Abstract
Mucus hypersecretion is a feature of several respiratory diseases and frequently leads to obstruction of small airways where the principal source of mucous glycoproteins (mucins), the major macromolecular constituents of mucus, are goblet cells. Hence, inhibition of mucin secretion from these cells may be clinically beneficial. In this study, we have developed a lectin-based assay for mucin secretion from ovine airway goblet cells and used this assay to investigate the regulation of these cells by endothelin (ET)-1. ET-1 inhibited baseline mucin secretion (maximum inhibition: 60.3 ± 4.2%, 50% inhibitory concentration: 0.8 ± 0.17 nM). This response was abolished by the ETA antagonist, BQ-123 (1 μM), but not by the ETB antagonist, BQ-788 (1 μM). ET-1 (1 μM) did not affect mucin secretion stimulated by ATP (100 μM) but secretion in response to ATP (10 μM) was inhibited by 63.3 ± 11.8%. This response could be eliminated by BQ-123, but not by BQ-788. Radioligand binding and immunohistochemistry indicated the expression of both ETA- and ETB-receptors on the epithelium. In summary, ET-1, acting via ETA-receptors, inhibits baseline and ATP-stimulated mucin secretion from ovine airway goblet cells. This represents the first report of a physiologic mechanism for inhibiting airway goblet cell mucin secretion; an understanding of this mechanism may provide opportunities for the treatment of obstructive airways disease.
Mucus secretion into the airway is a central component in the process of mucociliary clearance; the respiratory defense mechanism that entraps inhaled foreign material in mucus and removes it from the lung by the action of beating cilia. The physical properties of mucus are principally determined by the mucous glycoproteins (mucins), which are derived from two principle sources: mucous cells of the submucosal glands, and goblet cells situated in the epithelium lining the airways (1). The presence of these multiple sources of mucin in the respiratory tract has hindered investigations of the regulation of secretion from specific cell types, in particular goblet cells. Studies involving the regulation of secretion from goblet cells are of particular importance because goblet cells are the sole source of mucin in the small airways, which are the principle sites of obstruction in airway diseases associated with hypersecretion (2). Hence, the possibility of inhibiting the level of mucin secretion from airway epithelial goblet cells in hypersecretory diseases has therapeutic potential. However, to date, no physiologic mechanism for inhibiting mucin secretion from goblet cells has been identified, although previous studies have suggested that endothelin (ET)-1 can inhibit mucin secretion from cat and ferret tracheal submucosal glands (3, 4). An increasing amount of literature from in vitro and in vivo studies indicates that ET-1 mediates an array of effects in the lung, many of which have suggested a pathophysiologic role (5). Two distinct receptors, designated ETA and ETB, have been cloned and characterized, and both are widely distributed in the mammalian lung (6). Activation of ETA-receptors in human airways is responsible for ET-1–induced prostanoid release (7) and smooth muscle proliferation (8), whereas stimulation of ETB-receptors potentiates cholinergic nerve-induced contraction (9).
The effect of ET-1 on mucin secretion from airway goblet cells is unknown. We therefore investigated, in the present study, the effect of ET-1 on baseline and ATP-stimulated mucin secretion from goblet cells in ovine tracheal epithelial explants and the expression of ET-receptors in ovine tracheal epithelium.
In order to assess the mucin secretory activity of airway epithelial explants, a lectin-based assay for ovine goblet cell mucin was developed. To identify lectins with specificity for goblet cells in the epithelium, ovine tracheal tissue was obtained from the abattoir and tissues, or epithelial explants isolated therefrom, and fixed in 4% paraformaldehyde. Sections were cut from the paraffin-embedded fixed tissues and screened against a panel of 18 peroxidase-labeled lectins using standard immunohistochemical techniques, and the epithelial staining pattern was assessed. A number of lectins showed preferential staining of goblet cells in the epithelium, and of these, Helix pomatia agglutinin (HPA) was chosen for incorporation into a microplate-based mucin assay (see below), which was then used to monitor the mucin secretory activity of airway epithelial explants in subsequent studies.
The nature of the HPA-positive material released from airway epithelial explants was characterized further by cesium chloride density gradient ultracentrifugation analysis of material released from explants in response to the known mucin secretagogue, ATP (100 μM). Airway epithelial explants were prepared from ovine trachea as described below and, after periods for equilibration and assessment of baseline mucin secretion, were exposed to ATP (100 μM) in Ham's F12 medium for 30 min, with the medium being collected and replaced with fresh Ham's F12 containing ATP every 10 min. The ATP-stimulated samples from eight separate explants were pooled and subjected to isopycnic density gradient ultracentrifugation, according to the method of Carlstedt and colleagues (10). Briefly, guanidine hydrochloride was added to give a final concentration of 4 M and cesium chloride was added to give a density of 1.39 g/ml, and the mixture centrifuged at 120,000 × g for 72 h at 15°C. The resulting density gradient was fractionated into 1 ml fractions, which were then monitored for density by weighing and for HPA-positive material by lectin-based assay, as described below.
