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J. Biol. Chem., Vol. 279, Issue 37, 38854-38860, September 10, 2004

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Regulation of Antiprotease and Antimicrobial Protein Secretion by Airway Submucosal Gland Serous Cells^*

Nam Soo Joo, Dennis J. Lee, Kimberly M. Winges, Arjun Rustagi, and Jeffrey J. Wine

From the Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-2130

Received for publication, June 24, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors -1-antitrypsin and -1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na⁺ channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J_lyz) from 32 to 77 ng/hr/cm² (n = 36, p < 0.005). Thapsigargin increased J_lyz from 40 to 63 ng/h/cm² (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J_lyz by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J_lyz, but clotrimazole was without effect. CFTR_inh172 caused a small (15%) but significant inhibition of forskolin-stimulated J_lyz without affecting basal J_lyz. Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na⁺ channels via serine proteases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The main function of the airways is to conduct air to and from the respiratory bronchioles and alveoli for gas exchange. Airborne particles, including pathogens, are adsorbed by the mucus layer lining the airways, where they are inactivated by the innate mucosal defense system and removed via mucociliary and cough clearance. In cystic fibrosis (CF)¹ this process is defective, allowing bacteria and fungi to grow within static airway mucus. That leads to a process of inflammation that eventually occludes and destroys the airways.

Most of the mucus lining the upper airways is produced by submucosal glands, and gland secretion is defective in CF (1). Glands are complex structures that comprise a ciliated duct connected to a non-ciliated collecting duct into which multiple tubules empty. Each tubule is lined by mucous cells in its proximal part and serous cells at the distal ends. Serous cells are reported to secrete a rich mixture of proteins that have antimicrobial, antiprotease, antioxidant and anti-inflammatory functions, although there is to date relatively little direct evidence of such secretion. Within submucosal glands, serous cells are the main site for cystic fibrosis transmembrane conductance regulator (CFTR) expression.

Studies of submucosal gland secretion performed in animals, healthy human subjects, and humans with cystic fibrosis have led to a model in which cholinergic stimulation, which elevates [Ca²⁺]_i, leads to secretion from both mucous and serous cells, whereas stimulation with vasoactive intestinal peptide (VIP), which elevates [cAMP]_i, stimulates only serous cells. In cystic fibrosis, secretion to VIP or forskolin is completely lost (1), whereas the response to cholinergic stimulation is altered, which leads to thicker mucus (2, 3). Joo et al. interpreted their results with a model of gland secretion in which the lack of functional CFTR causes the electrolyte-driven fluid secretion from serous cells to be deleted, leaving protein secretion intact (1). However, because no mucus is observed leaving CF gland ducts after stimulation with VIP, these results imply that the proteins are trapped within the glands. An alternative view is that CFTR mediates the secretion of mucins and serous proteins and that its loss will cause CF mucus to have a diminished complement of defense molecules (4).

The inaccessibility of serous cells makes them difficult to study directly. Therefore, the Calu-3 cell line, a serous cell model, has been used as a surrogate to study fluid and protein secretion (5). Previous studies have shown that Calu-3 cells are capable of releasing proteins such as glycoproteins/mucins and lysozyme in response to cAMP agonists (4, 6-8).

In this study, we harvested uncontaminated mucus secreted by porcine glands and collected apical fluid from Calu-3 cells, analyzing bands via silver-stained SDS-PAGE gels. We then identified five proteins not previously known to be secreted by either glands or Calu-3 cells and reviewed evidence that they inhibit serine proteases and have antimicrobial functions. Because airway surface ENaCs are known to be activated by serine proteases, the secretion of serine protease inhibitors by gland serous cells could play a role in diminishing Na⁺-driven fluid absorption via ENaCs. We also demonstrate that proteins are released in response to elevations of either [Ca²⁺]_i or [cAMP]_i, with the latter pathway being more effective in Calu-3 cells. Our attempts to implicate CFTR in protein release were not conclusive; the most effective and specific CFTR inhibitor we used, CFTR_inh172 (3), produced only a slight (but significant) decrease in forskolin-stimulated lysozyme release.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Tissues and Mucus Collection—Porcine tracheas were harvested <1 h postmortem from adult Yorkshire pigs that had been killed after acute experiments unrelated to the present studies. Tissue was maintained until use in ice-cold Krebs-Ringer bicarbonate buffer bubbled with 95% O₂ and 5% CO₂. The Krebs-Ringer bicarbonate composition was 115 mM NaCl, 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgCl₂, 1.2 mM CaCl₂, and 10 mM glucose (pH 7.4), and osmolarity was adjusted to 290 mosM. To minimize endogenously generated prostaglandins, 1 µM indomethacin was present in the bath throughout the experiment. The experimental setup for the agonist-stimulated mucus secretion was as described previously (9-11). Secreted mucus bubbles were collected by aspiration and stored at -20 °C until use.

