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Published ahead of print on March 6, 2003, doi:10.1165/rcmb.2002-0104OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 245-251, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0104OC

Effects of Endothelin-1 on Epithelial Ion Transport in Human Airways

Sabine Blouquit*, Anouar Sari*, Alain Lombet, Michelle D'herbomez, Emmanuel Naline, Regis Matran and Thierry Chinet

Laboratoire de Biologie et Pharmacologie des Epithéliums Respiratoires, Boulogne; INSERM U339 UFR Saint Antoine, Paris; Département de Biophysique, University of Lille, Lille; Institut de Pharmacologie, Paris, France; and Département de Physiologie, University of Lille, Lille, France

Address correspondence to: Professeur Thierry Chinet, Laboratoire de Biologie et Pharmacologie des Epithéliums Respiratoires, UFR Paris Ile de France Ouest, Université de Versailles Saint Quentin en Yvelines, Hôpital Ambroise Paré, 9 avenue Charles de Gaulle, 92104 Boulogne cedex, France. E-mail: thierry.chinet{at}apr.ap-hop-paris.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10-7 M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10-7 M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1–induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1–induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.

Abbreviations: intracellular free Ca concentration, [Ca]i • cyclic adenosine monophosphate, cAMP • Dulbecco's modified Eagle's medium and Ham's F12 mixture, DMEM/F12 • endothelin-1, ET-1 • short circuit current, Isc • Kreb's bicarbonate Ringer, KBR • phosphate-buffered saline, PBS • potential difference, PD


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Endothelin 1 (ET-1) belongs to a family of potent peptidic vasoconstrictor agents that exert an array of biological effects in addition to constriction of the vascular smooth muscle cells (1, 2). In airways, several studies have shown that ET-1 causes sustained contraction and proliferation of bronchial smooth muscle cells. ET-1 also acts on airway epithelial cells. Animal studies have found that ET-1 increases the cilia beat frequency, activates mucus secretion by submucosal glands, and stimulates the proliferation of epithelial cells (3–7). In humans, ET-1 stimulates lactoferrin and mucous glycoprotein release from serous and mucous cells in cultured nasal mucosal explants (8), and may affect expression of genes in bronchial epithelial cells such as the fibronectin gene (9). The presence of immunoreactive ET-1 and abundant binding sites for ET-1 in airways suggests that this peptide is an important autocrine and/or paracrine neuromodulator of airway functions (10, 11).

In view of these data, we raised the hypothesis that ET-1 may also contribute to the regulation of transepithelial ion transport, another major function of human airway epithelial cells. Airway epithelial ion transport processes regulate the volume of airway surface liquid and airway secretions (12). The net movement of salt and fluid across human airway epithelium is generally regarded as the result of two opposite active ion transports: Na absorption and Cl secretion. Active Na absorption predominates in the basal state and induces fluid absorption from the lumen. Active Cl secretion is the driving force for fluid secretion in human airways and can be stimulated by various agents, including adenosine 3',5'-cyclic monophosphate (cAMP)-activating agents and purinergic agonists such as adenosine 5'-triphosphate (ATP).

ET-1 could affect Na absorption across human airway epithelium because recently published data suggest that ET-1 may be an important negative regulator of ENaC: adult rats lacking functional ETB receptor activity display enhanced Na absorption in the distal nephron (13); furthermore, in vitro studies in cell lines expressing ENaC have demonstrated that ET-1 potently inhibits ENaC via ETB receptors, and that this effect is mediated by Src family kinases (14).

ET-1 could also participate in the regulation of Cl secretion across human airway epithelium because intranasal administration of ET-1 in allergic and nonallergic subjects induces symptoms of rhinorrhea and increases the amount of secretions (15) and because this peptide regulates Cl secretion in other epithelia. However, the effects of ET-1 on epithelial Cl secretion–i.e., the nature of the effect (stimulation versus inhibition), its magnitude and its mechanism–differ between tissues and between species. In human gallbladder, ET-1 inhibits cAMP-induced Cl secretion (16), whereas in human intestine, ET-1 stimulates Cl secretion in part via the activation of enteric nerves (17). In dog airways, ET-1 increases electrogenic Cl secretion (3, 18, 19), and this effect is mediated via the secondary production of cyclooxygenase products such as PGE2.

