CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands

Stephen T. Ballard1, Laura Trout1, Zsuzsa Bebök2, E. J. Sorscher2, and Angela Crews1

1 Department of Physiology, College of Medicine, University of South Alabama, Mobile 36688; and 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl- and HCO-3 secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 ± 0.6 µl · cm-2 · h-1. Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl--channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl- channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in HCO-3 secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl- and HCO-3 and is mediated by the CFTR.

cystic fibrosis; cystic fibrosis transmembrane conductance regulator; bronchi; epithelium; exocrine glands


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYSTIC FIBROSIS transmembrane conductance regulator protein (CFTR) is a cAMP-regulated anion channel (1). In human, bovine, and ferret lungs, this protein is found in both surface and glandular epithelia of the airways, although the magnitude of expression is greatest in the serous cells of the airway submucosal glands (4, 11, 19). In persons afflicted with cystic fibrosis (CF), the CFTR is mutated, greatly reducing the ability of the airway epithelium to secrete Cl-. The myriad of pulmonary symptoms that characterize this disease, such as mucus plugging of airways, increased susceptibility to airway infection, and bronchiectasis, are thought to result in some way from impairment of transepithelial anion secretion. However, a definitive paradigm linking reduced anion transport with development of CF lung pathology has not been established. Indeed, the normal function of airway surface liquid as well as the site of origin of this fluid remains controversial.

Recent studies demonstrated that ACh-induced liquid secretion across porcine bronchial epithelium is driven by active transport of both Cl- and HCO-3 (22, 23). Because inhibitors of Cl- and HCO-3 secretion also cause mucins to accumulate within the ducts of the submucosal glands, it has been reasoned that these agents selectively blocked liquid but not mucus secretion from submucosal glands (7, 8). Whereas similar obstruction of airway gland ducts is one of the first signs of CF disease in newborns (15), these findings suggest that uncoupling of liquid and mucus secretion also occurs in this disease, possibly representing the primary pulmonary lesion in CF. The mechanism of ACh-induced Cl- and HCO-3 secretion from pig airways appears to be consistent with the model proposed by Smith and Welsh (21), who suggested that both Cl- and HCO-3 cross the apical membrane of airway epithelial cells through CFTR. Although evidence for CFTR involvement in anion secretion has been reported for primary cultures and cell lines derived from airway gland cells (12-14), to our knowledge, CFTR has not been definitively linked to physiological liquid secretion by submucosal glands in intact airways.

In the present study, we hypothesized that CFTR is present in the airway submucosal glands of pigs and plays a significant role in the secretion of both Cl- and HCO-3 by glandular epithelium. To test this hypothesis, we determined the immunohistochemical distribution of CFTR in pig airways and examined the liquid secretion response of these airways to a series of potential anion-channel blockers. Our results indicate that CFTR is present in pig airway glands and that it supports not only glandular Cl- and liquid secretion but HCO-3 secretion as well.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Airway excision. Young pigs (10-15 kg) were obtained from a local vendor. Animals were sedated with intramuscular injections of ketamine (80 mg) and xylazine (4 mg) and euthanized with an intravenous overdose of pentobarbital sodium. The chest was rapidly opened, and portions of both lungs were quickly excised and placed in Krebs-Ringer bicarbonate (KRB) at room temperature. Distal bronchi (external diameter 2-3 mm, length 25-35 mm) were dissected from the lung lobes. Airway branches were ligated with sutures adjacent to the central airway trunk. The isolated bronchi were then placed in fresh KRB at room temperature and warmed slowly to 37°C (~0.2°C/min).

Measurement of liquid secretion. After reaching physiological temperature, the bronchi were pretreated with the appropriate inhibitor (see below) for 45 min to ensure that complete channel or transporter blockade had occurred. During this pretreatment period, the inhibitors bathed both luminal and adventitial surfaces of the tissues. At the end of this pretreatment period, the airways were removed from the incubation solutions, and the lumens were cleared of all liquid and mucus. The tissues were quickly cannulated with polyethylene tubing and returned to their original incubation solutions containing the inhibitors. ACh (10 µM) was then added to the bath solutions to stimulate liquid secretion. This procedure was followed to ensure that the airway lumen was free of liquid at the start of the ACh exposure. After 2 h of exposure to ACh, the bronchi were removed from their solutions and sectioned lengthwise. Mucus liquid was collected from the lumen and placed in tared polyethylene microcentrifuge tubes (0.5-ml capacity). Two different methods were used to collect mucus liquid. One method utilized forceps and a small glass rod to collect the mucus while residual liquid was collected with a pipette. The other method consisted of aspirating all luminal liquid with polyethylene tubing attached to a 1-ml syringe. A previous study demonstrated that these two techniques resulted in equivalent volumetric recoveries (22). Samples were weighed with a Mettler H20 balance and frozen for later analysis. Secretory rate was calculated from total secretion volume and ACh incubation time and normalized to calculated surface area. Luminal surface area (SA) was calculated from airway length (L) and outer diameter (D) with the use of the following relationship
SA = (0.682)<IT>D</IT>&pgr;<IT>L</IT>
A previous morphometric analysis determined that the cross-sectional inner surface circumference of porcine small bronchi averages 68.2% of the outer surface circumference (Trout, unpublished observations). Liquid volume (in ml) was considered to be equivalent to wet weight (in g).