Superficial epithelium was isolated from airway tissues and explanted onto nitrocellulose substrates as described previously (11). Briefly, ovine tracheal tissue was obtained from a local abattoir and transported to the laboratory in minimal essential medium (MEM; GIBCO Laboratories, Paisley, UK), supplemented with penicillin (5,000 U/ml) (Sigma Chemical Co., Dorset, UK) and streptomycin (5 mg/ml) (Sigma Chemical Co.). The posterior membrane was removed and the tissue cut between and across the cartilage rings to give suitably sized pieces (∼ 8 cm2). The tissue was pinned, and sterile collagenase A (100 Mandl U/ml) (Roche Diagnostics UK Ltd., Lewes, UK) and dispase 2 (1 Wunsch U/ml) (Roche Diagnostics UK Ltd.) in phosphate-buffered saline (PBS) were injected underneath the epithelium. The preparation was covered in sterile MEM, containing DNase (0.1 mg/ml) (Sigma Chemical Co.) and dithiothreitol (0.15 mg/ml) (Sigma Chemical Co.) (MEM+). After a 30-min incubation at 37°C in a 5% CO2 atmosphere, the epithelium was removed as an intact sheet from the trachea by scraping a cover slip over the surface of the tissue at a 45° angle. The resulting epithelial sheet was suspended in MEM+, cut into suitably sized pieces (5–8 mm diameter) with fine scissors, and mounted, cilia-side up, onto nitrocellulose discs that were coated with collagen (Sigma Chemical Co.). Explants were placed in culture and allowed to attach to the nitrocellulose substrates by overnight incubation (37°C) at the air–liquid interface, with the luminal surface exposed to the humidified 5% CO2 atmosphere. The culture medium used was a 1:1 mixture of Ham's F12 (GIBCO Laboratories) and 3T3-conditioned medium, containing, insulin (10 μg/ml), endothelial growth supplement (7.5 μg/ml), transferrin (5 μg/ml), hydrocortisone (0.036 μg/ml), triiodothyronine (0.02 μg/ml) (all from Sigma Chemical Co.). The resulting explants were utilized the next day for mucin secretion studies.
After overnight incubation, suitable explants were cut to a consistent size with a cork borer (5 mm diameter), immersed in prewarmed Ham's F12 medium (0.5 ml), and allowed to equilibrate for 90 min with regular medium changes. After equilibration the medium was collected every 10 min for mucin assay and replaced with either fresh Ham's F12 medium or Ham's F12 medium containing reagents of interest.
Mucin content of samples was assessed by an enzyme-linked lectin-binding assay (ELLA). A 100-μl aliquot of sample was bound in triplicate into a 96-well high-binding microtitre plate (Fisher Scientific, Loughborough, UK) overnight at 4°C. Wells were washed three times with PBS containing 0.05% wt/vol gelatin (Sigma Chemical Co.), 0.05% vol/vol Tween 20 (Sigma Chemical Co.), and blocked by incubation with PBS containing 0.1% vol/vol Tween 20 at 37°C for 1 h. After further washing, wells were incubated at 37°C for 1 h with 100 μl of horseradish peroxidase–labeled lectin (Helix pomatia agglutinin; ICN Pharmaceuticals, Basingstoke, UK) (0.125 mg/ml in PBS). Finally, plates were washed repeatedly and developed by the addition of 150 μl of substrate solution (0.05% wt/vol O-phenylene diamine in 0.15 M citrate phosphate buffer containing 0.05% vol/vol H2O2) (all from Sigma Chemical Co.) for 2–3 min at room temperature, after which the reaction was terminated by the addition of 50 μl of 20% (vol/vol) H2SO4, and the absorbance at 492 nm was measured using a Titertek Multiskan plus plate reader (ICN Flow, Basingstoke, UK). Absorbance was compared with known mucin standards, purified by two rounds of density gradient ultracentrifugation, as described previously (10), from sputum from a patient with chronic obstructive pulmonary disease.
Three ovine tracheal epithelial sheets were isolated (see Epithelial Explant Preparation), rinsed in ice-cold PBS, and homogenized using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) (30 s at full speed) in 50 ml of ice-cold Tris buffer (25 mM Tris HCl, 135 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgSO4, pH 7.5) (all from Fisher Scientific). After the addition of a further 250 ml Tris buffer, the homogenates were centrifuged at 1,500 × g for 10 min at 4°C, the supernatant was removed, and the pellet was homogenized as before. The resulting homogenates were centrifuged for 30 min at 40,000 × g and the pellets resuspended in Tris buffer containing 10 mM ethylenediaminetetraacetic acid, 30 μM phenylmethylsulfonyl fluoride, and 50 μg/ml bacitracin (all from Sigma Chemical Co.). Aliquots were snap frozen and stored at −80°C. Protein concentration was determined by Pierce BCA method (Pierce Laboratories, IL).