The goal of this study was therefore to determine whether ET-1 regulates active Na absorption and/or active Cl secretion in human airway epithelium, and to describe the mechanisms of ET-1–mediated regulation of transepithelial ion transport. We used cultured human bronchial epithelial cells to ensure that the effect of ET-1 on ion transport would not be mediated by nonepithelial airway mucosal cells and by airway nerves.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Preparations
Human bronchi were obtained from pieces of lobectomy or pneumonectomy (usually removed for lung carcinoma) from 26 patients (age 61.7 ± 2.6 yr; 21 male, all smokers) in agreement with the current French legislation. Tissues used for experimental studies were taken from macroscopically normal areas distant from the pathological tissue. Bronchi were rinsed with Ham's F12 medium (Sigma Chemicals, Saint Quentin Fallavier, France), dissected free of adjacent parenchyma, washed again with F12, and incubated for 2–12 h at 4°C in Dulbecco's modified Eagle's medium (Gibco, Cergy Pontoise, France) and Ham's F12 (1:1) mixture (DMEM/F12) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamycin, and 5 µg/ml fungizone. The preparations were then washed with DMEM/F12 and incubated in DMEM/F12 with 0.1% protease and 0.01% DNase for 48 h at 4°C. Fetal bovine serum (10%) was then added to neutralize the enzymes, and cells were detached by gentle agitation. The resulting suspension was centrifuged (500 x g, 5 min, 10°C). The cell pellet was resuspended and plated on home-made collagen matrix supports affixed to an orifice drilled in polycarbonate cups at a density of 5 x 105 cells/cm2. The diameter of the aperture was 3.5 mm. The collagen matrix was made of calfskin type I collagen, which was diluted in 0.2% acetic acid at a concentration of 15 mg/ml, mixed 2:1 with glutaraldehyde (2.5% in phosphate-buffered saline [PBS]) and spread over the orifice of the polycarbonate cups. The culture medium consisted of Ham's F12 supplemented with: insulin (5 µg/ml), endothelial cell growth supplement (2 µg/ml), epithelial growth factor (25 ng/ml), hydrocortisone (10-6 M), transferrin (7.5 µg/ml), and triiodothyronine (3 x 10-8 M), L-glutamine (1 mM), penicillin/streptomycin (100 µg/ml), gentamycin (50 µg/ml), and amphotericin B (5 µg/ml). The preparations were fed every other day with culture medium and incubated at 37°C, 5% CO2–95% air, in a tissue culture incubator.

Transepithelial Electrical Recordings
Five to ten days after plating, the cell culture preparations were mounted in Ussing-type chambers. The apical and basolateral surfaces of the preparations were bathed by 10 ml of Kreb's bicarbonate Ringer (KBR) solutions gassed with 95% O2–5% CO2. Experiments were conducted at 37°C. The short-circuit current (Isc) was monitored continuously using a DVC1000 voltage clamp (WPI, Aston, UK), and the potential difference (PD) was measured every 5–10 min. Voltage-sensing electrodes consisted of 3 mol/liter KCl-agar bridges; the reference electrode was placed at the basolateral side. Current-passing bridges consisted of KBR-agar bridges. Transepithelial resistance (R) was determined by clamping the PD at +10 mV at 25-s intervals, recording the deflection in Isc, and applying Ohm's law. Cell preparations were allowed to equilibrate until stabilization of bioelectric variables which required ~ 20–40 min. Basal bioelectric activity was monitored for 10 min before addition of drugs.

