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Published ahead of print on July 1, 2004, doi:10.1165/rcmb.2003-0211OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 446-455, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0211OC

Nucleotide-Mediated Mucin Secretion from Differentiated Human Bronchial Epithelial Cells

Philip A. Kemp, Rosemary A. Sugar and Alan D. Jackson

Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom

Address correspondence to: Dr. Alan D. Jackson, Novartis Respiratory Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK. E-mail alan.jackson{at}pharma.novartis.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most current cell-based models for examining the regulation of mucin secretion demonstrate low signal-to-noise ratios, making experimental manipulation and data interpretation difficult. Using adenosine triphosphate (ATP) as a mucin secretagogue, we have developed a model of agonist-induced mucin secretion in differentiated human bronchial epithelial cells. Mucin secretory signals were estimated using enzyme-linked lectin assay, and typical signals of 300–400% of baseline were observed in response to a 30-min exposure to ATP (100 µM). ATP and uridine triphosphate equipotently stimulated mucin secretion consistent with mediation via P2Y2 receptor activation. Suramin and AR-C118925XX, a competitive P2Y2 receptor antagonist, inhibited adenosine 5'-o-(3-thiotriphosphate) (ATP-{gamma}S)–induced mucin secretion. A selective Gq G–protein antagonist (GP-ANT)–2A completely abrogated ATP-{gamma}S–induced mucin secretion. Pertussis toxin and the Gi/o-specific, GP-ANT-2, had no effect. The phospholipase C inhibitor, D609, and the protein kinase C inhibitor, calphostin C, substantially inhibited ATP-{gamma}S–induced mucin secretion. Phorbol myristate acetate also stimulated mucin secretion in a calphostin C–sensitive manner. ATP-{gamma}S–induced mucin secretion was inhibited by the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid tetra (acetoxymethyl) ester. Ionomycin and thapsigargin both stimulated mucin secretion. Our data are broadly consistent with known G-protein–coupling and downstream signaling events associated with the P2Y2 receptor. The exceptional signal-to-noise ratios obtained using this model have permitted clear evaluation of the involvement of these mechanisms in agonist-induced mucin secretion from differentiated human bronchial epithelial cells.

Abbreviations: alcian blue, AB • adenosine diphosphate, ADP • air–liquid interface, ALI • adenosine triphosphate, ATP • adenosine 5'-o-(3-thiotriphosphate), ATP-{gamma}S • 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid tetra (acetoxymethyl) ester, BAPTA-AM • bronchial–epithelial basal medium, BEBM • bronchial–epithelial growth medium, BEGM • collagen 1, Col I • cholera toxin, CTX • Dulbecco's modified Eagle's medium, DMEM • agonist concentration needed to produce 50% of the maximal response, EC50 • enzyme-linked immunosorbent lectin assay, ELISLA • enzyme-linked lectin assay, ELLA • G-protein antagonist, GP-ANT • GP–coupled receptor, GPCR • sequence scrambled GP-ANT-2A, SGP-2A • human bronchial epithelial cell, HBEC • horseradish peroxidase, HRP • concentration producing 50% inhibition, IC50 • lactate dehydrogenase, LDH • 2-methylthioadenosine 5'-triphosphate, 2MeSATP • affinity constant defined by the log of the concentration of antagonist required to shift an agonist concentration response curve 2-fold to the right, pA2 • periodic acid–Schiff, PAS • phosphate-buffered saline, PBS • phosphatidylcholine-specific phospholipase C, PC-PLC • phosphatidylinositol-specific PLC, PI-PLC • protein kinase C, PKC • phorbol myristate acetate, PMA • phospholipase C, PLC • pertussis toxin, PTX • sodium dodecyl sulfate, SDS • uridine diphosphate, UDP • Ulex europaeus agglutinin type 1, UEA-1 • uridine triphosphate, UTP • wheat germ agglutinin, WGA • HRP-conjugated WGA, HRP-WGA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Excess mucin secretion is an important feature of a number of respiratory disorders, including chronic obstructive pulmonary disease (1, 2) and asthma (3). Mucin hypersecretion has been associated with an excess decline in forced expiratory volume in 1 s, an increased frequency and duration of respiratory infections, hospitalization, morbidity, and mortality (1, 4, 5). In man, there are two dedicated sources of secretory gel–forming mucins in the airways: the mucous cells of the submucosal glands and the goblet cells of the surface epithelium. Submucosal glands are restricted to the large airways in both health and disease. In contrast, although goblet cells occur predominantly in large airways in healthy individuals, their numbers increase in small airways (< 2 mm internal diameter) in patients with asthma or chronic obstructive pulmonary disease. Because the bulk of airways obstruction occurs in the small airways, goblet cell–derived mucins are likely to be important with respect to mucus-related airways obstruction. Our current understanding of the regulation of surface epithelial goblet cell mucin secretion is poor, partly due to a lack of clinically relevant models with robust readouts. Those models that have been described generally rely on small animal paradigms (6) and transformed or immortalized human or animal cell lines (7–10) whose regulatory mechanisms may not closely resemble those of human epithelial cells in situ. The culture methods of Wu and colleagues (11, 12), later modified by Gray and colleagues (13), permit the routine generation of human bronchial epithelial cell (HBEC) cultures with well differentiated goblet and ciliated cells, providing an alternative to immortalized or transformed cell lines.

Nucleotide-regulated mucin release by primary airway goblet cell culture was first shown by Kim and Lee (14), who demonstrated a concentration-dependent release of 3H-labeled mucins from hamster tracheal epithelial cells (14). In this study, we have used the ubiquitously effective mucin secretagogue ATP (see review in Ref. 15) to develop a model of agonist-induced mucin secretion in HBECs. These cells histologically resemble epithelial cells present in the airways in situ. We have developed a model of mucin secretion in these cells with a substantial, robust signal-to-noise ratio. This model has allowed us to characterize the receptor responsible for ATP-induced mucin secretion, its G-protein–coupling and elements of the downstream signaling cascade initiated after receptor activation. We have examined nucleotide agonist potency profiles, suramin and AR-C118925XX (16) sensitivity, and blockers of Gq and Gi G-protein–coupling to characterize the receptor involved in ATP-induced mucin secretion. In addition, we have examined the roles of phospholipase C (PLC), protein kinase C (PKC), and calcium ions in HBEC mucin secretion.