Binding of [125I]–ET-1 (specific activity of 2,200 Ci/mmol) (Amersham Pharmacia Biotechnologies Ltd., Little Chalfont, UK), and the ETA-receptor–selective ligand [125I]–PD151242 (specific activity of 2,200 Ci/mmol) (Amersham Pharmacia Biotechnologies Ltd.) to membranes prepared from ovine tracheal epithelium was measured in triplicate after 2.5 h incubation at 23°C in binding buffer (10 mM ethylenediaminetetraacetic acid [GIBCO Laboratories], 50 μg/ml bacitracin [Sigma Chemical Co.], and 30 μM phenylmethylsulfonyl fluoride [Sigma Chemical Co.]). Binding was performed in 96-well filter binding plates (0.66 μm) (Millipore UK Ltd., Washington, UK). Total binding was assessed by measuring binding of the above ligands to membrane preparations (15 μg/well, 50 μl) in binding buffer only, and nonspecific binding was assessed in an excess of the nonselective ETA and ETB-receptor ligand UK240441 (10 μM) (kindly provided by Tony Chuck from Pfizer Global Research and Development, Sandwich, Kent, UK) in binding buffer, and competitive binding studies were performed in the presence of the appropriate competing ligand (20 μl) in binding buffer. Reactions were started by the addition of [125I]–ET-1 (125 fM–2 nM, 30 μl) or [125I]–PD151242 (8 pM–8 nM, 30 μl), with the exception of competitive binding studies, where the membrane homogenates were pretreated for 20 min with varying concentrations of the ETA-receptor–selective ligand BQ-123 or the ETB-receptor–selective BQ-788. After incubation, the reactions were terminated by rapid filtration using a vacuum filtration manifold (Millipore UK Ltd.). To separate membrane-bound radioactivity from free ligand, filters were washed four times in rapid succession with 200 μl of ice-cold Tris buffer and left to dry at 40°C. ‘Microscint’ (50 μl) (Packard Instrument Co., Cambridge, UK) was added to each well and bound radioactivity was measured. Typically, nonspecific binding was between 10–15% of total binding, and at the protein concentrations used [125I]–ET-1 and [125I]–PD151242 binding reached equilibrium by 2.5 h at 23°C. All binding experiments were conducted using three membrane preparations, from 3 different animals, pooled together.
Whole ovine tracheae were fixed in neutral buffered formalin (Fisher Scientific) for 48 h at room temperature, washed in Tris-buffered saline, and were subsequently embedded in paraffin wax. Tracheal sections were cut at 4 μm, mounted onto Superfrost Plus slides (Vector Laboratories Inc., Peterborough, UK), deparaffinized, and rehydrated. After washing in PBS for 5 min, sections were placed in boiling antigen-unmasking solution (10%), (Vector Laboratories Inc.) and placed into a microwave (Sharp Electronics Corp., Osaka, Japan) (1,000 W, defrost setting) for 4 min, after which sections were rinsed in distilled water (dH2O). Endogenous peroxidase was blocked with 3% (vol/vol) H2O2 in dH2O for 10 min. Sections were rinsed for 5 min in dH2O, followed by 5 min in PBS. All sections were incubated with 2% (vol/vol) normal blocking serum (Vector Laboratories Inc.) in dH2O for 20 min. Polyclonal rabbit anti–ETA or rabbit anti-ETB (Chemicon International Inc., Chandlers Ford, UK) were diluted 1:75 with 2% (vol/vol) normal blocking serum and applied to appropriate sections for 60 min at room temperature. In all cases negative controls were performed on adjacent sections; which were treated with 2% (vol/vol) normal blocking serum only, or appropriate control antigen (Chemicon International Inc.) in combination with the respective primary antibody. Sections were washed with PBS containing 1% (vol/vol) Tween 20 and immediately incubated at room temperature with 0.5% (vol/vol) biotinylated anti-rabbit IgG (Chemicon International Inc.) diluted with normal blocking serum for 30 min. After further washing, sections were incubated with Vectastain Elite ABC reagent, (Vector Laboratories Inc.) per manufacturers instructions. Sections were subsequently dehydrated and prepared for microscopy.
To assess the specificity of the ET-receptor antibodies, Chinese hamster ovary (CHO) cells, transfected to express either human ETA- or ETB-receptors (kindly provided by Tony Chuck from Pfizer Global Research and Development), were used as controls for immunohistochemical staining. CHO cells were cultured in Ham's F12 containing 10% fetal calf serum, 2 mM L-glutamine, and 500 μg/ml gentamicin (Sigma Chemical Co.). After passage, cells were resuspended in PBS and between 1.0 × 10−3 and 1.0 × 10−4 cells/ml were centrifuged onto Cytospin slides (Vector Laboratories Inc.). Slides were fixed in ice-cold 4% paraformaldehyde in dH2O, for 5 min at 4°C, washed 4 times in PBS, and air-dried. Slides were then probed with anti-ETA- and anti-ETB-receptor antibodies, as described above.
Stock solutions of ET-1 (100 μM), SRTX6b (50 μM), and SRTX6c (50 μM) were prepared in 0.1 M acetic acid, BQ-123 in 100 nM Na2CO3, and BQ-788 in dimethylsulphoxide. All vehicles used were shown to have no effect on mucin release.