The following pharmaceutical agents were used: ET-1, amiloride, forskolin, ATP, the ETA receptor-selective antagonist BQ123 and the ETB receptor-selective antagonist BQ788. Agents were added to the apical and/or basolateral bathing solutions, and bioelectric activity was monitored for at least 10 min thereafter. Amiloride (10-5 M), an Na channel inhibitor, was added first to inhibit Na absorption. The amiloride-sensitive Isc is a measure of Na absorption, and the residual amiloride-insensitive Isc is a measure of Cl secretion (12). The Cl secretagogues forskolin and ATP were added sequentially in the presence of amiloride. Forskolin (10-5 M), an activator of the cAMP pathway, was added to the apical and basolateral baths, whereas ATP (10-4 mol/liter), which increases the intracellular Ca concentration, was added to the apical bath only. ET-1 was added to the apical or the basolateral bath as indicated. BQ123 (10-6 M) and BQ788 (10-6 M) were added to the apical bath only. Changes in R and Isc were calculated as the variations between the values measured immediately before the addition of reagents and the values corresponding to the plateau phase after addition of amiloride, ET-1, and forskolin and corresponding to the maximal change after addition of ATP. To determine the contribution of Cl transport, some experiments were performed with a low-Cl solution on both the apical and basolateral sides of the preparations, and with bumetanide (10-5 M), an inhibitor of the Na/K/2Cl cotransporter, in the basolateral solution. To investigate whether Ca mobilization is important for ET-1–induced Cl secretion, we added the Ca chelator 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetomethoxy ester (BAPTA-AM, 10-5 M) to the apical and basolateral baths, 60 min before successive additions of amiloride, ET-1, and ATP.

Cyclooxygenase Inhibition
To determine the role of cyclooxygenase product formation in ET-1–stimulated Cl secretion, we added indomethacin 10-5 M (vehicle: ethanol) to the apical and basolateral baths after addition of amiloride. The bioelectric variables of the preparations were then recorded during at least 20 min before addition of ET-1 (10-7 M) to the apical bath.

Measurement of Intracellular cAMP Level
Freshly isolated cultured human bronchial epithelial cells were grown to confluence on polycarbonate surface coated with collagen. Preparations were exposed for 10 min on their apical side to ET-1 (10-7 M), vehicle (acetic acid 0.2%), or 3-isobutyl-1-methylxanthine (IBMX 10-6 M), a cAMP phosphodiesterase inhibitor. Cells were then lysed by apical addition of 40 µl of perchloric acid and centrifuged at 2,350 x g for 5 min. The excess perchloric acid in the supernatant was neutralized by potassium carbonate. cAMP was assayed in the supernatant by radioimmunoassay (Amersham Pharmacia Biotech, Buckinghamshire, UK). The protein content of cell suspension was quantified and intracellular cAMP levels were expressed as pmol/mg protein.

Measurement of Intracellular Free Ca Concentration
To measure intracellular free Ca concentration ([Ca]i), isolated bronchial epithelial cells were seeded at low density on glass coverslips coated with collagen and fed with culture medium. One or two days later, they were washed with PBS and loaded with 2.5 µmol/liter Fura-2/AM for 2 h at 37°C in PBS supplemented with (in mmol/liter): 1.3 CaCl2, 0.8 MgCl2, 20 HEPES/Tris, and 5 glucose (pH 7.3). After washing with this solution, the coverslips were placed on an inverted fluorescence microscope connected to a dynamic imaging system (QuantiCell 700; Visitech International Ltd, Sunderland, UK). Fluorescence images were recorded every 2 s, and [Ca]i were calculated from the ratio of the fluorescence intensities at 340 and 380 nm on a pixel basis. The Ca ionophore ionomycin was used to calibrate the Fura-2 fluorescence ratio signal. The 340 nm/380 nm ratio was converted to an actual [Ca]i measurement by using the formula derived from Grynkiewicz and coworkers (20). For each preparation, [Ca]i was measured on ~ 60 cells. After an initial 1-min long recording, ET-1 (10-7 M) or vehicle (acetic acid 0.2%) was added and fluorescence recorded for another 2 min. Thapsigargin (10-6 M), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, was then added and fluorescence recorded until a plateau was reached.

