. CFTRinh-172 (10 mM)and GPinh5a (200 pM) are used to inhibit CFTR. *** t-test vs. Fsk þ Gst stimulation.

C. Norez et al. / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e6 3

genistein (Gst) to stimulate its channel activity. To that end, wemeasured ionic transport by single-cell fluorescence imaging usingthe membrane potential-sensitive fluorescent probe, DiSBAC2(3).This method is well adapted to our study because with each humansample we isolated a low number of smoothmuscle cells. As shownin Fig. 1B, a sharp increase of fluorescence, corresponding to a

Please cite this article in press as: Norez C, et al., Bronchorelaxation ofTherapeutics (2013), http://dx.doi.org/10.1016/j.pupt.2013.06.008

membrane depolarization, was recorded in HBSMC after addition ofthe CFTR activators Fskþ Gst. These results confirm the presence ofa functional CFTResensitive transport on HBSMC. We tested thethiazolidinone compound CFTRinh-172, which has been developedas a selective CFTR blocker with no significant inhibitory action onother Cl� channels, and especially on the volume- and calcium-activated Cl� channels [10,21]. The decrease in the fluorescencelevel observed after addition of the CFTR inhibitor, CFTRinh-172,further confirmed the identity of CFTR (Fig. 1B, C). We also used thea-aminoazaheterocycle-methylglyoxal adduct 5a (denoted hereGPinh5a), a highly potent inhibitor of CFTR channels with pico-molar affinity [20]. We tested GPinh5a at 200 pM on HBSMCstimulated by CFTR activators Fsk/Gst and also observed a completeinhibition of CFTR activity (Fig. 1C).

In a next series of experiments, we directly activated muscularCFTR via a cAMP-independent pathway using the epithelial CFTRactivator: MPB-104 [9,10,19]. As shown in Fig. 2A and B, a sharpincrease of fluorescence, corresponding to a membrane depolari-zation, was recorded in HBSMC after addition of MPB-104. Thedecrease in the fluorescence level observed after addition of theCFTR inhibitors, CFTRinh-172 or GPinh5a, further confirmed theidentity of CFTR (Fig. 2). These results show that CFTR in HBSMCshares numerous pharmacological properties with that determinedfor epithelial and aortic CFTR [9,10,22,23].

3.3. Role of CFTR in agonist-dependent bronchodilation of smoothmuscle cells

In order to confirm our results at the ex-vivo level, we adaptedthe iodide influx (I125) assay to test the activity of CFTR directly onhuman bronchial tissue. To that end, we found that the stimulationby Fsk/Gst (Fig. 3A) or by MPB-104 (Fig. 3B) both induced a sig-nificant iodide influx on human bronchial tissue (gray bars). Thetwo CFTR inhibitors (CFTRinh-172 and GPinh5a) inhibited theseiodide influxes (Fig. 3A, B) in a concentration-dependent manner.Then, we performed experiments on human bronchi rings moun-ted in an organ bath apparatus and measured their muscular ac-tivity. Initial experiments were carried out to evaluate thecontractile response to carbachol (CCh). We obtained aconcentration-response curve for CCh used between 10�7 to 10�3Mand determined a half maximal response EC50 of 1.1�1.8 mM (n¼ 8,N ¼ 3). This concentration of CCh was then used in the next ex-periments described below. The CCh-induced constriction reachedamaximum, indicated by a plateau phase, and then declined slowlyduring 4 h. The vehicle DMSO had no effect on the maximumresponse (data not shown). We applied the benzo[c]quinoliziniumCFTR activator MPB-104, via cumulative application into the organbath (1e100 mM). MPB-104 induced a concentration-dependentrelaxation of human bronchi ring preconstricted by 1 mM CCh(Fig. 4A, B) that began at 3 mM and was complete for 100 mM. From25 different bronchi rings we determined half-maximal relaxationvalue IC50 for the CFTR activator MPB-104; IC50of 34.1 � 1.1 mM(n ¼ 25, N ¼ 7) (Fig. 4B). We also compared the relaxant effect ofMPB-104 on human bronchi rings to four classical CFTR activatorsi.e. Fsk, Gst, the xanthine theophylline (for review [22]) and the b2adrenergic agonist salbutamol [24] (Fig. 4B). Concentrationeeffectrelationships reveal the following order of potency: Salbutamol(IC50 ¼ 49.6 � 1.5 nM, n ¼ 13, N ¼ 3) > Fsk (IC50 ¼ 1.3 � 1.1 mM,n ¼ 7 , N ¼ 2) > MPB-104 (IC50 ¼ 34.1 � 1.1 mM, n ¼ 25,N ¼ 7) > genistein (IC50 ¼ 75 � 5 mM, n ¼ 10, N ¼ 3) > theophylline(IC50 ¼ 396 � 6 mM, n ¼ 7, N ¼ 2) (Fig. 4B).