Secretion data were expressed as percentage of the mean baseline value for that explant. Responses were analyzed by Students's two-tailed t test for paired observations, comparing the final point before application of a reagent with the maximum response after application of the reagent. To compare ATP-stimulated mucin secretion in explants treated with ET-1 with those treated with ATP alone, the increase in mucin secretion in response to ATP was calculated, as this took into account the lower level of baseline secretion seen in ET-1 pretreated explants. The increase in mucin secretion in response to ATP was calculated by subtracting the last pre-ATP value from the value giving the maximum response, and the result was expressed as a percentage of the mean baseline value for that explant. Control values were obtained from paired explants from the same animal.
Results from saturation binding experiments were analyzed with the iterative curve-fitting program LIGAND and are presented as means ± SEM. Data for competition experiments were analyzed using GraphPad Prism (GraphPad, San Diego, CA), comparing one-site and two-site models, and Hill coefficient (nH) values were determined for all experiments. Differences among values were tested for significance by comparing the residual variance using an F test and a significance level of P < 0.05.
Staining of sheep tracheal sections with a panel of lectins revealed that a number of these showed a degree of selectivity for goblet cells in the epithelium. In particular, Helix pomatia agglutinin stained goblet cells without staining the ciliated border of the epithelium (Figure 1A)
Figure 1. Detection of ovine airway mucins by HPA. (A) Staining of goblet cells in ovine tracheal tissue with peroxidase-labeled HPA (positive areas stain brown). (B) Negative control (no lectin) for A. (C) Standard curve for HPA ELLA using purified human sputum mucin as standard. Results are means ± SEM; n = 6. (D) Response of ovine tracheal explants to ATP. Explants were equilibrated and incubated in F12 medium (4 × 10 min) followed by F12 containing ATP (100 μM) (3 × 10 minutes) and samples analyzed for mucin content by HPA-binding assay. Results are means ± SEM; n = 6 (t = 40 min versus t = 50 min; P < 0.01). (E) Isopycnic density gradient centrifugation of ATP-stimulated ovine airway secretions. Ovine airway explants were stimulated with ATP (100 μM) for 3 × 10 min as in D, and samples were pooled and subjected to cesium chloride density gradient centrifugation as described in the text. The resulting gradient was fractionated and the fractions analyzed for density by weighing (closed circles) and by HPA-binding assay (open triangles).
[More] [Minimize]ET-1 (1 μM) inhibited baseline mucin secretion by 63.5 ± 9.6% within 10 min of exposure (P < 0.01) (Figure 2A)
Figure 2. Inhibition of baseline mucin secretion by ET-1. (A) ET-1 (1 μM) was applied to the explants for four 10-min periods after a 40-min baseline. Data are expressed as means ± SEM of 12 experiments (t = 40 min versus t = 50 min; P < 0.01). (B) Cumulative concentration–response curve of ET-1 on baseline mucin secretion. Explants were incubated with up to four sequentially increasing concentrations of ET-1 (1 pM–1 μM) for 3 × 10 min each. Data are means ± SEM of 6–12 experiments.
[More] [Minimize]In order to investigate the role of ET-receptors in the effect of 1 μM ET-1 on mucin secretion, the effects of the ETA antagonist BQ-123 and the ETB antagonist BQ-788 (both at a concentration of 1 μM) were studied. BQ-123 alone had no significant effect on baseline mucin secretion (Figure 3A)
Figure 3. Receptor involvement in the ET-1–mediated inhibition of baseline mucin secretion. (A) The effect of ET-1 on baseline secretion both in the presence and absence of the ETA-receptor antagonist, BQ-123 (1 μM). After a baseline period, explants were pretreated for 2 × 10 min with the BQ-123 (1 μM) (boxed “A”) followed by ET-1 (1 μM) in combination with BQ-123 (1 μM) (boxed “ET-1 + A”). After washout with F12 (boxed “W”) explants were exposed to ET-1 (1 μM) alone. Data are mean ± SEM of 6 experiments (t = 60 min versus t = 70 min, NS; t = 110 min versus t = 120 min, P < 0.01). (B) The effect of ET-1 on baseline secretion both in the presence and absence of the ETB-receptor antagonist, BQ-788 (1 μM). Experimental details as for A, but substituting BQ-788 for BQ-123. Data are means ± SEM of 6 experiments (t = 60 min versus t = 70 min, P < 0.01; t = 110 min versus t = 120 min; P < 0.01). (C) The effect of the ETA-receptor agonist, SRTX6b (100 nM), on baseline mucin secretion. Data are means ± SEM of 4 experiments (t = 40 min versus t = 50 min, P < 0.01). (D) The effect of the ETB-receptor agonist SRTX6c (100 nM) on baseline mucin secretion. Data are means ± SEM of 4 experiments (t = 40 min versus t = 50 min, NS).