Inhibitor pretreatments. To demonstrate the role of Cl- channels in ACh-induced airway liquid secretion, the bronchi were pretreated for 45 min with one of several agents known to inhibit Cl--channel activity: 100 µM diphenylamine-2-carboxylate (DPC), 1 mM DIDS, saturated (~350 µM) glibenclamide, and 300 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Glibenclamide and NPPB are thought to exhibit selectivity for the CFTR (12, 20). DPC and DIDS block a wide spectrum of Cl- channels, although DIDS reportedly does not inhibit the CFTR (2, 5, 10). For comparison, some airways were pretreated with both 10 µM bumetanide and 100 µM dimethylamiloride (DMA), a pretreatment combination that has been previously shown to block ~90% of ACh-induced liquid secretion from bronchi by selectively inhibiting Cl- and HCO-3 secretion, respectively (22). Control airways in each group were pretreated with equal volumes of the drug vehicle.

To ensure that the actions of DPC and NPPB were not the result of overt toxicity, one group of airways was pretreated for 2 h 45 min (the total duration of inhibitor exposure in the experiments above) with either DPC, NPPB, or the drug vehicle. Then the airways were placed in drug-free Krebs buffer for 15 min to wash out the inhibitors. The airways were then cleared of luminal liquid, cannulated, and treated with ACh for 2 h. Luminal liquid was collected and weighed as described above.

Epithelium removal. In some bronchi, the surface epithelium was removed to assess the contribution of these cells to the volume of secreted liquid. A wooden ream was inserted into the airway lumen and rotated both clockwise and counterclockwise to strip off the surface epithelium. Microscopic examination confirmed that this method effectively removed the surface epithelium while leaving the smooth muscle and submucosal structures intact. Epithelium removal was performed after dissection of the airways and before warming of the tissues.

Bicarbonate analysis and flux. Frozen samples of airway mucus liquid were thawed and assayed for HCO-3 with an Infinity CO2 kit (Sigma Diagnostics, St. Louis, MO). This assay is based on the enzymatic conversion of HCO-3 to oxaloacetate in the presence of phospho(enol)pyruvate and phospho(enol)pyruvate decarboxylase. Malate dehydrogenase then catalyzes reduction of oxaloacetate to malate and the oxidation of NADH to NAD. The reduction in absorbance at 380 nm is proportional to the amount of HCO-3 in the sample. Absorbance of the reaction product was measured with a Beckman DU65 spectrophotometer. Sample concentrations were determined by comparison to known standards. Because the airway lumen was nominally free of liquid at the start of the 2-h ACh incubation period, determination of the net flux of HCO-3 during this period could be made from the total volume of secreted liquid and the HCO-3 concentration in this volume.

Immunohistochemical detection of CFTR. Lung tissue was frozen in optimum cutting temperature (OCT) embedding medium. Thin (5-10 µm) sections were prepared with a cryostat and fixed at 4°C in acetone for 10 min. Sections were air-dried and stored at -20°C until further use. At the time of the assay, sections were rehydrated in PBS. Endogenous peroxidase activity was blocked with 0.1% (vol/vol) phenylhydrazine-HCl. After saturation of nonspecific protein binding sites with nonimmune goat serum (Vector Laboratories; diluted 1:50 in PBS for 20 min at room temperature), the specimens were incubated at room temperature with purified anti-CFTR (rabbit, polyclonal) first nucleotide-binding domain (NBD1) antibody (16) for 1 h. Preimmune serum at the same concentration was used as a negative control. After a wash with PBS, goat anti-rabbit horseradish peroxidase-labeled antibody was added at 1:200 dilution and incubated for 40 min at room temperature. Specific immune reaction was revealed with the use of 3-amino-9-ethylcarbazole as chromogen and 0.1% H2O2 as substrate, dissolved in 0.1 M sodium acetate buffer, pH 5.2. Mayer's hematoxylin was used as nuclear counterstain.