Our results indicate that ATP-induced mucin secretion is mediated via Gq coupling to the P2Y2 receptor, activation of phosphatidylcholine-specific PLC (PC-PLC), activation of PKC, and elevation of intracellular Ca2+ concentration consistent with known P2Y2-receptor signaling events. This is the first study to convincingly link these signaling mechanisms to mucin secretion in routinely cultured, differentiated HBECs. The signal-to-noise ratios, typically 3–4- and up to 7.5-times baseline generated using this model, are far in excess of those previously reported for nucleotides (17). This represents a substantial improvement over existing models and makes this model suitable for routine pharmacologic manipulation and evaluation of human goblet cell mucin secretory mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Bronchial–epithelial basal medium (BEBM), bronchial–epithelial growth medium (BEGM), SingleQuots, and HBECs were obtained from Biowhittaker (Workingham, UK). BIOCOAT collagen 1 (Col I) culture inserts 4.2 cm2 and BIOCOAT Col I culture inserts 0.9 cm2 were obtained from Becton Dickinson (Oxford, UK). GP-antagonist (GP-ANT)–2 was obtained from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). ATP, D609, and GP-ANT-2A were obtained from Calbiochem (Nottingham, UK). BioPorter was obtained from Cambridge Bioscience (Cambridge, UK). The scrambled analog of GP-ANT-2A (SGP-2A) was prepared by Sigma Genosys (Cambridge, UK). High-binding 96-well flat-bottomed plates and 75-cm2 vented flasks were obtained from Corning Costar (High Wycombe, UK). Horseradish peroxidase (HRP)–conjugated Ulex europaeus agglutinin (UEA) type 1 (UEA-1–HRP) was obtained from EY Laboratories (San Mateo, CA). Dulbecco's modified Eagle's medium (DMEM), Hams F12 w/o L-Glutamine, SeeBlue Plus2 Pre-Stained Standard, and 4–20% gradient Tris-glycine sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis gels were obtained from Invitrogen (Paisley, UK). DNase I was obtained from Qiagen (Crawley, UK). AR-C118925XX was synthesized at Novartis (Horsham, UK) according to the structure presented at the American Chemical Society (16). AR-C118925XX was inactive against the following receptors at 10 µM: human serotonin receptors 5-HT 1A, 5-HT 2A, 5-HT 2B, 5-HT 2C, 5-HT 3, 5-HT 6, and 5-HT 7, human adrenergic receptors {alpha}1, {alpha}2A, {alpha}2B, {alpha}2C, ß1, and ß2, Human cannabinoid receptor CB1, human dopamine receptors, D1, D2L, D3, and D4.4, human GABA-B receptor, human histamine receptors H1 and H2, human muscarinic receptors M1, M2, M3, M4, and M5, human nicotinic receptor Nins, human tachykinin receptors NK1 and NK2, human opiate receptors OpD, OpK, and OpM, L-type and N-type human calcium channels, human potassium channel K+ATP, human purinergic receptor P2Y2, and rat purinergic receptor P2Y1. Periodic acid–Schiff reagent and 10% vol/vol neutral-buffered formalin were obtained from Surgipath (Peterborough, UK). Mouse anti-human MUC5AC (45M1) antibody was obtained from Neomarkers (Lab-Vision-Neomarkers, Fremont, CA). RALwax was obtained from Raymond A. Lamb Ltd. (Eastbourne, UK). Slide-A-Lyser dialysis cassettes were obtained from Peribo UK Science Ltd. (Tattenhall, UK). Ultra-Free-15 centrifuge unit with BIOMAX-50K membrane were obtained from Millipore (Billerica, MA). All other reagents were obtained from Sigma (Poole, UK).

Cell Culture
The apical surfaces of Col I–coated cell culture inserts were prehydrated for 30–60 min at room temperature with either 1 ml or 0.5 ml of Hams F12 for 6-well (4.2 cm2) and 12-well (0.9 cm2) inserts, respectively. HBECs were cultured according to the methods of Gray and colleagues (13). Briefly, cells from a P2 stock were seeded at 5 x 105 cells into a 162 cm2 flask in Clonetics growth medium (500 ml BEBM + SingleQuots; Cambrex, East Rutherford, NJ) and incubated at 37°C and 5% CO2. All SingleQuots (bovine pituitary extract, insulin, hydrocortisone, gentamycin/amphotericin, all-trans retinoic acid, transferrin, epinephrine, tri-iodothyronine, and human epidermal growth factor) were added to the growth medium at the recommended concentrations. Cells were harvested when 90–95% confluent (assessed by light microscopy) and seeded at P3 into either 6-well Col I inserts at 2 x 105 cells/insert or into 12-well Col I inserts at 8.25 x 104 cells/insert in differentiation medium (500 ml BEBM/DMEM [50:50] + BEGM SingleQuots (except tri-iodothyronine)). Additional all-trans retinoic acid was added to give a final concentration of 5 x 10–8 M. The cells were grown in foil-covered plates in submerged culture for 7 d. The medium was changed every 2–3 d. After 7 d, the medium was removed from the apical side of the inserts and cells were cultured at air–liquid interface (ALI). Cells were fed with differentiation medium basally every 2–3 d after establishment of ALI. Cells were used for the study of mucin secretion between 14–17 d after ALI establishment.

Apical Cell Surface Washing to Remove Accumulated Mucin
Mucin secreted onto the apical surface of cells during culture was removed using the following protocol: apical surfaces of cells in 6-well or 12-well plates were rinsed by addition of 1 or 0.5 ml medium prewarmed to 37°C, respectively, and allowed to stand for 2–3 min. The plates were gently rocked manually, and the medium in the apical chamber was aspirated. The apical surfaces of the cells were then washed by adding 1 or 0.5 ml minimal medium (6- or 12-well plates, respectively) to each well, followed by incubation for 1 h at 37°C and 5% CO2. The plates were gently rocked and the media in the apical chambers was aspirated. This process was repeated to give a total of 1 rinse and four 1 h washes before using the cells for the study of agonist-induced mucin secretion.

Effect of Culture Medium on Agonist-Induced Mucin Secretion
To determine the effect of supplements (BEGM SingleQuots) on ATP-induced mucin secretion, cells were exposed to minimal media (50/50 BEBM/DMEM + L-glutamine without retinoic acid or Clonetics supplements) at various times from 48–2 h before agonist exposure. A parallel set of inserts was treated identically, but with complete differentiation medium rather than minimal medium. Cells were then challenged with ATP or vehicle as described below.

Experimental Modulation of HBEC Mucin Secretory Responses
Unless otherwise stated, in all experiments, the differentiation medium in the basolateral chamber of the inserts was replaced with minimal medium 48 h before beginning the experiment. Four hours before each experiment, the medium in the basolateral compartment was replaced with fresh minimal medium. All culture inserts were washed with minimal medium as described in the apical cell surface wash procedure (above) to remove mucin that had accumulated during the differentiation period. All reagents were dissolved in relevant media (i.e., either differentiation medium or minimal medium) unless otherwise stated. Nonaqueous reagents were first dissolved in dimethyl sulfoxide, then diluted using either differentiation or minimal medium to give a final concentration of dimethyl sulfoxide not exceeding 0.1% vol/vol. Total apical volume was always 1.0 and 0.5 ml for 6- and 12-well inserts, respectively, to ensure consistency of measurements of mucin concentration. Where both apical agonist and apical modulator of agonist responses were used in the same experiment, each was added at half of the final required volume (0.50 or 0.25 ml for 6- and 12-well inserts, respectively) and double the final required concentration. Agents used to modulate agonist-induced mucin secretion were added to the apical surface of inserts 20–30 min before stimulation at 37°C and 5% CO2. In experiments that examined the effect of hexokinase on nucleotide agonists, nucleotide agonists were dissolved in either minimal medium or minimal medium containing 10 U/ml hexokinase with 25 mM glucose and incubated at room temperature for 10 min before addition to the inserts. In experiments involving G-protein–inhibiting peptides, to facilitate peptide entry into the HBECs, the GP-ANT-2A, GP-ANT-2, and SGP-2A peptides were mixed with a lipid delivery agent (BioPorter) before addition to the cells. The BioPorter was resuspended according to the manufacturer's instructions and dispensed into microcentrifuge tubes (120 µl/tube) and then air-dried. GP-ANT-2A, GP-ANT-2, and SGP-2A (500 µM in phosphate-buffered saline [PBS]) were added to separate tubes of BioPorter (125 µl/tube), allowed to stand at room temperature for 5 min, vortexed, then diluted 1/10 in minimal medium. The BioPorter/peptide mix was then added (250 µl/insert) to the apical chamber of washed cells (as previously described) and incubated for 4 h at 37°C and 5% CO2 before the addition of agonist. Where used, pertussis toxin (PTX) was added to the basolateral chamber in a 1 ml volume for 22 h at 37°C and 5% CO2 before the addition of agonists. Where used, cholera toxin (CTX) was added to the basolateral chamber (10 ng/ml) in a 1 ml volume of differentiation media, in line with the standard feeding protocol (i.e., the basolateral medium was changed every 2–3 d) throughout the 14 d differentiation period. Agents used to stimulate mucin secretion were added to the apical surface of inserts for 30 min at 37°C and 5% CO2. At the end of each exposure period, supernatants were removed and stored at –80°C before analysis for mucin content by enzyme-linked lectin assay (ELLA). All experimental reagents were tested in the ELLA at the highest concentration at which they were used. No reagent used interfered with the ELLA (data not shown). The cytotoxicity of each experimental reagent at the highest concentration used was assessed using the CytoTox 96 nonradioactive cytotoxicity assay (Promega UK, Southampton, UK) performed according to manufacturer's instructions. Over the exposure periods used in these studies, lactate dehydrogenase (LDH) release never exceeded 2% of total cellular LDH levels.