[More] [Minimize]The role of ET-receptors in the regulation of baseline mucin secretion was investigated further with the ET-receptor agonists sarafatoxin S6b (SRTX6b) and sarafatoxin S6c (SRTX6c). The ETA-selective agonist, SRTX6b (100 nM) resulted in a 56.8 ± 5.1% inhibition of baseline mucin secretion (P < 0.01) (Figure 3C), a response similar to that observed with ET-1 (1 μM). This inhibitory effect was sustained for the 30-min agonist exposure period. However, the ETB-receptor–selective agonist, SRTX6c (100 nM), did not significantly affect baseline mucin secretion levels throughout the 30-min treatment period (Figure 3D). Thus, the ETA-receptor and not the ETB-receptor appears to mediate the inhibitory effect on mucin secretion.
To determine whether ET-1 inhibited stimulated as well as baseline mucin secretion, the effect of ET-1 in combination with the mucin secretagogue, ATP, was studied. Exposure of the explants to ATP (100 μM) caused an increase in mucin secretion over and above the baseline level of 199.1 ± 45.1%, a response that was not significantly altered by the addition of ET-1 (1 μM) (Figure 4A)
Figure 4. Inhibition of ATP-stimulated mucin secretion by ET-1. (A) The increase in mucin secretion in response to ATP (100 μM), either in the presence or absence (control) of ET-1 (1 μM). After a 4 × 10-min baseline period, test explants were exposed to ET-1 (1 μM) for 4 × 10 min, followed by ATP (100 μM) in combination with ET-1 (1 μM). Control explants received the same treatment, but with the omission of ET-1, at all stages. In each case, the value for the final pre-ATP sample was subtracted from the value giving the maximum response, and the result was expressed as a percentage of the mean baseline. Data are means ± SEM; n = 12. (B) As in A, but with the concentration of ATP reduced to 10 μM. In addition, in antagonist studies, explants were pretreated with either BQ-123 (1 μM) or BQ-788 (1 μM) and the appropriate antagonist was also added during the ET-1 and ET-1 + ATP incubations. Data are means ± SEM; n = 6; *P < 0.05; **P < 0.01 compared with control (ATP alone).
[More] [Minimize]Binding of [125I]–ET-1 (nonselective ligand interacting with ETA and ETB-receptors) and [125I]–PD151242 (ETA-receptor–selective ligand) to membranes prepared from ovine tracheal epithelium was dependent on the membrane protein concentration as well as the incubation time (data not shown). Analysis of saturation binding data revealed that [125I]–ET-1 and [125I]–PD151242 bound with high affinity to membranes prepared from ovine tracheal epithelium, with apparent dissociation constants (KD) of 0.16 ± 0.09 nM and 0.66 ± 0.06 nM, respectively (Table 1)
KD | Bmax | ||
|---|---|---|---|
| Radioligand | (nM) | (fmol mg-1 protein) | nH |
| [125I]–ET-1 | 0.16 ± 0.09 | 1170 ± 120.5 | 1.1 ± 0.4 |
| [125I]–PD151242 | 0.66 ± 0.06 | 280 ± 20.1 | 0.99 ± 0.09 |
The ETA-selective ligand BQ-123 (12.8 pM–25 μM) competed for [125I]–ET-1 binding in a monophasic manner, a one-site fit being preferred to a two-site model (Figure 5A)
Figure 5. Radioligand binding of ET-receptor ligands to ovine tracheal epithelial membranes. (A) Inhibition of [125I]–ET-1 binding to ovine tracheal epithelial homogenates by the ETA-selective BQ-123 and (B) the ETB-selective BQ-788. Epithelial homogenates were exposed to varying concentrations of either BQ-123 or BQ-788 for 20 min and then incubated with 150 pM [125I]–ET-1 for 2.5 h before termination of the reaction and measurement of bound radioactivity. (C) Inhibition of [125I]–PD151242 (150 pM) binding by ETA-selective BQ-123 and (D) ETB-selective BQ-788. Protocols were as in A and B, but substituting [125I]–PD151242 for [125I]–ET-1. All results are means ± SEM; n = 4.
[More] [Minimize]Similar competition binding experiments were performed using the selective ETA-receptor ligand [125I]–PD151242. As expected, BQ-123 (64 pM-25 μM) competed for [125I]–PD151242 binding (KD, 0.89 ± 0.19 nM) in a monophasic manner, with close to 100% inhibition of binding (Figure 5C), and a one-site fit was preferred to a two-site fit. The ETB-selective ligand, BQ-778 (40 nM–10 μM), at the highest concentration competed for 60% of the ETA-selective [125I]–PD151242 binding, and KD value could not be calculated (Figure 5D).