Statistics. Where paired comparisons are indicated, data were analyzed with dependent t-tests. Unpaired data were compared with independent t-tests. Data are reported as means ± SE, with n referring to the number of airways (each from a different animal) in each group. P < 0.05 was considered significant. Values that fell more than two standard deviations from the mean within each group were considered statistical outliers and were omitted from analysis.

Solution composition and drugs. KRB contained 112 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.6 mM glucose in aqueous solution. The pH of KRB was maintained at 7.4 by continuous gassing of solutions with 5% CO2 in O2. PBS (pH 7.4) contained 8.0 mM Na2HPO4, 2.0 mM KH2PO4, 120 mM NaCl, and 2.7 mM KCl. To make stock solutions, inhibitors were dissolved in DMSO. All drugs and chemicals except for glibenclamide (RBI), DPC (N-phenylanthranilic acid; Fluka Chemika), OCT (Fisher Scientific), and NPPB (Calbiochem) were purchased from Sigma Chemical.


    RESULTS
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INTRODUCTION
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DISCUSSION
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Immunohistochemical distribution of CFTR. Peroxidase staining in the airway sections of pig bronchi shows that the purified anti-CFTR (rabbit, polyclonal) NBD1 antibody localizes to both the surface epithelium and the submucosal glands (Fig. 1A). The most intense labeling appears to be within the ducts of the submucosal glands. Tissue sections incubated with preimmune serum show only nonspecific labeling with minimal signal (Fig. 1B).


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Fig. 1.   Localization of the cystic fibrosis transmembrane conductance regulator protein (CFTR) antibody in porcine bronchi. Airway sections were exposed to rabbit polyclonal antibodies raised against first nucleotide-binding domain (NBD1) of human CFTR. Goat anti-rabbit antibody labeled with horseradish peroxidase localized anti-CFTR antibodies to both submucosal gland and surface epithelial cells (A; arrows). Tissue sections treated with preimmune serum show only nonspecific antibody binding (B).

Effect of epithelium removal. In the presence of ACh, the bronchi secreted 15.6 ± 0.6 µl · cm-2 · h-1 (n = 67) (Fig. 2). The secretion response to ACh in denuded airways (13.4 ± 1.7 µl · cm-2 · h-1; n = 17) was not significantly different from that in epithelium-intact airways. The rate of liquid flux (JV) was significantly inhibited by pretreatment with 10 µM bumetanide and 100 µM DMA [a drug combination previously shown to inhibit Cl-, HCO-3, and liquid secretion across porcine bronchi (22)] in both epithelium-intact (2.1 ± 0.3 µl · cm-2 · h-1; n = 12) and epithelium-denuded airways (3.2 ± 0.6 µl · cm-2 · h-1; n = 8) (Fig. 2). The effect of epithelium removal was also insignificant in inhibitor-treated tissues. These results indicate that most of the liquid secreted by bronchi in response to ACh arises from submucosal glands.


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Fig. 2.   Effect of surface epithelium removal on ACh-induced liquid secretion by porcine bronchi. Epithelium-intact or epithelium-denuded airways were exposed to 10 µM ACh after pretreatment with either 10 µM bumetanide + 100 µM dimethylamiloride (DMA) or drug vehicle. JV, rate of liquid volume secretion. +, Presence; -, absence. * Significant difference from vehicle pretreatment (independent t-tests). Epithelium removal did not significantly affect response to either ACh or inhibitor pretreatment (independent t-tests).

Effect of channel blockers. Similar to previous studies (22), pretreatment with the combination of 10 µM bumetanide and 100 µM DMA, which selectively target the Na+-K+-2Cl- cotransporter and the Na+/H+ exchanger, respectively, inhibited 86.5% of the ACh-induced liquid secretion from porcine bronchi (Table 1). This action has been attributed to respective inhibition of transepithelial Cl- and HCO-3 secretion by these agents (22). Comparable inhibition of ACh-induced liquid secretion was observed when airways were pretreated with 100 µM DPC or 300 µM NPPB. Glibenclamide (~350 µM) pretreatment significantly blocked 38.5% of the secretion response, whereas 1 mM DIDS pretreatment had no effect.