Calu-3 Cell Culture and Apical Fluid Collection—Calu-3 cells were grown in a T₂₅ tissue culture flask containing a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture supplemented with 15% fetal bovine serum (Sigma), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were passaged once a week with 1:8 dilutions and plated at 5 x 10⁵ cells/cm² onto Costar Snapwell filters (12-mm diameter, 0.4-µm pore size, 1 cm² growing area) that had been coated with human placental collagen (Sigma). Cells on filters were grown at an air/liquid interface after 2-3 days of an initial submerged culture and used within 2-4 weeks when they reached confluence.

A day before apical fluid collection, the medium was replaced with serum-free medium (minimal medium), and the apical surface was washed multiple times with minimal medium. Transepithelial resistance and transepithelial potential difference of the cell monolayers were measured with Millicell chopstick electrodes (Millipore). Confluent cells were incubated in the presence of apical 200-µl minimal medium and basolateral medium with or without a vehicle or an agonist in a highly humidified 5% CO₂ incubator for 2 h. To investigate the potential involvement of CFTR in the process of protein secretion by Calu-3 cells, Calu-3 monolayers were apically pretreated with a vehicle or a known inhibitor of ion transport/fluid secretion for 20 min and then followed by 2 h of incubation with the basolateral medium containing a vehicle or an agonist with or without an inhibitor.

Secreted apical fluid was collected after incubation, and another 200 µl of minimal medium was added apically to complete the fluid collection. The collected fluid was briefly centrifuged at 3000 rpm (Eppendorf 5415 centrifuge) to remove any cellular debris. The supernatant was stored at minus 20 °C until use.

SDS Gel, Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Time-of-flight Tandem Mass Spectrometry (LC-MS), and Enzyme-linked Immunosorbent Assay (ELISA)—Secreted proteins from gland mucus or Calu-3 apical fluid were separated and visualized on silver-stained 10-20% gradient Tris-Tricine SDS polyacrylamide gels (Ready Gel; Bio-Rad). Selected bands were excised from the SDS gels and digested by trypsin.

Peptide mixtures were analyzed using LC-MS. The acquired mass spectra from trypsin-digested products were compared with the known trypsin digest protein sequence data base using Mascot software (Matrix Science).

An ELISA for human lysozyme was initially carried out with a kit from Biomedical Technologies Inc. We then developed an in-house ELISA for human lysozyme. Briefly, rabbit anti-lysozyme antibody (Zymed Laboratories Inc.) was coated onto 96-well ELISA plates at 5 µg/ml using 50 mM carbonate-bicarbonate buffer (Sigma). Coating was done by overnight incubation at 4 °C in the presence of 1% nonfat dried milk in phosphate buffered saline (PBS) containing 0.5% Tween. After the removal of the blocking solution and the washing of the ELISA plate 3-5 times with PBS containing 0.1% Tween (PBS-Tween), Calu-3 apical fluid samples diluted 1:3 in PBS and human neutrophil lysozyme standards (Sigma) were incubated in the plate for 2 h at room temperature. The plate was washed 3-5 times with PBS-Tween and then incubated with diluted (1:1500 in blocking buffer) secondary antibody (Calbiochem sheep anti-human lysozyme) for 1 h at room temperature. After 3-5 washes with PBS-Tween, the plate was incubated with a diluted (1:5000 blocking buffer) detection antibody (Zymed Laboratories Inc. rabbit anti-sheep IgG (H+L) horseradish peroxidase conjugate) for 1 h. The plate was washed 3-5 times with PBS-Tween and incubated with the substrate, 3,3',5,5'-tetramethylbenzidine (Zymed Laboratories Inc.) for 20 min in the dark. Absorbance at 450 nm was recorded using an automated ELISA microplate reader (Molecular Devices).