The localization of ET-receptors in ovine tracheal epithelium was investigated by immunohistochemistry using antibodies against either ETA- or ETB-receptors, which confirmed that both ETA- and ETB-receptors are expressed on ovine tracheal epithelium. ETA-receptor–like immunoreactivity was detected throughout the epithelial layer, with the most intense immunoreactivity detected in the apical region of the epithelium, but not on the cilia (Figure 6A)
Figure 6. Expression of ETA- and ETB-receptors in ovine tracheal epithelium. Ovine tracheal sections were stained with either, (A) anti–ETA-receptor antibody or, (C) anti–ETB-receptor antibody followed by peroxidase conjugated secondary antibody (ETA- and ETB-receptor–positive areas stain brown; blue is hematoxylin counterstain; magnification ×400). Control sections were incubated with either (B) anti–ETA-receptor antibody or, (D) anti–ETB-receptor antibody in the presence of the appropriate immunogen peptide. The specificity of the ET-receptor antibodies was assessed using CHO cells expressing either ETA- or ETB-receptors. CHO cells expressing ETA-receptors were immunostained with (E) anti–ETA-receptor antibody or (F) anti–ETB-receptor antibody. (G) ETB-receptor–expressing CHO cells immunostained with anti–ETB-receptor antibody and (H) with anti–ETA-receptor antibody (magnification ×200).
[More] [Minimize]A major limitation of studies of airway mucin secretion using whole tracheobronchial tissue has been the difficulty in distinguishing between the mucins secreted by goblet cells and submucosal glands. The results presented in this study indicate that the technique of isolating and explanting intact sheets of epithelium, as described in previous studies of mucin secretion from goblet cells in canine (11) and human tracheae (12), is also applicable to ovine airways. This explant preparation has the advantage over mucin-producing primary cultures of airway epithelium in that cellular differentiation occurs through the normal processes in the host organism. Furthermore, the unchanging morphology (11) during the isolation and explantation procedure suggests that tissue damage is minimal, increasing the likelihood that the explants accurately model the characteristics of the native tissue.
To use the ovine airway epithelial explants for studies of mucin secretion, it was necessary to develop an assay that would selectively measure mucin production from the explants. This was achieved using an ELLA with HPA as the lectin of choice. The choice of HPA for the assay was based on the staining pattern observed in ovine tracheal tissues, its reactivity with a human mucin standard in a microtitre plate assay, its ability to detect changes in mucin production in explants treated with a known secretory stimulus, and the migration pattern of the HPA-positive material in a cesium chloride density gradient.
Staining of ovine airway epithelial tissues with HPA indicated that the lectin bound to goblet cells within the epithelium without binding to the ciliated border, as has been reported for a number of antimucin antibodies (12, 13). This pattern of staining is consistent with the lectin binding selectively to mucins, a finding that is supported by the ability of HPA to recognize a human respiratory mucin standard in the lectin-binding assay. Further evidence of lectins usefulness in measuring mucin secretion from explants can be found in the fact that it allowed the detection of changes in mucin secretion by explants in response to the known mucin secretagogue, ATP. Explants incubated with F12 alone showed a stable level of baseline mucin secretion; however, the absolute values for the amount of mucin produced by each explant was highly variable and ranged from 1.3–33.8 ng mucin per 10 min period. The reason for this variability is not clear, and could possibly reflect differences in the secretory activity of different explants, but could also be a reflection of differences in the reactivity of mucins from different explants with the HPA. Differences in the reactivity of different mucins with HPA should also be considered with respect to the use of the human mucin standard in the assay. It is quite possible that the HPA has a different sensitivity for the human mucin standard than for the ovine mucins produced by the explants; hence, care should be taken in interpreting the values for the absolute amounts of mucin produced by the explants. Irrespective of the levels of baseline mucin secretion observed, all explants responded similarly to stimulation by ATP with a transient increase of ∼ 250% in mucin secretion within 10 min of application. This pattern of mucin secretory response is similar to that reported for a number of other airway epithelial systems and mucin assays (12–14) and is therefore consistent with HPA measuring mucin release from the ovine explants. In addition, the material produced in response to ATP represented a single peak on the cesium chloride density gradient, with a buoyant density generally similar to that reported previously for mucins (10, 15). The fact that the upper end of this peak was at a slightly higher density than in previous studies may represent species differences between ovine mucins and the human mucins that have been previously investigated. The possibility that the HPA assay also measures other nonmucin macromolecules cannot, however, be completely ruled out, although the data obtained in the present study are consistent with mucins being the predominant, if not the sole, species of molecules recognized.
The present study has established that ET-1 dose-dependently inhibits baseline mucin secretion from ovine tracheal epithelial goblet cells (50% inhibitory concentration, 0.8 ± 0.17 nM; maximum inhibition, 60.3 ± 4.2%) and represents the first report of a physiologic inhibitor of airway goblet cell mucin secretion. Previous studies by Shimura and colleagues (3) investigating the effect of ET-1 on feline tracheal submucosal glands found that, although ET-1 stimulated mucin secretion in isolated glands, it inhibited mucin secretion in the presence of epithelial cells, suggesting the involvement of an epithelium-derived inhibitory factor. Furthermore, the facts that indomethacin could partially inhibit this effect and that ET-1 has been reported to induce the release of phospholipase A2 products (16), suggest that this factor may be an eicosanoid. It is not known from the present study whether ET-1 inhibits baseline mucin secretion from ovine tracheal goblet cells indirectly, via the release of epithelium-derived inhibitory factor(s), or through a direct action of ET-1 on mucin-secreting cells. In addition, the intracellular signaling pathways responsible for this action are not known. ET-receptors are coupled to a number of effectors to produce an extensive network of second messengers; for example, stimulation of ETA-receptors and/or ETB-receptors in various cell types have been linked to various intracellular pathways, including phospholipase C (PLC) (17), phospholipase A2 (16, 18), guanosine 3′5'-cyclic monophosphate (cGMP) (19), and phospholipase D (20). In addition, calcium (Ca2+) signaling appears to be an almost universal response to ET-induced receptor activation (4, 21, 22). However, previous studies suggesting that rises in intracellular Ca2+ stimulate mucin secretion from airway goblet cells (13) call the role of Ca2+ in ET-1–mediated inhibition of stimulated mucin secretion into question.