In a next series of experiments, to clarify the role of muscularCFTR in the bronchi relaxation, we induced bronchodilation byMPB-104 [25] and evaluated the effect of the selective CFTR in-hibitors CFTRinh-172 and GPinh5a to antagonize the effect of MPB-

the human bronchi by CFTR activators, Pulmonary Pharmacology &

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Fig. 2. Activation of CFTR by MPB-104 in HBSMC using DisBAC2(3) assay. A. Example oftypical time course obtained with HBSMC stimulated by 100 mM MPB-104 used toactivate CFTR. CFTRinh-172 (10 mM) is used to inhibit CFTR. B. Example of typical timecourse obtained with HBSMC stimulated by 100 mM MPB-104 used to activate CFTR.GPinh5a (200 pM) is used to inhibit CFTR. C. Histograms report the mean of the relativefluorescence collected from two separate experiments (N ¼ 2) with a total of cells from14 to 25. CFTRinh-172 (10 mM) and GPinh5a (200 pM) are used to inhibit CFTR. *** t-testvs. MPB-104 stimulation.

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Fig. 3. Measurement of the activity of CFTR stimulated by Fsk þ Gst or MPB-104 usingiodide influx (I125) on human bronchial tissue. A. Stimulation by forskolin þ genistein(Fsk þ Gst) (gray bar) induced a significant iodide influx on human bronchial tissueinhibited by two CFTR inhibitors: GPinh5a and CFTRinh-172. * ¼ t-test vs unstimulated,# ¼ t-test stimulated vs. stimulated þ CFTR inhibitors. B. Stimulation by MPB-104 (graybar) induced a significant iodide influx on human bronchial tissue inhibited by the twoCFTR inhibitors: GPinh5a and CFTRinh-172. * ¼ t-test vs. unstimulated, # ¼ t-teststimulated vs. stimulated þ CFTR inhibitors. AeB. The number of experiments isindicated below each bar graph as number of rings, number of patients.

C. Norez et al. / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e64

104. Fig. 5A shows that in the presence of 100 mM CFTRinh-172 or200 pMGPinh5a, the concentration response of MPB-104 is shifted.Moreover, we observed a partial inhibition of the relaxation withthe concentration of inhibitors used. Then, to access the role of theepithelium, experiments were performed with intact or denudedepithelium. No significant difference was observed with or withoutepithelium (Fig. 5B) suggesting that MPB-104 induced a bronchirelaxation by an epithelial-independent pathway. All these exper-iments highlighted that the activation of muscular CFTR leads torelaxation of human bronchi.

Please cite this article in press as: Norez C, et al., Bronchorelaxation ofTherapeutics (2013), http://dx.doi.org/10.1016/j.pupt.2013.06.008

4. Discussion

The present study highlights that human bronchi SMC express afunctional CFTR chloride channel and that its activation leads to abronchodilation after muscarinic contraction. Based on our exper-iments, a number of important findings could be summarized asfollow: (i) in vitro, the human smooth muscle CFTR can be activatedby different pharmacological agents activators of CFTR i.e. Fsk, Gst,MPB-104, salbutamol and theophylline and inhibited by the twoselective inhibitors CFTRinh-172 and GPinh5a, (ii) ex vivo, using io-dide influx technique on human bronchial tissue, we confirmed theactivity of CFTR following a stimulation by Fsk/Gst or by MPB-104and its specific inhibitory profile, (iii) using isometric contraction

the human bronchi by CFTR activators, Pulmonary Pharmacology &

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Fig. 4. Bronchodilation effect of the CFTR activator MPB-104 on human bronchi andcomparaison with several CFTR activators. A. Typical trace from experiments per-formed with human bronchial rings preconstricted with 1 mM carbachol (CCh), andrepresenting the effect on tension of various concentrations of MPB-104. B. Therelaxant activity of MPB-104 (n ¼ 25, N ¼ 7) was compared against Fsk (n ¼ 7, N ¼ 2),Gst (n ¼ 10, N ¼ 3), salbutamol (n ¼ 13, N ¼ 3), and theophylline (n ¼ 7, N ¼ 2) inairways contracted with 1 mM carbachol. Concentrationeeffect relationships reveal anorder of potency: Salbutamol (>) > Fsk (*) > MPB-104 (C) > genistein(,) > theophylline (7).

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Fig. 5. Effects of CFTR inhibitors and epithelium on CFTR-dependent bronchodilation ofhuman bronchial smoothmuscle cells. A. Concentration-dependent curves showing thebronchodilation of bronchial rings precontracted by 1 mM CCh for MPB-104 in absence(IC50¼ 34.1�1.1 mM, n¼ 25,N¼ 7) or inpresence of 100 mMCFTRinh-172 (IC50>150 mM,n ¼ 7, N ¼ 2) or in presence of 200 pM GPinh5a (IC50 > 150 mM, n ¼ 12, N ¼ 3). B.Concentration-dependent curves showing the bronchodilation of bronchial rings pre-contracted by 1 mM CCh for MPB-104 in intact (IC50 ¼ 35.1 � 2.1 mM, n ¼ 4 N ¼ 2) or inepithelium denuded (IC50 ¼ 36 � 1.1 mM, n ¼ 4 N ¼ 2) bronchial rings.