Reagents—All chemicals were obtained from Sigma unless indicated otherwise and maintained as aliquots of stock concentrations. A stock solution for carbachol was made in deionized water, and forskolin, thapsigargin, 1-ethyl-benzimidazolinone (1-EBIO), CFTR_inh172, and clotrimazole were dissolved in dimethyl sulfoxide (Me₂SO). CFTR_inh172 was kindly provided by Drs. Alan Verkman (University of California, San Francisco) and Jack Stutts (University of North Carolina).

Statistics—Data are expressed as mean ± S.E. unless indicated otherwise. Student's t test for paired data was used to compare the means of different treatment groups. The difference between the two means was considered to be significant when p < 0.05.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Identification of Secreted Proteins—Uncontaminated submucosal gland mucus from porcine tracheas was evaluated with the silver staining of Tris-Tricine SDS gradient gels (Fig. 1). Under the conditions used, at least 40 bands, from 5 to 200 kDa, could be resolved. No differences were found between forskolin- or carbachol-induced mucus in either the number or intensities of the bands, indicating that these two methods of stimulation have equivalent effects on the protein secretion by submucosal glands (Fig. 1a).

View larger version (84K): [in this window] [in a new window]   FIG. 1. Protein analyses from mucus/apical fluid collection. a, top section, side view of gland mucus bubbles secreted into an oil layer covering a porcine tracheal preparation. Bottom section, silver-stained protein bands from uncontaminated porcine mucus on a Tris-Tricine 10-20% gradient SDS polyacrylamide gel. Mucus was produced either in response to 10 µM forskolin (Fsk) or 10 µM carbachol (Carb). Protein molecular weight standards are shown on the right. b, top section, schematic of apical fluid collection from confluent Calu-3 cells growing on a filter coated with human placental collagen. Bottom section, silver-stained protein bands from control wash out (WO) of the apical surface with a minimal medium, a 2-h incubation with a vehicle (Bs, basal), or a 2-h exposure to 10 µM forskolin (Fsk). Molecular weight markers are on the left. c, identification of secreted proteins from Calu-3 by LC-MS. Results for -1-antitrypsin are shown as representative. Predicted mature 394-amino acid residues plus an N-terminal 24-amino acid signal peptide (underlined) (30) of -1-antitrypsin are shown here. A total of 11 tryptic fragments (boxed, boldface) matched the -1-antitrypsin 47 kDa protein.

A comparison of forskolin-stimulated gland and Calu-3 secretions indicated that our collection methods (see "Materials and Methods") yield less total protein from Calu-3 cells versus glands. Nevertheless, although not immediately apparent in Fig. 1, many bands of comparable molecular weight and comparable relative intensities were observed in the silver-stained SDS gels from glands and Calu-3 cells, and some of these were selected for further analysis using LC-MS.

Six proteins secreted by Calu-3 cells were identified using LC-MS, namely lysozyme, -1-antichymotrypsin, -1-antitrypsin, siderocalin (represented here by two proteins, the neutrophil gelatinase-associated lipocalin precursor lipocalin-2 and its processed form, NGAL), and the heat shock cognate protein HSC71. Because the 198-amino acid NGAL precursor lipocalin-2 ( 22.8 kDa) and the 178-amino acid NGAL (20.7 kDa) are not clearly distinguished in terms of function, we discuss them together as siderocalin (12). A representative LC-MS analysis for -1 antitrypsin is shown in Fig. 1c. Analysis for two bands was inconclusive.

Reverse transcriptase PCR analysis of Calu-3 lysates was used to confirm the presence of transcripts for these five proteins (counting NGAL and lipocalin-2 as one protein) using primers designed on reported sequences of each protein. We detected clear evidence for the expression of all of them in Calu-3 lysates (data not shown). The complementary results from reverse transcriptase PCR and LC-MS provide evidence that these proteins were correctly identified and are indeed secreted into the apical media by Calu-3 cells. We consider it significant that antimicrobial, anti-inflammatory. or anti-proteolytic actions have been reported for each of these proteins (see below)

Lysozyme was the first natural antimicrobial to be discovered and is abundant and well studied. It is secreted by human submucosal glands and Calu-3 cells (7) and was used in this study as an archetype to quantify the effects of agonists and inhibitors on protein secretion (see next section).