                              
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Table 1.   Effect of transport inhibitors on ACh-induced liquid volume secretion

Inhibitors that reduced liquid secretion also reduced the net secretory flux of HCO-3 and the HCO-3 concentration in the luminal liquid (Table 2). Compared with the subset of airways for which HCO-3 analysis was performed, the residual HCO-3 flux after pretreatment with DPC, NPPB, and bumetanide+DMA was approximately one-half of the JV when expressed as a percentage of the control response (Table 2). Even in glibenclamide-pretreated tissues, the relative inhibition of HCO-3 flux exceeded that of the JV. The inhibitory effect on HCO-3 flux was significantly greater than JV inhibition for NPPB, DPC, and glibenclamide pretreatments but not for bumetanide+DMA pretreatments. Even in airways denuded of their surface epithelium, the residual HCO-3 flux after bumetanide and DMA pretreatment (12.4 ± 5.8%; n = 5) was approximately one-half of the residual JV (28.4 ± 7.6%; n = 5), although this difference was not significant. Inhibitor-induced reductions in HCO-3 secretion are reflected in the significantly lower concentrations of HCO-3 in the secreted liquid, which range from 8.1 ± 3.2 meq/l (NPPB pretreatment) to 13.4 ± 2.0 meq/l (glibenclamide pretreatment) (Table 2). Although glibenclamide pretreatment had less effect on the JV than DPC, NPPB, and bumetanide+DMA, it produced comparable reductions in the HCO-3 concentration in secreted liquid (Table 2). DIDS pretreatment had no significant effect on HCO-3 flux or concentration.

                              
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Table 2.   Effect of transport inhibitors on ACh-induced bicarbonate and liquid secretion

Possible toxicity of DPC and NPPB was evaluated by exposure of airways to these inhibitors for a period equivalent to that described in METHODS (2 h 45 min). The airways were then washed with drug-free Krebs, cannulated, and treated with ACh to induce secretion. Under these conditions, similar rates of liquid secretion were observed in control (14.0 ± 2.0 µl · cm-2 · h-1; n = 4), DPC-treated (13.1 ± 1.6 µl · cm-2 · h-1; n = 4), and NPPB-treated airways (11.6 ± 5.2 µl · cm-2 · h-1; n = 4). These data indicate that DPC and NPPB exposure do not significantly affect tissue viability.


    DISCUSSION
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INTRODUCTION
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We made the following observations during this study. First, antibodies to the CFTR densely stained the epithelial cells of the airway submucosal glands of porcine bronchi. Second, porcine bronchi when treated with ACh secreted liquid at similar rates in both the presence and absence of the surface epithelium. Third, Cl--channel blockers that are known to inhibit the CFTR were effective blockers of ACh-induced liquid secretion and disproportionately reduced HCO-3 secretion. These findings reasonably support the hypothesis that CFTR is present in submucosal glands of pig airways and participates in the secretion of Cl-, HCO-3, and liquid from these structures.

Earlier studies by Inglis et al. (9) of the bioelectric properties of porcine bronchi provided evidence that ACh stimulates both Cl- and HCO-3 secretion from these tissues. Subsequent studies demonstrated that inhibitors of Cl- and HCO-3 secretion not only blocked the liquid secretion response to ACh (22, 23) but caused mucins to accumulate within the ducts of the submucosal glands (9, 16). The authors of these studies reasoned that this secreted liquid likely originated from submucosal glands, although they could not rule out significant contributions from nonglandular airway regions. The results of the present study confirm these earlier notions by showing that similar quantities of liquid are secreted into the lumen of airways in the presence or absence of the surface epithelium. Inhibitors of Cl- and HCO-3 secretion substantially reduce this secretion response even when the surface epithelium is removed. Consequently, we conclude that the secreted liquid emanates largely from submucosal epithelium (i.e., glands) and is driven by active anion secretion.