Solutions and Drugs
The composition of the regular KBR solution was (in mmol/liter): 120 NaCl, 0.7 Na2HPO4, 1.5 NaH2PO4, 2 CaCl2, 0.5 MgCl2, 0.45 KCl, 15 NaHCO3, and 1 glucose (pH 7.3). All but 4–6 mM Cl were replaced with gluconate in the low-Cl solution. The composition of PBS medium was (in mmol/liter): 140 NaCl, 4 KCl, 0.5 Na2HPO4, 0.15 KH2PO4 (pH 7.4). ET-1 was used at a final concentration of 10-9 M to 10-6 M (stock solution: 10-4 M in acetic acid 1N). Fura-2 was obtained from Molecular Probes (Leidin, Netherlands). All salts and drugs were purchased from Sigma Chemicals, except BQ123 and BQ788, which were obtained from Bachem (Voisins-Le-Bretonneux, France).

Data Analysis
Results are expressed as means ± SEM for n preparations. Comparisons were made using the Wilcoxon's matched pair test and the unpaired or paired Student's t test as appropriate. A P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transepithelial Electrical Recordings
The baseline PD, Isc, and R of cultured human bronchial epithelial cells were, respectively, 8.4 ± 1.9 mV, 52.9 ± 8.4 µA/cm2, and 160.5 ± 28.9 {Omega}.cm2 (n = 10). Amiloride induced a significant decrease in Isc by 40.3 ± 8.5 µA/cm2 (n = 10; P < 0.05), whereas subsequent additions of forskolin and then ATP increased Isc by 5.8 ± 1.2 and 32.2 ± 8.2 µA/cm2, respectively (n = 10; P < 0.05 for both).

When ET-1 (10-7 M) was added to the apical and basolateral baths, we observed a significant increase in Isc (2.7 ± 0.5 µA/cm2, n = 9, for ET-1, versus 0.2 ± 0.3 µA/cm2, n = 7, for vehicle; P < 0.05). However, we observed no significant effect of ET-1 on the decrease in Isc induced by the subsequent addition of amiloride (-25.9 ± 7.3 µA/cm2, n = 9 for ET-1, versus –20.4 ± 5.2 µA/cm2, n = 7 for vehicle, not significant [NS]).

In the presence of amiloride, apical addition of ET-1 (10-7 M) was typically characterized by an increase in Isc followed by a slow decrease toward initial values over ~ 10 min (Figure 1). In some instances, a transient decrease in Isc below initial values followed the initial peak. Addition of vehicle (acetic acid 0.2%) had no effect on Isc ({Delta}Isc = -0.5 ± 0.9, n = 4, NS). Table 1 summarizes the results of similar experiments. To ascertain that the ET-1–induced increase in Isc in the presence of amiloride could be attributed to stimulation of Cl secretion, we performed similar experiments with low-Cl solutions on both sides and in the presence of bumetanide in the basolateral bath. Under these conditions, the ET-1–induced increase in Isc was significantly inhibited, indicating that ET-1 stimulates Cl secretion when added to the apical side (Table 1). The responses to forskolin and ATP were also significantly reduced under these experimental conditions.



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  		Figure 1. Representative tracings of Isc in cultured human bronchial epithelial cells exposed to amiloride (10-5 M, apical side), ET-1 (10-7 M, apical side), or vehicle, forskolin (10-5 M both sides) and ATP (10-4 M, apical side). Drugs were added sequentially after stabilization of baseline Isc.

 

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  		TABLE 1 Baseline Isc and changes in Isc induced by sequential additions of amiloride, ET-1, forskolin and ATP in cultured human bronchial epithelial cells

 
Addition of ET-1 (10-7 M) to the basolateral bath of amiloride-pretreated preparations had no significant effect on Isc ({Delta}Isc= 0.0 ± 0.3 µA/cm2, n = 5, NS). This indicates that ET-1 stimulates Cl secretion only through receptors located in the apical side of cultured bronchial epithelial cells.

To construct the dose–effect curve, bronchial epithelial cell cultures were exposed to concentrations of ET-1 ranging from 10-9 M to 10-6 M in the presence of amiloride. The dose–response curve is displayed in Figure 2. The 10-6 M concentration yielded a biphasic response with an increase in Isc immediately followed by a transient decrease in Isc. This curve yields an half-maximal effective concentration (EC50) of ~ 10 nM.



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  		Figure 2. Changes in Isc induced by various concentrations of ET-1 added in the apical bath of bronchial epithelial cell cultures in the presence of amiloride (n = 6). *Significantly different from 10-9 M (P < 0.05). {dagger}Significantly different from 10-7 M (P < 0.05).