Preparation of Human Mucin Standard
A human mucin standard was prepared from the sputum of a healthy smoker according to the methods of Goswami and colleagues (18). Sputum was predigested sequentially with hyaluronidase, chondroitinase ABC, heparinase, and DNase I and separated on a sepharose CL-4B column in the presence of 4 M guanidine-HCl. Slot blots and densitometry were used to determine the reactivity of each column fraction with reagents known to react with mucins. UEA-1 (specific for detection of {alpha}-L-fucose) and periodic acid–Schiff (PAS) reactivity was almost exclusively associated with void volume material. A mouse anti-human respiratory mucin antibody (17Q2), a mouse anti-human MUC5AC antibody (45M1), and wheat germ agglutinin (WGA; specific for detection of (GlcNAc)4/NeuNAc/(GlcNAc)2-NeuNAc) detected a major peak of reactivity in the void volume coinciding with the peak detected by PAS and UEA-1. They also detected two additional peaks of material in lower molecular weight fractions (19). The void volume fractions from this column separation were pooled and then dialyzed against PBS (4 x 250 ml PBS for 22 h at 4°C) using a Slide-A-Lyzer dialysis cassette with 10K membrane, concentrated using an UltraFree-15 centrifuge unit with BIOMAX-50K membrane, lyophilized, reconstituted in 50% glycerol, and stored at –20°C until used. Strong reactivity of all reagents with void volume material together with preferential staining of goblet cells in sections of differentiated HBECs by UEA-1, WGA, and 45M1 is consistent with mucin reactivity. In addition, UEA-1 selectively stained very high molecular weight material with a similar migration profile to thyroglobulin (670kD) on Western blots of the void volume material taken from 1% agarose gels. Both UEA-1 and WGA detected material from the pooled void volume fractions with a molecular weight much greater than the highest molecular weight marker (200kD) by Western blots of 4–20% w/v gradient Tris-glycine SDS–polyacrylamide gel electrophoresis gels. Because this standard cannot be directly compared with HBEC derived mucin or pure preparations of respiratory gel forming mucins, all data are expressed as U/ml rather than absolute concentrations.

ELLA
In all experiments mucin was measured using a sandwich ELLA with UEA-1 as capture lectin and HRP-conjugated WGA (WGA-HRP) or UEA-1–HRP as the detection lectin. Ninety-six–well plates were coated with UEA-1 at 1.25 µg/ml in 0.1 M bicarbonate-carbonate buffer (pH 9.6; 100 µl), sealed, and incubated overnight at 4°C. Plates were washed three times with 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.05% gelatin (200 µl) using a Platewash 96/384 (Packard Bioscience, Pangbourne, UK). Plates were tapped dry and residual binding sites were blocked with 10 mM PBS (pH 7.4) + 0.1% Tween-20 (150 µl) for 1 h at 37°C. The plates were washed three times and tapped dry. Plates were stored at –80°C until required or for up to 6 mo. Before the addition of samples, plates were washed an additional two times. Samples or standards diluted in 10 mM PBS (pH 7.4) were added to duplicate wells (100 µl) and incubated for 2 h at room temperature. The plates were washed four times, tapped dry, then WGA-HRP (100 µl) was added to each well and incubated for 2 h at room temperature. Plates were washed four times and tapped dry. Bound HRP-conjugated UEA-1 or WGA-HRP was detected using o-phenylenediamine dihydrochloride solution (100 µl) and stopped by addition of 4M H2SO4 (50 µl). Absorbance was read at 490 nm and mucin concentrations were calculated using SoftMax Pro v3.0 software (Molecular Devices, Workingham, UK). Mucin concentrations were reported as U/ml.

Enzyme-Linked Immunosorbent Lectin Assay
For selected experiments, data obtained using ELLA was confirmed using an enzyme-linked immunosorbent lectin assay (ELISLA). The ELISLA used UEA-1 as capture reagent and 45M1 as detection reagent. Ninety-six–well plates were coated, washed, and blocked as described in the ELLA assay section (above) and the plates were stored at –80°C until required or for up to 6 mo. Before the addition of samples, plates were washed an additional two times. Samples or standards diluted in 10 mM PBS (pH 7.4) were added to duplicate wells (100 µl) and incubated for 1 h at 37°C. The plates were washed three times, tapped dry, then 150 µl 1% bovine serum albumin (in PBS pH 7.4 + 0.05% Tween-20) was added to each well and incubated for 1 h at 37°C. The plates were washed three times, tapped dry, then 100 µl 45M1 (PBS pH 7.4 + 1% bovine serum albumin + 0.05% Tween-20) was added to each well and incubated for 1.5 h at 37°C. The plates were washed five times, tapped dry, then 100 µl goat anti-mouse IgG-biotin (PBS pH 7.4 + 1% bovine serum albumin + 0.05% Tween-20) was added to each well and incubated for 1 h at 37°C. Plates were washed five times and tapped dry, then 100 µl HRP-streptavidin (PBS pH 7.4 + 1% bovine serum albumin + 0.05% Tween-20) was added to each well and incubated for 20 min at room temperature. Plates were washed five times and tapped dry. Bound streptavidin-HRP was detected using o-phenylenediamine dihydrochloride solution (150 µl) and stopped by addition of 4M H2SO4 (50 µl). Absorbance was read at 490 nm and mucin concentrations were calculated using SoftMax Pro v3.0 software (Molecular Dynamics). Mucin concentrations were reported as U/ml In all cases, ELLA and ELISLA data gave comparative results.