ET-1 had no effect on mucin secretion in the presence of BQ-123, indicating that ETA-receptors mediate the inhibitory action of ET-1—a finding supported by the fact that the ETA-receptor–selective agonist SRTX6b inhibited mucin secretion from the ovine goblet cells to an extent similar to that observed with ET-1. BQ-788 was also capable of significantly reducing the effect of ET-1 on baseline mucin secretion, although to a much lesser extent than BQ-123. However, the role for ETB-receptors that this suggests is not supported by the studies with SRTX6c (Figure 3D), which indicated that activation of ETB-receptors had no effect on baseline mucin secretion. Although it is possible that this contradiction might be the result of an inability of SRTX6c to mediate an effect from ETB-receptors at this concentration, this seems unlikely, because previous studies, although in other species, have suggested that SRTX6c is active at ETB-receptor sites at the concentration used here (22). It seems more likely that the ability of BQ-788 to inhibit the action of ET-1 is the result of lack of selectivity for ETB-receptors. This hypothesis is supported by the radioligand binding studies in which BQ-788 competed for almost 100% of [125I]–ET-1 binding in a biphasic manner, and also competed with the ETA-selective ligand [125I]–PD151242 (23) (Figures 5B and 5D). The selectivity of BQ-788 has similarly been questioned in previous studies in rat lung, where the binding of [125I]–ET-1 to both high-affinity ETB-receptor sites and low-affinity ETA-receptor sites was inhibited by BQ-788 (24). Furthermore, the affinity of BQ-788 for ETB-receptors has been reported to differ between rat and human tissues (25), raising the possibility of species differences in the binding characteristics of BQ-788 to ET-receptors. Such species differences might explain the results obtained with ovine tissues in the present study.
ET-1 (1 μM) did not affect the ability of the explants to respond to 100 μM ATP; however, although the lower concentration of 10 μM ATP was still able to elicit a significant increase in mucin secretion from the explants, ET-1 (1 μM) was able to inhibit this response. The reason for the difference in the effect of ET-1 at the two ATP concentrations is not clear. However, studies by Abdullah and colleagues (26) showed that ATP dose-dependently stimulated mucin secretion from the rat tracheal epithelial cell line, SPOC 1, with an apparent K0.5 of 4 μM, and maximum stimulatory response was achieved at 100 μM ATP. Similarly, Kim and Lee (14) concluded that ATP dose-dependently stimulated mucin secretion from hamster tracheal epithelial cells, with an apparent EC50 of 20 μM and near-maximal mucin stimulation at 100 μM ATP. It is possible, therefore, that the lack of an action of ET-1 on mucin secretion stimulated by ATP at 100 μM was due to the near maximal stimulus of mucin secretion at this concentration, a stimulus that overshadowed the inhibitory effect of ET-1. At the lower concentration of ATP, the stimulatory drive for secretion would have been reduced, thus unmasking the inhibitory effect of ET-1. The ability of ET-1 to inhibit stimulated mucin secretion is in agreement with previous studies in ferret submucosal glands, which indicated that ET-1 was capable of inhibiting methacholine- and phenylephrine-stimulated mucus secretion (4).
In the present study, the inhibitory action of ET-1 on ATP-stimulated mucin secretion could be blocked by BQ-123 but not by BQ-788, indicating that the effect of ET-1 was mediated by ETA-receptors but not ETB-receptors. The mechanism by which ET-1 inhibits ATP-stimulated mucin secretion is not clear. In a number of systems, stimulation of ETA-receptors has previously been linked to activation of PLC and mobilization of intracellular Ca2+ (4, 21). However, the role of PLC in the effect of ET-1 on ATP-stimulated mucin secretion is questionable, as purinergic stimulation of goblet cell mucin secretion is also mediated, in part, by a PLC and Ca2+ pathway in a number of systems (27, 28).