C. Norez et al. / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e6 5

measurement on human isolated bronchial rings, we found that theactivation of CFTR by either Fsk, MPB-104, Gst, theophylline orsalbutamol leads to a concentration-dependent bronchodilationand (iv) the relaxation induced by CFTR activation is not dependenton the bronchial epithelial cells.

The airway is a complex systemwithmore than twenty differentcell types, a smooth muscle layer surrounding an epithelial layerfacing the lumen. In this multicellular organ, CFTR is functionallyexpressed both in epithelial and in smooth muscle cells [1,9e11,13].However, the link between CFTR-mediated ion transport and thelung physiology has been the subject of intense debate and remainspoorly understood. The role of the airway epithelium in modifyingthe contractility of the underlying smooth muscle has been sug-gested but is not yet fully demonstrated. Indeed some studiessuggest that epithelium can be an active source of mediators, asCa2þ, substance P or NO, that relax constricted airways [26e28]. Inthe same way, another study demonstrated an epithelium-dependent relaxation of mouse tracheal smooth muscle via achloride e but not CFTR-dependent pathway via the release ofendogenous relaxing factors [14]. In the present study, we proposedthat activation of CFTR induced a bronchorelaxation independent ofthe bronchial epithelium.

In disorders of the conducting airways like asthma, COPD andCF, understanding the molecular mechanisms controlling the con-tractile state of the airway smooth muscle cell may generate newtherapeutic opportunities. In CF, chronic endobronchial infection isa primary feature of the pulmonary disease. In addition, defectivefunction of CFTR in airway epithelial cells and submucosal glandsresults in chronic involvement of the respiratory tract, manifestedby progressive airway obstruction that begins early in life [2,29].Asthma pathogenesis is characterized by progressive airway wallremodeling that includes, in part, local inflammation and fibrosis aswell as increased airway smooth muscle mass [2,30]. Hays et al.demonstrated structural changes of airway smooth muscle in CF[2,31]. In particular it was noted an increased smooth muscle

Please cite this article in press as: Norez C, et al., Bronchorelaxation ofTherapeutics (2013), http://dx.doi.org/10.1016/j.pupt.2013.06.008

content of the airway in subjects with CF compared to healthycontrols, due to smooth muscle cell hyperplasia without hyper-trophy [2,31]. These findings imply that smooth muscle cell pro-liferation is a characteristic of airway remodeling in CF [2,31].Although further studies will be required, these information,together with our findings on CFTR expression and function inairway smooth muscle, suggest that CFTR in the airways may havecomplex functions depending of the cell type in which it is func-tional as a chloride channel.

Recent progress into the pharmacology of chloride channels andparticularly of CFTR channels provided interesting new tools tostudy the contribution of these transport proteins into organphysiology. We took advantage of these new CFTR activators orinhibitors MPB-104 [19], CFTRinh-172 [21] and GPinh5a [20] tomonitor the muscular reactivity of isolated human bronchial rings.Using these agents we observed that the relaxation induced byMPB-104 in a dose-dependent manner with precontracted bronchiis strongly inhibited by the two potent CFTR inhibitors i.e. CFTRinh-172 or GPinh5a. These results indicate that CFTR is the major ionicchannel responsible for the cAMP-regulated Cl� transport inHBSMC and demonstrate an unexpected role of CFTR in broncho-dilation after muscarinic stimulation. It is well known that airwaysmooth muscle relaxation is predominantly brought about bystimulation of adenylyl cyclase-coupled receptors (e.g. b2-adreno-ceptor) resulting in elevation of cell cyclic adenosine mono-phosphate content. Importantly, this signaling pathway is central inactivating CFTR-mediated chloride transport in epithelial [32],aortic and airway smoothmuscle cells. Taken together these resultsilluminate a direct implication for CFTR in the bronchodilation ofthe human bronchi.

This pharmacological evidence set the stage for the discovery ofa new and innovative approach to identify novel bronchodilators

the human bronchi by CFTR activators, Pulmonary Pharmacology &

C. Norez et al. / Pulmonary Pharmacology & Therapeutics xxx (2013) 1e66

and to develop new therapeutics for individuals suffering from adisorder of the contracting airways.

Acknowledgments

This work was supported by CNRS and the University of Poitiers.The authors thank Yvette Mettey for the obtention of MPB-104 andJean-Luc Decout for providing the GPinh5a.

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the human bronchi by CFTR activators, Pulmonary Pharmacology &