Histology
Differentiated HBECs on inserts were gently rinsed with PBS at 37°C to remove accumulated mucus and/or debris. Basolateral medium was aspirated and the cells fixed by the addition of 0.5 ml 10% neutral buffered formalin to the apical surface followed by 1 ml neutral buffered formalin to the basolateral surface. After 24 h fixation, the inserts were bisected and placed in processing capsules between two sheets of foam (Surgipath Europe Ltd., Peterborough, UK) to keep them flat during the subsequent processing. The inserts were processed to RALwax through graded industrial methylated spirit and chloroform using a closed VIP tissue processor (Bayer plc, Newbury, UK). Both halves of the insert were embedded on edge using fresh RALwax. The paraffin blocks were sectioned at 4 µm using a RM2135 rotary microtome (Leica Microsystems UK Ltd., Milton Keynes, UK). The sections were stained using alcian blue (AB)/hematoxylin and eosin according to standard histologic protocols to enable detection of mucin and discrimination of individual cells by nuclear staining. The stained slides were examined using an Axioplan2 microscope (Carl Zeiss Ltd., Welwyn Garden City, UK). The total numbers of apical epithelial cells were counted across the entire length of both pieces of insert in 2 or 3 sections at 20x magnification. The AB-stained goblet cells present on the same inserts were counted and expressed as a percentage of the total apical epithelial cells.

Statistical Analysis
All data are reported as means ± SEM from a single experiment with n = 6 inserts per group unless otherwise stated. All experiments were repeated 2–3 times. Statistical analyses were made on single experiments with Kruskal-Wallis one-way ANOVA using Systat v.10 (SPSS Inc, Chicago, IL). Differences were considered significant when P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Wash Procedure and Culture Medium on Agonist-Induced Mucin Secretion
Mucin was visible to the naked eye on the apical surface of unwashed HBECs and high concentrations (1,210 ± 81 U/ml) were detected in apical surface washings of the inserts by ELLA. Apical exposure of unwashed cultures to ATP (1 mM, 30 min) did not result in apparent mucin secretion compared with vehicle control (Figure 1A). A washing procedure was developed to remove accumulated mucin to optimize signal-to-noise ratio. This procedure consisted of five steps, one rinse (2–3 min), and four 1 h washes. The mucin content of cell surface washings was high in the first rinse (1,210 ± 81 U/ml) and progressively reduced with each subsequent washing step (Figure 1B; n = 3, P = 0.05) such that, after this procedure, a baseline mucin content was obtained (36 ± 8 U/ml). In contrast to the results observed in the absence of washing, ATP (1 mM, 30 min) triggered a mucin secretory response of ~ 250% of baseline after washing (data not shown), suggesting that mucin that had accumulated during culture obscured the specific mucin secretory response to ATP. This washing procedure was used for all subsequent experiments.



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  		Figure 1. (A) High levels of mucin were present in the supernatants from sham-stimulated cells and no change in these levels was observed after a 30-min exposure to 1 mM ATP. (B) A single 2–3-min apical rinse and four 1-h washes removed progressively lower concentrations of mucin until a baseline level of mucin was achieved. (C) Omission of Clonetics SingleQuot culture supplements for 18–48 h before a 30-min exposure to 1 mM ATP significantly increased ATP-induced mucin secretion as a percentage of baseline, compared with inserts in which the basal differentiation medium was replaced with fresh differentiation medium (#P = 0.05 versus rinse; n = 3, *P < 0.05 versus differentiation media; n = 6). Experiments were repeated three times. Veh = vehicle; ATP = ATP alone. Data are reported as means ± SEM. Open bars, differentiation media; closed bars, minimal media.

 
The effect of media supplements on HBEC mucin secretion was determined by exposing cells to minimal media for periods of between 0–48 h before ATP exposure. Exposure to minimal media for 2 or 6 h had no significant effect on ATP-induced mucin secretion. Exposure to minimal media for 18, 24, or 48 h reduced baseline and ATP-induced mucin secretion by 53 ± 3% and 24 ± 9% (P < 0.05), 44 ± 13% and 20 ± 10% (P < 0.05), and 51 ± 1% and 28 ± 5% (P < 0.05), respectively (data not shown), leading to an enhancement of signal-to-noise ratio from ~ 2.5 to ~ 4 (Figure 1C). Cytotoxicity (LDH release) in response to exposure to minimal media never exceeded 0.70 ± 0.06% of total cellular LDH levels (data not shown), the resistance of the cultures measured in Ussing chambers did not change (data not shown), and beating cilia were observed at all times. In contrast, cells in which differentiation medium was replaced with fresh differentiation medium to control for the effects of media change itself, the signal-to-noise ratio remained constant at ~ 2.5 (Figure 1C). These experiments were consistent when run on three separate occasions. We have not defined the components of the SingleQuot supplements responsible for these changes. For all subsequent experiments HBECs were fed with minimal media for 48 h before experimental manipulation. The increased mucin secretion measured by ELLA after exposure to ATP was associated with a reduction in number of AB-positive goblet cells as a percentage of total apical epithelial cell number as assessed by light microscopy (Figures 2A and 2B), suggesting that some goblet cells exocytosed all of their contents. This observation provides evidence that data generated using this ELLA represents a surrogate measure of goblet cell exocytosis. For selected experiments, samples were analyzed using both the ELLA and an ELISLA. The ELISLA utilized the mouse anti-human MUC5AC–specific monoclonal antibody 45M1 (20) as a detection reagent to confirm results obtained using the lectin WGA as a detection reagent. Data obtained using these assays to determine mucin concentrations in samples derived from HBECs treated with vehicle, 1, 10, and 100 µM ATP-{gamma}S were statistically indistinguishable (Figure 2C).





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  		Figure 2. (A) AB/(hematoxylin and eosin)–stained sections of HBEC cultures before (top) and 30 min after (bottom) apical exposure to 100 µM ATP, demonstrating a reduction in the number of AB-stained goblet cells. (B) AB-positive goblet cells as a percentage of total apical epithelial cells in sections of HBEC cultures before and 30 min after apical exposure to 100 µM ATP. (C) ELLA (closed bars) and ELISLA (open bars) assay determination of mucin secretion (% vehicle control) from HBECs exposed to ATP-{gamma}S for 10 min, demonstrating both assays measure mucin secretion in a concentration-dependent fashion (*P < 0.05 versus vehicle; n = 6). Data are reported as means ± SEM.