 
Determination of the ET Receptors Mediating the ET-1–Induced Increase in Cl Secretion
To determine which receptors were involved in the stimulation of Cl secretion by ET-1, the ETA receptor inhibitor BQ-123 or the ETB receptor inhibitor BQ-788 were added in the apical bath in the presence of amiloride. Neither BQ-123 (10-6 M) nor BQ-788 (10-6 M) significantly modified Isc ({Delta}Isc= 0.5 ± 0.4 µA/cm2 and 0.3 ± 0.3 µA/cm2, respectively; n = 6 for both). Preparations were exposed to ET-1 15 min later. Pre-incubation with BQ-123 had no effect on the subsequent ET-1–induced increase in Isc ({Delta}Isc= 4.6 ± 1.4 µA/cm2, n = 6). In contrast, in the presence of BQ-788, ET-1 failed to increase Isc ({Delta}Isc= -0.1 ± 0.2 µA/cm2, n = 6). These data indicate that ETB, receptors but not ETA receptors, mediate ET-1–induced Cl secretion.

Effect of Indomethacin on the ET-1–Induced Increase in Isc
Because previous studies have suggested that stimulation of epithelial Cl secretion by ET-1 could be attributed to the secondary production of PGE2 in some tissues, we tested the effects of indomethacin, an inhibitor of cyclooxygenase product formation, on the ET-1–induced increase in Isc in the presence of amiloride. Indomethacin 10-5 M (or vehicle) was added to the apical and basolateral baths 20 min before addition of ET-1 10-7 M to the apical bath. As shown in Table 2, we found no significant effect of indomethacin on the ET-1–induced increase in Isc.


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  		TABLE 2 Effects of indomethacin on the changes in Isc induced by ET-1 in cultured human bronchial epithelial cells

 
Effect of ET-1 on cAMP Production
We next tested the effect of ET-1 on the production of intracellular cAMP in this epithelium. Figure 3 shows that ET-1 increased cAMP from basal levels of 0.8 ± 0.2 (n = 5) to 10.3 ± 2.2 pmol/mg protein (n = 6; P < 0.01). Apical addition of IBMX increased cAMP levels to 11.0 ± 1.5 pmol/mg protein (n = 6; P < 0.01 compared with control, NS compared with ET-1).



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  		Figure 3. Intracellular concentration of cAMP in cultured human bronchial epithelial cells under baseline conditions and after addition of ET-1 (10-7 M) or IBMX (10-6 M) (n = 6). *Significantly different from baseline value (P < 0.01).

 
Effect of ET-1 on [Ca]i
We then studied the effect of ET-1 on [Ca]i. Nine preparations of cultured human bronchial epithelial cells were loaded with Fura-2 and exposed to ET-1. Baseline [Ca]i was 144.2 ± 2.3 nM. No significant change in [Ca]i was observed after addition of ET-1. For instance, 30 s after addition of ET-1, [Ca]i was 145.2 ± 2.8 nM (NS as compared with baseline value). Subsequent addition of thapsigargin increased [Ca]i to 233.8 ± 14.4 nM (P < 0.01).

Effect of BAPTA-AM on the ET-1–Induced Increase in Isc
To further investigate the contribution of a Ca second messenger component in the ET-1 effect on Cl secretion, we exposed the preparations to the Ca chelator BAPTA-AM for 60 min before addition of amiloride, ET-1, and ATP. As shown in Table 3, pre-incubation with BAPTA-AM had no significant effect on basal Isc, amiloride-induced decrease in Isc, and ET-1–induced increase in Isc. In contrast, the ATP-stimulated Cl secretion was significantly inhibited by BAPTA.