-1-Antichymotrypsin and -1-antitrypsin are members of the serine protease inhibitor (serpin) family and are major protein components of the lung epithelial lining fluid, where they may also function as innate defense molecules against bacteria and viruses (13). However, the cellular origin within the lung of these inhibitors of serine proteases has not been demonstrated. Our results suggest that gland serous cells are one source.

The presence of -1-antitrypsin and -1-antichymotrypsin is of particular interest, because the activity of the ENaC is activated, at least in part, by serine proteases (14). It is well documented that serine protease inhibitors protect the respiratory tract from proteolytic damage (15). Loss of functional CFTR in airways increases Na⁺-driven fluid absorption by surface epithelial cells because of the increased activity of ENaC. Extracellular serine proteases such as trypsin and chymotrypsin can increase ENaC activity, whereas a serine protease inhibitor, aprotinin, decreases ENaC activity (16). We hypothesize that a balance between serine proteases, (such as trypsin, channel-activating serine proteases, neutrophil elastase, and cathepsin G) and serine protease inhibitors (such as -1-antichymotrypsin and -1-antitrypsin) may be important for maintaining optimal properties of airway mucus. This hypothesis provides an additional mechanism to link CFTR and ENaC; in CF, the diminished surface availability of gland-derived serine protease inhibitors might further enhance fluid absorption and further dehydrate airway mucus.

HSC 71 is also called lipopolysaccharide-associated protein 1 or heat shock 70 kDa protein 10. In general, heat shock proteins are regarded as intracellular chaperones that aid the folding and assembly of oligomeric proteins and as intracellular proteases that mediate ubiquitin-dependent degradation of certain proteins via proteosomes. However, heat shock proteins, including HSC 71, can be released into the extracellular milieu under physiological conditions within small membrane-bound vesicles termed exosomes (17) by a number of cell types, including epithelial cells (18). Heat shock proteins are found in the peripheral circulation of healthy people, where they are thought to function as anti-inflammatory molecules because of their immune responses to pathogenic microbes (19). Our results indicate that they may serve the same function in airway surface liquid. The occurrence of HSC71 in exosomes raises the possibility that its release may be entirely constitutive. However, the band identified as HSC71 was clearly darker in stimulated preparations, and we found no evidence of any bands that were unaffected by stimulation (data not shown).

Siderocalin (NGAL), originally identified as a protein stored in neutrophil granules, is a member of the lipocalin superfamily, so named because of the ability of its members to bind the lipophilic components of ligands. However, whereas NGAL binds lipids with only millimolar affinity, it was recently shown (in a remarkable study that included crystallographic evidence for binding) that NGAL binds bacterial catecholate-type ferric siderophores with subnanomolar affinity (12). As pointed out by Goetz et al. (12), the binding of siderophores will complement iron sequestering by lactoferrin to starve bacteria of iron and is potentially much more efficient because it targets iron already bound for bacterial use. They showed directly that NGAL is bacteriostatic and argue that the recognition machinery of NGAL should allow a large range of catecholate-type siderophores to be bound. Because siderophores and not lipids are the natural ligands of NGAL, the authors propose that NGAL be renamed siderocalin (12).

Quantification of Protein Secretion Using Lysozyme as a Marker Protein—As shown above, lysozyme is one of many proteins secreted into the apical fluid by unstimulated Calu-3 cells. Because relative band densities were maintained across different stimulation conditions, we used lysozyme as a representative protein for the quantification of apically secreted proteins. The lysozyme secretion rate in ng/hr/cm² was measured by ELISA in the presence or absence of an agonist or an antagonist. The confluent Calu-3 monolayers used in the ELISA experiments had a mean transepithelial resistance of 600 ± 16 ·cm² and a mean transepithelial potential difference of 9.2 ± 0.2 mV (n = 205, apical negative). Basal and agonist treatments were done for 2-h periods in a highly humidified CO₂ incubator. Unstimulated Calu-3 cells secreted lysozyme basally at an average rate of 31.7 ± 2.3 ng/h/cm² (range 8-91 ng/h/cm², n = 51).