 
Nucleotide Agonist Potency Profile
ATP and UTP (1–1,000 µM, 30 min) concentration-dependently and -equipotently increased mucin secretion. Responses at the highest concentrations of ATP and UTP examined were 755 ± 180% (P = 0.05, n = 4) and 714 ± 157% (P = 0.05, n = 4) of vehicle-induced secretion, respectively. Significant increases in mucin secretion were observed with ATP and UTP concentrations as low as 1 µM. ADP and uridine diphosphate (UDP; 1–1,000 µM, 30 min) also concentration-dependently and approximately equipotently increased mucin secretion to 355 ± 90% (P = 0.05, n = 4) and 400 ± 56% (P = 0.05, n = 4) at 1 mM, respectively (Figure 3A). However, the lowest concentration at which both nucleotide diphosphates caused significant increases in mucin secretion was 100 µM, indicating that they were ~ two orders of magnitude less potent than their nucleotide triphosphate counterparts. Pretreatment of ATP, ADP, or UDP with hexokinase (10 U/ml; 25 mM glucose; 10 min) substantially reduced their mucin secretory capacity by 66 ± 4% (P < 0.05), 55 ± 3% (P < 0.05), and 65 ± 2% (P < 0.05), respectively, suggesting that nucleotide triphosphate contaminants were at least partly responsible for the observed mucin secretion in response to nucleotide diphosphate preparations (Figure 3B). In contrast, the P2Y1 and a P2Y11 receptor agonist, 2-methylthioadenosine 5'-triphosphate (2MeSATP; 1–1,000 µM, 30 min), which is inactive at the P2Y2 receptor (see review in Ref. 21), and adenosine (1 mM, 30 min), a P1 receptor agonist, did not elevate mucin secretion in this model (data not shown). The agonist potency profile ATP = UTP > > ADP = UDP (2MeSATP- and adenosine-inactive) for nucleotide-induced mucin secretion is consistent with the secretory response in HBECs being mediated by the P2Y2 receptor (21). A number of other mediators that have previously been associated with mucin secretory responses were also examined. Acute exposure (30 min) to methacholine, bradykinin, or endothelin-1 did not significantly alter HBEC mucin secretion even at the highest concentrations examined (100 µM, 100 nM, and 1 µM, respectively; data not shown).



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  		Figure 3. (A) Effect of 30-min exposure to nucleotides on mucin secretion from HBECs. The nucleotide agonist potency profile ATP = UTP > > ADP = UDP is consistent with nucleotide-induced mucin secretion in HBECs being mediated through the P2Y2 receptor. Significant mucin secretion was observed at nucleotide triphosphate concentrations as a low as 1 µM. Dotted bars, UDP; closed bars, ADP; open bars, UTP; hatched bars, ATP. (B) Pretreatment of ATP, ADP, and UDP preparations with hexokinase (10 U/ml + 25 mM glucose; closed bars) for 10 min resulted in a substantial and significant reduction in mucin secretion, suggesting that nucleotide triphosphate contamination of diphosphate preparations is responsible for much of their stimulatory capacity (*P = 0.05 versus vehicle, n = 4; #P < 0.05 versus vehicle, n = 6; §P < 0.05 versus untreated [open bars]; n = 6). Veh = vehicle. Data are reported as means ± SEM.

 
P2Y2 Receptor Antagonism and G-Protein Coupling
There are no commercially available selective antagonists of the P2Y2 receptor; however the nonselective compound suramin is known to antagonize this receptor. Pre-exposure (30 min) of HBEC cultures to suramin inhibited ATP-{gamma}S–induced (500 µM, 30 min) mucin secretion in a concentration-dependent manner, reaching a maximum of 66 ± 5% inhibition at 500 µM (P < 0.05) (Figure 4A). In the absence of ATP, the highest concentration of suramin examined (500 µM) did not affect baseline mucin secretion over the 60-min exposure period studied. The concentration producing 50% inhibition (IC50) of suramin against ATP-{gamma}S–induced (500 µM, 30 min) mucin secretion was ~ 90 µM, consistent with the relatively weak P2Y2 receptor antagonism reported in the literature (pA2 = 4.3) (22). Although no P2Y2 receptor antagonists are commercially available, the structure of AR-C118925XX, a selective P2Y2 receptor antagonist with submicromolar activity was described recently (16). In our hands, AR-C118925XX (10 µM) was inactive against a panel of 37 receptors except the human P2Y2 receptor. AR-C118925XX (30 min pretreatment) concentration-dependently antagonized ATP-{gamma}S–induced (100 µM, 30 min) mucin secretion in HBECs by 41.7 ± 8.9 (P < 0.05), 58.5 ± 10 (P < 0.05), and 101.1 ± 2.8% (P < 0.05) at 0.1, 1.0, and 10.0 µM, respectively (Figure 4B). The IC50 of AR-C118925XX against ATP-{gamma}S–induced (100 µM, 30 min) mucin secretion was ~ 1 µM.



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  		Figure 4. (A) After a 30-min apical pre-exposure, suramin concentration-dependently inhibited ATP-{gamma}S–induced (100 µM, 30-min exposure) mucin secretion. Inhibition was significant at a suramin concentration of 500 µM (IC50 ~ 90 µM). (B) A 30-min apical pre-exposure to AR-C118925XX, a selective P2Y2 receptor antagonist, concentration-dependently inhibited ATP-{gamma}S–induced (100 µM, 30-min exposure) mucin secretion at 0.1, 1.0, and 10.0 µM. Complete abrogation of the ATP-{gamma}S response was observed at 10 µM, and the IC50 was ~ 1 µM. (C) Mucin secretion was measured from cultures exposed to vehicle (open bars) or ATP-{gamma}S (closed bars). GP-ANT-2A (GP-2A) (50 µM, 4-h apical pretreatment) introduced into the cells using a lipid BioPorter, inhibited ATP-{gamma}S–induced (500 µM, 30-min exposure) mucin secretion. In the same experiment, the Gi/o peptide inhibitor GP-ANT-2 (GP-2) (50 µM, 4 h pretreatment) and SGP-2A (50 µM, 4-h apical pretreatment) did not inhibit ATP-{gamma}S–induced mucin secretion. BioPorter itself did not alter ATP-{gamma}S–induced mucin secretion. (D) Mucin secretion was measured from cultures in the absence (open bars) or in the presence (closed bars) of CTX. PTX (100 ng/ml; 22-h basolateral pretreatment) did not inhibit ATP-{gamma}S–induced (100 µM, 10-min exposure) mucin secretion from cells cultured in the absence of CTX. Cells cultured in the presence of CTX were inhibited by PTX (100 ng/ml; 22-h basolateral pretreatment) from secreting mucin in response to ATP-{gamma}S (100 µM, 10-min exposure) stimulation. (*P < 0.05 versus ATP-{gamma}S; #P < 0.05 versus relevant vehicle; $P < 0.05 versus relevant BioPorter and ATP-{gamma}S–exposed group; §P < 0.05 versus relevant PTX and ATP-{gamma}S–exposed group; n = 6). CTX = cholera toxin; PTX = pertussis toxin. Data are reported as means ± SEM.

 
To investigate the functional G-protein–coupling involved in ATP-{gamma}S–induced mucin secretion in HBECs, we used GP-ANT-2A, a peptide antagonist that selectively inhibits coupling of G-protein–coupled receptors (GPCRs) to Gq (23, 24) in combination with a lipid BioPorter to facilitate cell entry. GP-ANT-2A (50 µM, 4 h pretreatment) completely inhibited ATP-{gamma}S–induced (100 µM, 30 min) mucin secretion (Figure 4C). In the same experiment, SGP-2A, a sequence-scrambled analog of GP-ANT-2A (50 µM, 4 h pretreatment) had no effect on ATP-{gamma}S–induced (100 µM, 30 min) mucin secretion. The related peptide, GP-ANT-2 (50 µM, 4 h pretreatment), which selectively blocks coupling of Gi/o G-protein to GPCRs did not inhibit ATP-{gamma}S–induced mucin secretion. PTX, which causes the dissociation of the {alpha}-subunit of Gi from the ß{gamma}-complex, thus preventing guanosine 5'-diphosphate/guanosine triphosphate exchange and subsequent signaling, did not inhibit ATP-{gamma}S–induced (100 µM, 10 min) mucin secretion after basolateral pre-incubation for 22 h at concentrations of 100 ng/ml (Figure 4D). Apical pre-incubation with PTX (300 ng/ml, –22 h, 20 µl) was also ineffective (data not shown). Because these data were at odds with previously published reports (25, 26), we examined whether differences in the culture medium used could account for these differences. When HBECs were cultured in medium containing CTX (10 ng/ml) for the 14 d differentiation period, the ATP-{gamma}S–induced (100 µM, 10 min) mucin secretory response became PTX-sensitive (100 ng/ml, 22-h basolateral preincubation) (Figure 4D).