The radioligand binding studies reported here demonstrate that ovine tracheal epithelium expresses both ETA- and ETB-receptors. [125I]–ET-1 binds with high affinity to ovine tracheal epithelium. The KD value for [125I]–ET-1 (0.16 ± 0.09 nM) was in a range similar to that reported for other tissues (e.g., rat lung, 0.15 nM [24], and canine tracheal epithelial cells, 0.2nM [29]). The ETA-receptor antagonist, BQ-123, competed for almost 60% of [125I]–ET-1 binding and the BQ-123 insensitive component of [125I]–ET-1 binding was abolished by concomitant treatment with BQ-788, suggesting the presence of both ETA- and ETB-receptor sites. However, the ETB-receptor–selective ligand, BQ-788, competed for almost 100% of [125I]–ET-1 binding in a biphasic manner, indicating the presence of high-affinity (subnanomolar range) ETB-receptor sites (71.6% of sites) and lower affinity (nanomolar range) non–ETB-receptor sites. This receptor ratio is comparable with the saturation binding studies with [125I]–ET-1 and [125I]–PD151242, where the percentage difference between the Bmax values for the nonselective ([125I]–ET-1) and ETA-selective ([125I]–PD151242) ligands revealed a receptor ratio of 24:76 (ETA:ETB) (Table 1). However, in competition binding studies, the ETA-selective antagonist BQ-123 inhibited almost 60% of [125I]–ET-1 binding, and inhibition curves were monophasic, suggesting binding to only one receptor subtype. These results are difficult to explain without postulating the existence of additional receptor subtypes, or may alternatively be explained by the different agonist versus antagonist binding affinity profiles observed with G-protein–coupled receptors; where only agonist binding is affected by the G-protein–coupling state or the absence or presence of guanine nucleotides (30). It is also interesting to note that, although BQ-788 was able to almost completely inhibit [125I]–ET-1 binding in the binding assay, it was not able to block the effect of ET-1 on mucin secretion from the explants (Figure 3B). This apparent discrepancy is probably a reflection of the different systems used in these two assays, and also of the fact that in the mucin secretion studies, ET-1 and BQ-788 were present in equimolar concentrations, whereas in the ligand binding studies, BQ-788 was present in a large excess compared with the concentration of [125I]–ET-1.
The ETA-selective linear tetrapeptide radioligand [125I]–PD-151242 (23) was used to characterize ETA-receptors in ovine tracheal epithelium. Saturation binding assays with [125I]–PD-151242 revealed a single population of high affinity ET-receptors: (KD = 0.66 ± 0.06 nM). The dissociation constant for [125I]–PD-151242 was in a range similar to other tissues that contain mainly ETA-receptors, such as human vasculature and heart (aorta [0.8 nM], coronary artery [0.5 nM], pulmonary artery [1.8 nM], and ventricle [1.1 nM]) (23). BQ-123 and BQ-788 competed for [125I]–PD151242 binding in a monophasic manner, but the former with high-nanomolar affinity and the latter with micromolar affinity. BQ-123 competed for almost 100% of [125I]–PD-151242 binding, and BQ-788 (1 μM) competed for almost 60% of ETA-selective [125I]–PD151242 binding, once again calling in to question the selectivity of the ETB-receptor antagonist in this preparation. Pharmacologic studies in animal tissues have suggested the presence of an atypical ETA-receptor (e.g., in rat kidney) (31). However, as BQ-123 competed for almost all of the binding of [125I]–PD151242 to ETA-receptors, and inhibition curves were monophasic, there is nothing to suggest that atypical ETA-receptors are present in ovine tracheal epithelium.
Immunohistochemical studies indicated both ETA- and ETB-receptor like immunoreactivity throughout the epithelial layer. ETA-receptor–like immunoreactivity was expressed predominantly on the apical cell layer and on the basal cells of the epithelium, with lateral bands of immunoreactivity through the epithelial layer also being evident. Ninomiya and colleagues (32) found a similar expression pattern of ETA-receptors in basal cells from guinea pig tracheal epithelium and suggested that ET-1 activates ETA-receptors and stimulates the proliferation of the basal cells. This mitogenic effect is consistent with previous observations in porcine tracheal epithelial cells (33), as well as other cell types that express the ETA-receptor subtype (34). ETB-receptor–like immunoreactivity was expressed on cilia and along the apical surface of the epithelial layer. Similarly, in guinea pig trachea, expression of ETB-receptors was located on the cilia (32). The localization of ETB-receptors on cilia and ciliated cells suggests that ETB-receptors are important in controlling ciliary beat frequency in ovine trachea. ET-1 depresses tracheal mucus velocity in ovine airways (35); however, antagonist studies suggest that this response is mediated via ETA-receptors. Mucus velocity is dependent on other factors in addition to ciliary beat frequency (e.g., the composition of airway surface liquid [36]), which may explain this finding.
In summary, this study has shown that ET-1 is a potent inhibitor of baseline and stimulated mucin secretion from ovine airway epithelial goblet cells, and that this inhibitory effect is mediated via ETA-receptors. This finding represents the first physiologic mechanism for inhibiting mucin secretion from goblet cells, and, in view of the central role of goblet cells in obstructive pulmonary disease, an understanding of this mechanism may provide novel opportunities for the treatment of these diseases.
The authors are grateful to Tony Chuck, Emma Levett, and Julie Owen for their technical expertise with the radioligand binding and immunohistochemical studies. The advice given by Anthony Davenport was also appreciated. These studies were supported by the Biotechnology and Biological Sciences Research Council and by Pfizer Global Research and Development.
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