Forskolin Stimulated Lysozyme Secretion—Forskolin (10 µM), which directly activates adenylate cyclase to increase [cAMP]_i, significantly increased the lysozyme secretion rate from 31.7 ± 3.0 to 77.3 ± 9.1 ng/h/cm² (160.5 ± 28.8%, n = 36, p < 0.005; Fig. 2a). The percentage increase in lysozyme secretion is much larger than the percentage increases in I_sc (20) or fluid secretion (21) induced by forskolin in Calu-3 cells. The rates for both unstimulated and forskolin-stimulated lysozyme secretion were relatively constant during the 2-h measurement periods (Fig. 2b). The constant rate of forskolin-stimulated lysozyme secretion is consistent with the rates of forskolin-stimulated gland mucus secretion, which are also constant over time (1, 10). We did not find any evidence that mechanical stimulation during the apical fluid collection process caused increased lysozyme secretion.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Basal and forskolin-stimulated lysozyme secretion. a, rate of lysozyme secretion (determined by ELISA) from unstimulated (basal) and forskolin-stimulated (Fsk) confluent Calu-3 monolayers. Lysozyme secretion rate is shown as ng/h/cm2. Data are mean ± S.E., *, p < 0.005, n = 36. b, a representative 2-h time course for basal () and forskolin-stimulated () lysozyme secretion. c, correlation between basal and forskolin-stimulated lysozyme secretion rates. Each symbol plots basal lysozyme secretion rate on the x-axis and forskolin-stimulated lysozyme secretion rate on the y-axis, n = 36. Correlation coefficient, r2 = 0.4.

Carbachol- and Thapsigargin-stimulated Lysozyme Secretion—The cholinergic agonist carbachol is a potent secretagogue for mucus secretion from airway submucosal glands (Fig. 1a). Cholinergic agents cause a fast and transient burst of mucus secretion followed by sustained secretion at a rate that is much slower than that for the initial transient burst but is still usually 2-3 times greater than the rate of sustained secretion to forskolin or VIP (10). However, in Calu-3 cells lysozyme secretion was only modestly stimulated by carbachol (100 µM), increasing from 24.1 ± 4.1 to 31.6 ± 5.7 ng/h/cm² (40.3 ± 24.5%, n = 11, p < 0.05; Fig. 3a). In I_sc experiments with Calu-3 cells, the I_sc response to carbachol is large but transient, with little or no sustained increase (5). The reason for the rapid adaptation of the Calu-3 I_sc response to carbachol is not known, but if the I_sc spike represents a spike in [Ca²⁺]_i and if carbachol-stimulated lysozyme secretion is mediated by increases in [Ca²⁺]_i, then any initial large spike in lysozyme secretion would be averaged over the 2-h sample period of our experiments. However, an alternative possibility is that the lysozyme secretion from Calu-3 cells is driven by some aspect of muscarinic stimulation other than an increase in [Ca²⁺]_i.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Agonist-stimulated lysozyme secretion from Calu-3 cells. a-c, basal and agonist-stimulated lysozyme secretion rates to three agonists: 100 µM carbachol (Carb) (a); 1 mM 1-EBIO (b); and 333 nM thapsigargin (Tg) (c). Bar graphs show mean ± S.E.; *, p = 0.017, n = 11 (a); *, p = 0.04, n = 10 (b); and *, p = 0.001, n = 16 (c). d, lysozyme secretion rates to 10 µM forskolin (Fsk) or 10 µM forskolin plus 333 nM thapsigargin (Fsk + Tg). Bar graphs show mean ± S.E.; *, p = 0.028, n = 9. Note that different scales were used, depending upon the agonist.

Thapsigargin in the presence of forskolin produces additive or synergistic effects on fluid secretion by Calu-3 cells (21). To see if protein secretion is also increased by combining cAMP and Ca²⁺ agonists, lysozyme secretion was measured for cells treated with 10 µM forskolin alone or with 10 µM forskolin plus 333 nM thapsigargin. As shown in Fig. 3d, thapsigargin plus forskolin was a more effective stimulus than either used alone, with thapsigargin causing a further increase of almost 50% over forskolin alone (from 61.1 ± 6.7 to 85.9 ± 11.1 ng/h/cm², 47.9 ± 25.6%, n = 9, p < 0.05).