Signaling Downstream of P2Y2 Receptor Activation
P2Y2 receptor–mediated mucin secretion has previously been shown to occur through downstream activation of PLC, activation of PKC, and elevation of intracellular calcium concentrations in rat goblet cells (8, 9). In the present study, the PC-PLC inhibitor D609 (30 min pretreatment), concentration-dependently inhibited ATP-{gamma}S–induced (100 µM, 30 min) mucin secretion by up to 72 ± 1.5% at 10 µM (P < 0.05) (Figure 5A). By contrast, the phosphatidylinositol-specific PLC (PI-PLC) inhibitor U73122 (27, 28) did not inhibit ATP-{gamma}S–induced mucin secretion at any concentration examined (0.1, 1.0, and 10.0 µM, 30 min pretreatment) (data not shown). The light-activated PKC inhibitor calphostin C (30 min pretreatment), concentration-dependently inhibited ATP-{gamma}S–induced (500 µM, 30 min) mucin secretion by 35 ± 8% (P < 0.05), 46 ± 6% (P < 0.05) and 94 ± 3% (P < 0.05) at 5, 50 and 500 nM, respectively (IC50 {cong} 50 nM) (Figure 5B). Calphostin C did not affect baseline levels of mucin secretion at a concentration of 500 nM and did not demonstrate overt cytotoxicity at 500 nM over the 1-h exposure period examined (data not shown). Phorbol myristate acetate (PMA, 30 min), which directly activates PKC, concentration-dependently increased mucin secretion by 190 ± 15% (P < 0.05), 274 ± 10% (P < 0.05), and 317 ± 17% (P < 0.05) of baseline at 100, 300, and 1,000 nM (the highest concentration examined), respectively (Figure 6A). PMA concentrations between 1 and 30 nM were ineffective (data not shown). The mucin secretory response to PMA at 300 nM and 1 µM was equivalent to that observed after exposure to a submaximal ATP-{gamma}S concentration of 100 µM in the same experiment with no evidence of a maximal response having been achieved. Calphostin C (30-min pretreatment) partially inhibited PMA-induced (300 nM, 30 min) mucin secretion in a concentration-dependent manner by 27 ± 8% and 62 ± 5%, respectively, at 50 and 500 nM (P < 0.05 and 0.05, respectively) (Figure 6B).



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  		Figure 5. (A) Pre-exposure to D609 (30-min apical pretreatment) concentration-dependently inhibited ATP-{gamma}S–induced (100 µM, 30-min exposure) mucin secretion. Inhibition was significant at 10 µM. (B) ATP-{gamma}S–induced (500 µM, 30-min exposure) mucin secretion was concentration-dependently inhibited by a 30 min apical pretreatment with the potent selective PKC inhibitor, calphostin C. Inhibition was significant at 5 nM and almost complete at 500 nM (*P < 0.05 versus ATP-{gamma}S; n = 6). Data are reported as means ± SEM.

 


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  		Figure 6. (A) Exposure of culture inserts to PMA for 30 min concentration-dependently induced mucin secretion. Exposure to PMA at 1 µM gave comparable responses to an equivalent exposure time to ATP-{gamma}S at 100 µM. (B) PMA-induced (300 nM, 30-min exposure) mucin secretion was concentration-dependently inhibited by a 30 min apical pretreatment with calphostin C. Inhibition was significant, but not complete at 500 nM (*P < 0.05 versus vehicle; #P < 0.05 versus PMA; n = 6). Data are reported as means ± SEM.

 
The Role of Ca2+ in P2Y2 Receptor–Mediated HBEC Mucin Secretion
The extracellular calcium chelator EGTA had no effect on ATP-{gamma}S–induced mucin secretion at 0.5–2 mM (data not shown). In contrast the intracellular Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra (acetoxymethyl) ester (BAPTA-AM) (60-min pretreatment), demonstrated a trend toward concentration-dependent inhibition of ATP-{gamma}S–induced (500 µMM, 30 min) mucin secretion by 11 ± 14%, 38 ± 6% and 57 ± 6% (P < 0.05) at 5, 50, and 250 µM, respectively (Figure 7A). The Ca2+ ionophore, ionomycin (30 min), increased HBEC mucin secretion by 193 ± 8% (P < 0.05) and 221 ± 14% (P < 0.05) at 1 and 2.5 µM, respectively. The ionomycin response observed at 2.5 µM was equivalent to that seen in the same experiment in response to a submaximal ATP-{gamma}S concentration (100 µM, 30 min) (Figure 7B). Although ionomycin concentrations of up to 10 µM were examined (data not shown), maximum achievable ionomycin responses were observed at ~ 2.5–3 µM. Thapsigargin (30 min), which inhibits Ca2+-ATPase–mediated reuptake of Ca2+ into intracellular stores, thereby acutely increasing the cytosolic Ca2+ concentration, caused an increase in mucin secretion of 145 ± 4% (P < 0.05) and 215 ± 19% (P < 0.05) at 50 and 500 nM, respectively (Figure 7C). Suramin (30 min pretreatment) did not inhibit ionomycin-induced (3 µM, 30 min) mucin secretion at concentrations of up to 500 µM (data not shown) despite inhibiting ATP-{gamma}S–induced (500 µM, 30 min) mucin secretion by 66 ± 5% in the same experiment, indicating that ionomycin-induced mucin secretion occurs independently of P2Y2 receptor activation.



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  		Figure 7. (A) Apical pre-exposure 60 min) to the cell-permeable Ca2+ chelator BAPTA-AM concentration-dependently inhibited ATP-{gamma}S–induced (500 µM, 30-min exposure) mucin secretion. Inhibition was significant, but not complete at 250 µM. (B) The calcium ionophore, ionomycin (30-min exposure) increased HBEC mucin secretion in a concentration-dependent manner, reaching significance at 1 µM. (C) Exposure to the Ca2+–ATPase inhibitor thapsigargin for 30 min increased HBEC mucin secretion in a concentration-dependent manner, reaching significance at 50 nM (*P < 0.05 versus ATP-{gamma}S; §P < 0.05 versus vehicle; n = 6). Iono = Ionomycin; Thap = Thapsigargin. Data are reported as means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mucin hypersecretion contributes to morbidity and mortality associated with respiratory diseases, including chronic bronchitis and asthma (1, 4, 5). The underlying mechanisms that regulate mucin production and secretion are poorly understood, in part due to a lack of robust, routine in vitro models that closely resemble human respiratory epithelial cell function. We report here the development of an in vitro agonist-induced mucin secretion model using routinely cultured, well differentiated HBECs. Mucin released into the apical media of culture inserts was measured using a UEA-1/WGA ELLA. The reactivity of these reagents with mucin was validated by comparison with mucin specific antibodies and PAS using dot blots, Western blots of 1% agarose, and 4–20% SDS gels of a human mucin standard. Samples from selected experiments were also analyzed using an ELISLA with UEA-1 as capture and 45M1 as a detection reagent to confirm that the ELLA was detecting mucin. In addition, AB staining of histologic sections was reduced after ATP-{gamma}S exposure, and mucin detection in the apical medium was increased. These data together indicate that UEA-1/WGA assay format detected mucins in our system. All mucin assays, whether directed at carbohydrate or peptide epitopes, are vulnerable to changes in the expression of, for example, enzymes involved in glycosylation and/or protein processing. Therefore, any agent that changes the expression of mediators involved in these processes has the potential to alter the apparent detection of mucins. The experiments described in this article are largely derived from acute studies using 10–60-min exposures to various reagents. It is unlikely that such short exposure periods could lead to substantial qualitative or quantitative changes in stored mucin glycoproteins.