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  		TABLE 3 Effects of BAPTA-AM on the basal Isc and on the changes in Isc induced by subsequent additions of amiloride, ET-1, and ATP in cultured human bronchial epithelial cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that ET-1 has no significant effect on Na absorption, but activates Cl secretion across cultured human bronchial epithelial cells. ET-1 evoked a sustained increase in Isc, indicating stimulation of active ion transport. This effect could be attributed to stimulation of Cl secretion because ET-1 increased Isc in the presence of amiloride, an inhibitor of Na absorption, the other major active transepithelial ion transport in airways, and because the increase in Isc was significantly inhibited by depleting bathing solutions in Cl. In human airways, transepithelial Cl secretion can be activated by agents that raise the intracellular concentration of cAMP and by agents that raise the intracellular Ca concentration (12). We herein provide evidence that ET-1 activates Cl secretion through an increase in cAMP but not in Ca. The ET-1–induced increase in Cl secretion was concentration-dependent, and the threshold concentration for responsiveness to ET-1 was in the nanomolar range, which supports a physiologic role of the endogenous peptide in the regulation of human airway ion transport.

Previous studies indicate that the effects of ET-1 on epithelial Cl transport are heterogeneous and vary among tissues and species in an unpredictable manner. In humans, for instance, ET-1 inhibits cAMP-dependent anion secretion in gallbladder epithelial cells, but potently stimulates transepithelial Cl secretion in intestinal epithelial cells (16, 17). The effect of ET-1 on airway epithelial ion transport had not been studied previously in humans but in animals. Evidence for ET-1–induced stimulation of transepithelial Cl secretion was obtained in dog and sheep trachea (3, 18, 19, 21). Our results are consistent with these studies and suggest that ET-1 acts as a Cl secretagogue in mammalian airways. The mechanism for this effect may, however, differ between species. Studies in dog trachea suggest that ET-1 acts through multiple pathways to induce Cl secretion, including release of cyclooxygenase products, increase in intracellular Ca levels, and accumulation of cAMP (3, 18, 19). In human airways, we observed that ET-1 increases intracellular cAMP but not intracellular Ca levels, and that the ET-1–induced stimulation of Cl secretion is not reduced by pretreatment with the cyclooxygenase inhibitor indomethacin or with the Ca chelator BAPTA-AM.

ET-1 actions are mediated via specific G protein–coupled cell-surface receptors. There are at least two receptor subtypes, ETA and ETB receptors. In excised human lungs, one study found low levels of ETA but not ETB receptors in the bronchial epithelium using a quantitative autoradiographic technique (22). However, expression of ETB receptors was detected in native human bronchial epithelial cells by using a reverse transcription-polymerase chain reaction–based assay (23). The signaling mechanism that mediates the effect of ET-1 on Cl secretion in human airways is likely to involve ETB receptors, because the ETB receptor inhibitor BQ788, but not the ETA receptor inhibitor BQ123, prevented the ET-1–induced stimulation of Cl secretion. In addition, the EC50 in our study was ~ 10 nM, which is consistent with the dissociation constant of ETB receptor as determined in other cell types (24). Because ET-1 affected Cl secretion only when added in the apical bath and not the basolateral bath, the ETB receptors that mediate the ET-1–induced Cl secretion are presumably located in the apical membrane. Interestingly, in cultured guinea pig tracheal epithelial cells, studies using anti-ETB immunostaining and [125I]ET-1–binding assay indicated almost exclusive expression of ETB by the ciliated columnar cells, and within the cells, localization of the receptors in the apical side (6). Cell culture techniques may affect the expression of ET-1 receptors. For instance, primary culture of sheep tracheal smooth muscle cells is associated with a selective increase in the density and function of the ETB receptor, a receptor subtype not present in intact sheep tracheal smooth muscle, with no change in ETA receptor density. However, feeding smooth muscle cells with serum-free medium completely abolishes the increase in ETB receptor number (25). Although we used serum-free media, we cannot rule out that culture conditions may have affected the expression of endothelin receptors in our cells. To our knowledge, neither the subcellular localization of ETA and ETB receptors in human airway epithelial cells nor the effect of cell culture conditions on the distribution of endothelin receptors in human airway epithelial cells have been determined. Immunolocalization studies are needed to confirm the apical localization of ETB receptors in native and cultured human bronchial epithelial cells, as suggested in this study.