1-EBIO Is a Weak Agonist for Lysozyme Secretion—The results with forskolin and thapsigargin point to a dissociation between protein and fluid secretion in Calu-3 cells. To explore this possibility further, we used 1-EBIO, a benzimidazolone that was shown by Devor et al. to powerfully activate I_sc in Calu-3 cells by coordinate action on both apical CFTR and basolateral Ca²⁺-activated K⁺ channels, with both effects possibly being mediated by direct interactions with the respective channels (22). 1-EBIO also strongly stimulates fluid secretion by Calu-3 cells, increasing net fluid secretion by >170% (21). In contrast to its powerful effects on electrolyte-mediated fluid secretion, 1-EBIO (1 mM) stimulated only a small increase of lysozyme secretion, from 34.3 ± 8.2 to 44.4 ± 11.6 ng/h/cm² (31.4 ± 11.9%, n = 10, p < 0.05; Fig. 3b).

Summary of Agonist Effects—These results lead to three conclusions. First, Calu-3 cells have both cAMP- and Ca²⁺-mediated protein secretory pathways, similar to what has been shown for exocytosis by pancreatic cells (23) and some epithelial cells (for a review, see Ref. 24). Second, the cAMP pathway appears to be more robust than that of Ca²⁺, at least under the conditions of our experiments, and, finally, the effects of the agonists on the rate of protein secretion do not correspond to their effects on I_sc and net fluid secretion.

Effects on Protein Secretion of Three Antagonists of Fluid and Electrolyte Secretion—Although there is ample evidence that fluid and protein secretion can be independently regulated, coordinate action is the rule, raising the question of what common pathways participate in such linkage. Of most interest is the possibility that CFTR plays a critical role in the process such that protein secretion would be altered, at least in some cells, by the loss of functional CFTR (4).

To investigate the potential involvement of CFTR in the process of protein secretion by Calu-3 cells, protein secretion by Calu-3 monolayers was assessed in the presence of various inhibitors (see "Materials and Methods" for details of these experiments). We tested three inhibitors known to affect ion transport: (i) glibenclamide, a nonspecific inhibitor of CFTR and other transporters; (ii) clotrimazole, a specific inhibitor of Ca²⁺-activated K⁺ channels; and (iii) CFTR_inh172, the most potent and specific inhibitor of CFTR presently available (25). The rationale was that a comparison of the effects of these three inhibitors might support or refute the specific involvement of CFTR in protein secretion.

Glibenclamide (0.6 mM) inhibited basal and stimulated lysozyme secretion (Fig. 4a). Basal lysozyme secretion was reduced 25%, from 26.1 ± 3.4 to 20.2 ± 4.2 ng/h/cm² (n = 14, p < 0.05). Forskolin-stimulated secretion was inhibited 41%, from 74.0 ± 13.7 to 45.5 ± 10.4 (n = 19, p < 0.0005); and thapsigargin-stimulated secretion was inhibited 47%, from 57.5 ± 6.9 to 30.7 ± 4.5 ng/h/cm² (n = 6, p < 0.0005).

View larger version (14K): [in this window] [in a new window]   FIG. 4. Effects of three antagonists on lysozyme secretion by Calu-3 cells. a, effects of 600 µM glibenclamide (G) on basal secretion (Bs) and secretion stimulated by forskolin (Fsk) or thapsigargin (Tg). Histograms show mean ± S.E.; *, p < 0.05, n = 14 (Bs); *, p < 0.0005, n = 19 (Fsk + G); and *, p < 0.0005, n = 6 (Tg + G). b, effects of 30 µM clotrimazole (C) on basal secretion (Bs) and secretion stimulated by forskolin (Fsk) or thapsigargin (Tg). Histograms show mean ± S.E., n = 3-5 of each condition. All comparisons were non-significant. c, effects of 10 µM CFTRinh172 (172) on basal (Bs) or forskolin-stimulated (Fsk) lysozyme secretion. Histograms show mean ± S.E.; *p = 0.008, n = 7 (Bs + 172) and 11 (F + 172). In all conditions an inhibitor was present for 20 min before the agonist was introduced and remained throughout the 2-h incubation period.

CFTR_inh172 (10 µM) did not inhibit basal lysozyme secretion (39.6 ± 4.0 to 42.7 ± 3.9 ng/h/cm², n = 7), and was not tested with thapsigargin. It produced a small (15%), but significant decrease in forskolin-induced lysozyme secretion from 63.2 ± 4.7 to 53.3 ± 4.4 ng/h/cm² (n = 11, p = 0.008; Fig. 4c).