We have used nucleotide triphosphates to develop this model because of their apparently ubiquitous efficacy in stimulating secretion from dedicated mucin-producing cells. Using this model, we have demonstrated that ATP and UTP, but not methacholine, bradykinin, or endothelin-1, which have also previously been implicated in regulating mucin secretory responses, induced mucin secretion. The agonist potency profile ATP = UTP > > ADP = UDP (2MeSATP and adenosine ineffective), together with hexokinase and suramin sensitivity are consistent with a P2Y2 receptor–mediated response. In addition, we have demonstrated sensitivity to AR-C118925XX, a selective P2Y2 receptor antagonist recently reported in the literature, and have also examined elements of the downstream signaling pathways involved in ATP-induced mucin secretion in HBECs.

A critical factor in establishing this model was the development of a wash procedure permitting removal of mucin that had accumulated during cell differentiation at ALI. It has previously been reported that even relatively minor disturbance to cells in culture can trigger the release of nucleotide triphosphates (29), leading to a potential for activation of nucleotide receptors, receptor desensitization, and depletion of mucin granules before experimental manipulation. Following the wash procedure described above, substantial increases in mucin secretion in response to exogenously added ATP were observed at concentrations as low as 1 µM. These responses indicate that this wash procedure did not result in substantial receptor desensitization or depletion of intracellular mucin granules. In addition to removal of accumulated apical mucin during differentiation, it was also found that exposure of the cultures to media free of the supplements present in the Clonetics SingleQuots for 18–48 h before ATP-{gamma}S exposure consistently and significantly increased the signal-to-noise ratio. The specific components of the SingleQuots responsible for this were not investigated. In addition to previously published validation of the ELLA reagents used in the current article (19), the increases in mucin secretion detected by this assay were associated with a decrease in the amount of stored AB-positive material within the goblet cells. This provides additional evidence that the readout from the ELLA is a surrogate measure of goblet cell exocytosis. The substantial responses observed using this model, together with the consistency observed between repeated experiments, provide clear advantages in data interpretation over models demonstrating lower signal-to-noise ratios (10, 17). No other model utilizing routinely cultured, differentiated HBECs consistently demonstrates signal-to-noise ratios in response to ATP (100 µM) of 3–4 times baseline.

After establishment of the basic experimental conditions permitting agonist-induced mucin secretion, we examined the acute effects of ATP, UTP, ADP, UDP, 2MeSATP, adenosine, methacholine, bradykinin, and endothelin-1 on HBEC mucin secretion. ATP, UTP, and their corresponding nucleotide diphosphate preparations were the only agents tested that significantly altered baseline mucin secretion. It has been shown previously that cholinergic stimuli trigger mucin secretion from human submucosal glands and the surface epithelial goblet cells and submucosal glands of some small animal species (see review in Ref. 30). However, there is no convincing evidence that cholinergic stimuli trigger mucin secretion from human surface epithelial goblet cells. The lack of a prosecretory effect of methacholine in this model supports the hypothesis that in humans, cholinergic stimuli do not control surface epithelial goblet cell mucin secretion. An alternative explanation for this lack of response may be that, although relevant muscarinic receptors are expressed in situ in the airways, their expression is reduced after in vitro cell culture. The cells used in this study were obtained at passage 1, expanded to passage 2, and used at passage 3. It is conceivable that receptor expression could change over the course of three passages, especially because the cells are cultured for 4 wk before experimentation. The increase in mucin secretion in response to ATP was substantial, reaching up to 755 ± 180% of baseline. The nucleotide agonist potency profile for mucin secretion, ATP = UTP > > ADP = UDP (2MeSATP and adenosine ineffective), has been classically considered to indicate activation of the P2Y2 receptor. More recent reports, however, have shown that nucleotide diphosphate preparations are often contaminated with nucleotide triphosphates, and it is these triphosphates that are responsible for P2Y2-mediated events (see review in Ref. 21). To determine whether the nucleotide diphosphate–induced mucin secretion observed in the present study was due to nucleotide triphosphate contaminants, they were pretreated with hexokinase. Hexokinase, which breaks down nucleotide triphosphates, but not nucleotide diphosphates, reduced the mucin secretory response of both ADP and UDP, suggesting that a considerable proportion of the nucleotide diphosphate response is mediated by nucleotide triphosphate contaminants.

There are no commercially available P2Y2 receptor–selective antagonists, but the nonselective compound suramin is known to antagonize this receptor (22). In our model, it concentration-dependently inhibited ATP-{gamma}S–induced mucin secretion consistent with mediation via P2Y2 receptor activation. The apparent IC50 (~ 90 µM) for inhibition of ATP-{gamma}S–induced mucin secretion in this study is consistent with the reported low potency (pA2 = 4.3) of suramin against P2Y2 receptor–mediated inositol triphosphate generation and release of Ca2+ from intracellular stores (22, 31). We also examined the activity of AR-C118925XX, a recently described selective P2Y2 receptor antagonist (16). AR-C118925XX concentration-dependently inhibited ATP-{gamma}S–induced mucin secretion from HBECs with complete blockade at 10 µM and an IC50 of ~ 1 µM.

The P2Y2 receptor is a GPCR generally considered to couple to Gq (see review in Ref. 21), although some authors have reported functional responses, including mucin secretion, to be mediated at least partially by Gi G-proteins (26, 32). We investigated whether, in our model, nucleotide triphosphate–induced mucin secretion signaled exclusively through either Gq or Gi/o G-proteins or whether there was a dual signaling pathway, as has been suggested by Chen and colleagues (26) and Kim and colleagues (32). The selective peptide antagonist of Gq, GP-ANT-2A (23, 24), completely inhibited ATP-{gamma}S–induced mucin secretion from HBECs, although the sequence-scrambled control had no effect. Consistent with these results, GP-ANT-2, a peptide antagonist that selectively inhibits Gi G-protein signaling (23, 24), had no effect on ATP-{gamma}S–induced mucin secretion. Furthermore, a 22 h apical or basolateral pre-incubation with the Gi/o G-protein inhibitor PTX failed to inhibit ATP-{gamma}S–induced mucin secretion. These observations suggest that ATP-{gamma}S–induced mucin secretion in this model is mediated entirely via Gq coupling with no role for Gi G-protein–coupling. In contrast, when cells were differentiated in the presence of CTX in accordance with the culture protocol of Chen and colleagues (26), ATP-{gamma}S–induced mucin secretion from HBECs was inhibited by a 22-h basolateral pre-incubation with PTX. This suggests that the PTX used in this study was functionally active in this HBEC system, and also that in the absence of CTX P2Y2 receptor–mediated mucin secretion is Gq-coupled, but that in the presence of PTX it may become at least partially Gi-coupled.