ETA and ETB receptors may initiate several intracellular signal transduction events, such as activation of Ca influx, of phospholipase C, modulation of cAMP, and activation of protein kinases (26). In agreement with the present set of experiments, other investigators found no effect of ET-1 on intracellular Ca concentration in human bronchial epithelial cells (27, 28). In contrast, we found that ET-1 induced accumulation of intracellular cAMP to the same level as did IBMX, a phosphodiesterase inhibitor. ET-1 has been reported not to affect, decrease, or increase intracellular cAMP, depending on the cell studied. In most expression systems, ETB is associated with inhibition of adenylate cyclase, whereas ETA is associated with stimulation of adenylate cyclase (26). However, exceptions have been reported: ETA activation can lead to inhibition of adenylate cyclase, as shown in guinea pig ventricular myocytes, and ETB activation can lead to activation of adenylate cyclase in specific cell types, as shown in rabbit tracheal smooth muscle (29, 30). The mechanisms by which ET receptors activate or inhibit adenylate cyclase activity are also variable among the tissues studied. Such interactions may occur directly, via GTP-binding proteins, or through intermediary signaling components (30, 31). In excised human bronchi, Hay and colleagues provided evidence for ET-1–induced prostanoid release that was mediated via ETA receptor activation; however, their experiments do not indicate whether the source of prostanoid release was the epithelial cells or other cell types (32). Takimoto and colleagues found that application of ET-1 on cultured primary human bronchial epithelial cells dose-dependently stimulated the secretion of PGE2 via ETA receptor activation (28). Taken together, these studies indicate that in human bronchial epithelial cells, ET-1 may provoke the release of prostanoids and that this release is likely mediated via ETA rather than ETB receptor activation. Although prostanoids may induce cAMP activation of Cl secretion in human airways (33), the stimulation of Cl secretion by ET-1 in our study was probably not mediated via prostanoids because the effect was (i) mediated via ETB rather than ETA receptors, and (ii) not inhibited by the cyclooxygenase inhibitor indomethacin.

Sources of production of ET-1 in the lung are multiple. ET-1 is synthesized and released by endothelial cells, type II pneumocytes, and tissue macrophages, and may therefore act as a paracrine mediator (2, 12). In addition, ET-1 may also be produced and secreted by airway epithelial cells (2, 12). Animal studies suggest that the nonciliated secretory cells rather than the ciliated columnar or basal cells are the major site of ET-1 production in the surface airway epithelium (6). Interestingly, both animal and human studies demonstrate that ET-1 can be secreted to both the luminal and serosal sides of the airway epithelium (2, 6, 34). These data together with our results imply that ET-1 may act as an autocrine modulator of airway epithelial Cl transport at the luminal side of the mucosa. Similar autocrine effects of ET-1 have been implicated in other organs, for instance in the inhibition of cAMP-dependent anion secretion in human gallbladder epithelial cells (16).

ET-1 is thought to play an important role in the pathophysiology of various lung diseases such as asthma, chronic obstructive pulmonary disease, pulmonary hypertension, and pulmonary fibrosis (2, 12). Production and release of ET-1 are induced by numerous factors including hypoxia, bacterial endotoxins, growth factors, and cytokines (2, 12, 34). In asthma, for instance, the production and release of ET-1 in airways increases dramatically (2, 12). In chronic obstructive pulmonary disease, sputum levels of ET-1 rise during exacerbations (35). Our observations, together with these reports, suggest that enhanced local production of ET-1 can be expected to participate to the airway hypersecretion observed in patients with inflammatory airway disease. Hence, antagonists for ETB receptors and inhibitors of ET production may provide a useful therapeutic tool for the treatment of inflammatory airway disease.

In conclusion, ET-1 stimulates transepithelial Cl secretion across cultured human bronchial epithelial cells. This effect is mediated via ETB receptors located in the apical membrane and probably involves activation of the cAMP pathway. These results support the notion that ET-1 exerts multiple functions in airways and may be involved in the pathophysiology of airway inflammatory diseases.


   Acknowledgments
 
This study was supported by grants from the Association Vaincre la Mucoviscidose, from the Direction à la Recherche Clinique, Assistance Publique Hôpitaux de Paris (projet CRC94162), and from CHU Lille (grant CH&U 99-12352).


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form July 5, 2002

Received in final form January 29, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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