Summary of Inhibitor Effects—The mechanisms responsible for the various effects observed on protein secretion are unknown. At the levels used here, glibenclamide inhibits at least CFTR, ATP-sensitive K⁺ channels, and carnitine palmitoyltransferase 1 (26).² Although clotrimazole is a specific blocker of intermediate conductance Ca²⁺-activated K⁺ channels, its K_d for blocking those channels is in the nanomolar range, and other effects could operate at the doses used here. Nevertheless, none of the clotrimazole treatments reached significance, although they were consistently associated with increases in protein release, which, in the case of thapsigargin, was a 50% increase that approached significance. Thus, a powerful inhibition of ion transport does not necessarily translate into inhibition of protein secretion. Finally, the small but significant inhibitory effect of 10 µM CFTR_inh172 on forskolin-mediated lysozyme secretion might be mediated via CFTR, but CFTR_inh172 is a new compound with micromolar potency, and it is possible that it has other targets.

Protein Secretion by Calu-3 Cells in the Context of Submucosal Gland Mucus Secretion—In their model of submucosal gland secretion (1), Joo et al. hypothesized that serous cells secrete electrolyte-driven fluid secretion and protein secretion in response to both acetylcholine and VIP. In cystic fibrosis, they showed that electrolyte-driven fluid secretion is deleted, and hypothesized that protein secretion was intact. Because in their experiments no mucus escaped the CF glands in response to VIP, the inference is that any secreted proteins are trapped in the gland until partial secretion is produced via acetyl choline (1).

In the present work, Calu-3 cells were used as a surrogate for serous cells, whereas forskolin and thapsigargin were used to mimic the effects of VIP and acetylcholine. Although clearly imperfect as models, Calu-3 cells allow the study of electrolyte transport and protein secretion in a relatively homogeneous, serous like cell, whereas mucus secreted by glands is an admixture of multiple cell types.

To the extent that the cell model and pharmacological tools are valid, these results support a model of gland secretion in which serous cells provide a component of mucus that is rich in antimicrobial and anti-proteolytic compounds. Whereas the lack of CFTR may have only a modest effect on protein secretion from serous cells, the deletion of the HCO₃^--rich fluid secretion from serous cells could cause secreted proteins to be entrapped in underhydrated, viscous mucus (27, 28). That result should have profoundly negative consequences for the bio-availability of these proteins within the airway surface mucus, where they are essential to slow the proliferation of inhaled pathogens. In addition, the lack of serine protease inhibitors will render the airways more vulnerable to damage, and the unchecked proteolytic activation of ENaC may further dry and thicken the already underhydrated airway secretions, further crippling mucociliary and cough clearance (29). Thus, we hypothesize that the chronic airway infections of cystic fibrosis arise as the consequence of static mucus with reduced antimicrobial capacity.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK-51817 and the Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Cystic Fibrosis Research Laboratory, Rm. 516, Bldg. 420, Main Quad, Stanford University, Stanford, CA 94305-2130. Tel.: 650-725-2468; Fax: 650-725-5699; E-mail: nsjoo{at}psych.stanford.edu.

¹ The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; CFTR_inh172, a CFTR inhibitor; 1-EBIO, 1-ethyl-benzimidazolinone; ELISA, enzyme-linked immunosorbent assay; ENaC, epithelial Na⁺ channel; I_sc, short circuit current; J_lyz, lysozyme secretion rate; LC-MS, liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry; NGAL, neutrophil gelatinase-associated lipocalin (siderocalin); PBS, phosphate-buffered saline; VIP, vasoactive intestinal peptide.

² We have observed inhibitory effects of glibenclamide on mucus secretion from CF submucosal glands (N. S. Joo and J. J. Wine, unpublished results), further supporting the idea that it has effects on processes other than CFTR.

	ACKNOWLEDGMENTS

 
We thank Rui Yun and for excellent technical assistance and Mauri E. Krouse for critical reading of the manuscript. We are grateful to Alan S. Verkman and Jack Stutts for providing CFTR_inh172 and to Allis Chien for helpful discussion on our Ms-spec analyses.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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