Changes in intracellular calcium and PKC activation have been shown to be the effective activators of the PLC signal transduction system (33). Activation of the P2Y2 receptor has previously been shown to trigger activation of PLC, and subsequently PKC, and both of these enzymes have been linked to mucin secretion in hamster (34), human tracheobronchial cultures (17), or the spontaneously transformed rat goblet cell line, SPOC1 cells (8, 9). In the model described here, the PC-PLC inhibitor D609 inhibited ATP-{gamma}S–induced mucin secretion in a concentration-dependent manner, but the PI-PLC inhibitor U73122 was ineffective. These observations and our PTX data are in contrast to those of Kim and colleagues (32) and Chen and colleagues (26), who demonstrated that, in their model systems utilizing primary hamster or human tracheal epithelial cells, PTX and U73122 inhibited nucleotide triphosphate–induced mucin secretion. We have been unable to confirm roles for Gi G-protein–coupling to the P2Y2 receptor or PI-PLC (26) in our model system in the absence of CTX in the culture medium. However, by culturing the cells in the presence of CTX as reported by Wu and colleagues (25) and Chen and colleagues (26), we were able to make the mucin-secretory responses sensitive to PTX. In addition, we were able to make the mucin-secretory responses sensitive to U73122 (data not shown). The discrepancies between the data published here and those of previous reports seem to stem from differences in components of the culture medium. The non–isoform-specific PKC inhibitor calphostin C inhibited ATP-{gamma}S–induced mucin secretion in a concentration-dependent manner, which is consistent with previously published data (33). PMA, which directly activates PKC, also stimulated HBEC mucin secretion, in contrast to the results published by Li and colleagues (17), and this response was at least partially sensitive to calphostin C, further supporting an involvement of PKC in the regulation of mucin secretion. Calphostin C significantly inhibited 500 µM ATP-{gamma}S–induced mucin secretion at 5 nM and almost completely blocked it at 500 nM, independent of overt cytotoxicity. Calphostin C was less effective against mucin secretion triggered by 300 nM PMA than mucin secretion triggered by 500 µM ATP-{gamma}S, giving no inhibition at 5 nM and only 62 ± 5% at 500 nM. Calphostin C is a highly selective inhibitor that competes for the phorbol ester binding site of PKC. These data suggest that either calphostin C at 500 nM is unable to compete effectively with PMA at 300 nM for the PKC binding site or that PMA-induced mucin secretion includes a PKC-independent process. In fact, it is known that PMA directly activates MUNC13/18, a fundamental part of the secretory apparatus (35), which may account for the calphostin C–insensitive portion of the PMA-induced mucin secretory response.

It has previously been shown that activation of the P2Y2 receptor leads to increases in intracellular Ca2+ concentration and that this elevation of intracellular Ca2+ concentration is important in the mucin-secretory response in the rat cell line SPOC1 (33). In this model, we have demonstrated that BAPTA-AM, a membrane permeable form of the Ca2+ chelator BAPTA, inhibited ATP-{gamma}S–induced mucin secretion in a concentration-dependent manner by up to 57 ± 6%. In addition, the calcium ionophore ionomycin, which causes an increase in intracellular Ca2+ concentration, induced a substantial concentration-dependent increase in mucin secretion from HBECs. In contrast to ATP-induced mucin secretion, suramin did not inhibit ionomycin-induced mucin secretion. This indicates that ionomycin does not operate via indirect activation of P2Y2 receptors for example by stimulating the release of nucleotide triphosphates from cells. Thapsigargin, which blocks uptake of Ca2+ into intracellular stores, also caused a concentration-dependent increase in mucin secretion. These observations support a role for the involvement of changes in intracellular Ca2+ concentrations in the signaling pathway that leads to mucin secretion in HBECs.

The apparent agonist concentration needed to produce 50% of the maximal response (EC50) for ATP/UTP at the P2Y2 receptor varies between studies. Values of from 200 nM up to 15 µM have been reported in transformed and immortalized cell lines independent of whether the readout is proximal or distal to receptor activation. In more complex models, using differentiated, nontransformed, nonimmortalized cells, such as colonic mucosa and conjunctival epithelial cells, both proximal Ca2+ mobilization and terminal mucin secretion exhibit EC50s of 3–6 µM (36, 37). Although we are not able to estimate an EC50 because we did not have evidence of a maximal response at 1 mM ATP, concentrations of ATP as low as 1 µM resulted in significant increases in mucin secretion, which is consistent with literature reports for nontransformed, nonimmortalized cells. The physiologic relevance of the concentrations of nucleotide triphosphates used in the current studies is difficult to assess. Nucleotide triphosphates are rapidly broken down by specific extracellular enzymes, which limits accurate estimation of concentrations in most microenvironments. However, circumstantial evidence suggests that the concentrations used here are physiologic. The concentration of extracellular ATP on the apical surface of epithelial cells in vitro and in airway surface liquid in vivo has been estimated at ~ 0.5–1 µM (38) and as high as 20 µM. The concentration of ATP in the cytoplasm of all cells is ~ 3–5 mM, and the concentration of ATP in the secretory granules of some cells (e.g., medullary chromaffin cells, platelets, mast cells, and goblet cells) has been estimated to be as high as 170 mM (39). Release of ATP from these sources after cell lysis or exocytosis could achieve local ATP concentrations in excess of even the highest concentrations used in the current study. The physiologic relevance of stimulated ATP release was recently highlighted in a study demonstrating that physical stimulation of a single epithelial cell, without rupturing its membrane, caused release of sufficient ATP to trigger functional responses in cells up to 300 µm away (40).

In summary, we have developed a robust, physiologically relevant in vitro model of substantial agonist-induced mucin secretion from routinely cultured, differentiated, HBECs. This model has an exceptional signal-to-noise ratio compared with those described in other reports in the literature, making it the first model of agonist-induced mucin secretion in HBECs that is readily amenable to pharmacologic manipulation. In addition, this is the first report of the activity of a selective P2Y2 receptor antagonist, AR-C118925XX, against ATP-{gamma}S–induced mucin secretion. The results reported here using this model are largely consistent with known mechanisms of P2Y2 receptor activation, G-protein–coupling, and downstream signaling, and also with the small number of reports describing ATP-induced mucin secretion.


   Acknowledgments
 
The authors thank June Giddings, Gareth Jones, and Francis Schindler for their histology support, and Paul Kirkham and Claire Foulkes-Jones for their support during preparation of the human mucin standard. The authors also thank Robin Fairhurst, Darryl Jones, and Andrew Tuffnell for the synthesis of AR-C118925XX, and Henry Danahay for the epithelial resistance measurements.

Received in original form June 5, 2003

Received in final form June 8, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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