Unfortunately, potent, highly selective inhibitors of the CFTR are presently unavailable. Therefore, demonstration of CFTR involvement in complex tissues must be inferred from the pattern of responses to several semiselective Cl--channel inhibitors. DPC blocks the CFTR, Ca2+-activated Cl- channels, outwardly rectifying Cl- channels, and a variety of other Cl- channels (2, 10). In the present study, DPC was an effective inhibitor of the ACh-induced liquid secretion response, consistent with Cl--channel involvement. DIDS inhibits Ca2+-activated Cl- channels, outwardly rectifying Cl- channels, and many of the other DPC-sensitive Cl- channels but not the CFTR (2, 5, 10, 24). Consequently, blockade of ACh-induced secretion by DPC but not by DIDS implies a functional role for the CFTR. Both NPPB and glibenclamide are also known to inhibit the CFTR (12, 20). In the present study, NPPB was as effective as DPC in blocking liquid secretion, but glibenclamide inhibited only ~40% of the response. The reason why incomplete inhibition of the JV occurred with glibenclamide is unclear. This was probably not a dose effect, since maximum (i.e., saturating) concentrations of glibenclamide were used. It is more likely that this effect reflects the lower intrinsic efficacy of glibenclamide compared with the other CFTR blockers. Vandorpe et al. (24) showed in inner medullary collecting duct cells that the order of effectiveness for absolute reductions in the product of CFTR-channel number and open probability was NPPB > DPC > glibenclamide, with DIDS having no effect. This same order of inhibitor effectiveness is seen in the present study for reductions in both liquid volume and HCO-3 secretion. Blockade of liquid secretion with DPC and NPPB is unlikely to be the result of overt toxicity because we observed that the ACh secretion response was fully restored after washout of these inhibitors. We conclude that the responses to this group of inhibitors are most consistent with participation of the CFTR in the liquid secretion response to ACh.

Cl--channel inhibitors that would be expected to block the CFTR also appear to inhibit HCO-3 secretion. DPC and NPPB reduced HCO-3 secretion more than predicted based on reductions in liquid volume secretion, whereas DIDS had no effect. Even glibenclamide, which reduced liquid secretion by only 40%, caused a fractionally greater and significant inhibition of HCO-3 flux. This inhibitory action of these agents on HCO-3 secretion clearly targets glandular epithelium and not surface epithelium, since reductions in HCO-3 flux were also observed in bumetanide- and DMA-pretreated airways with the epithelium removed. These data suggest that HCO-3 secretion from glands is mediated through the CFTR. Involvement of the CFTR in the secretion of HCO-3 in airways is supported by reports that the CFTR is permeable to this anion (5, 17) and that it mediates HCO-3 secretion by duodenal epithelia (3, 6).

In addition to providing functional evidence for CFTR involvement in gland secretion, we showed by immunohistochemistry that CFTR is present in gland epithelium of pigs. Although the distribution is shown for polyclonal antibodies to CFTR, we saw a similar expression pattern for monoclonal antibodies as well (data not shown). These findings support those of others reporting high levels of CFTR expression in airway glands of humans, cows, and ferrets (4, 11, 19) and imply a functional role for CFTR in anion transport by these structures.

Cl- concentrations and fluxes were not reported in the present study because the quantity of Cl- present in the smallest (<5 µl) sample volumes was too low for accurate determination with conventional potentiometric Cl- titration. Trout et al. (22) showed in previous studies that ~70% of ACh-induced liquid secretion from pig bronchi is driven by bumetanide-sensitive Cl- secretion, whereas HCO-3 secretion accounts for only ~10-20%. Because DPC and NPPB block ~85-90% of the liquid secretion response, Cl- secretion must have been nearly abolished by these agents. Partial inhibition of Cl- secretion must have occurred even with glibenclamide because the observed 40% inhibition of the JV exceeds the estimated maximum contribution of HCO-3 secretion by at least twofold. Therefore, we conclude that these agents exert a large fraction of their inhibitory effects on liquid volume secretion through inhibition of active transepithelial Cl- secretion, which accounts for most of the active anion secretion response to ACh by this tissue.

In previous studies, we observed that bumetanide and DMA, inhibitors of epithelial Cl- and HCO-3 secretion, respectively, induced changes in porcine bronchi that resembled those expected to occur in CF disease. First, we saw abolishment of ACh-induced electrogenic anion secretion (9), an expected consequence of defective CFTR expression. Second, we found that these inhibitors greatly reduced ACh-induced airway liquid secretion (22), confirming that this process is dependent on active anion secretion. Again, this would be the anticipated result of defective CFTR expression as occurs in CF. Third, we observed that the bronchial mucus that is secreted after the Cl- and HCO-3 secretion inhibitors is less hydrated and more rigid and exhibits less recoil than control mucus (23). This finding supports the commonly held yet controversial notion that the CF condition leads to secretion of relatively dehydrated mucus with altered rheological properties. Fourth, we found that bumetanide and DMA together cause mucin accumulation and an obstruction of submucosal gland ducts of airways (7, 8) that closely resembles the gland duct obstruction that occurs very early in CF disease (15). Bumetanide and DMA produce their inhibitory effects on Cl- and HCO-3 secretion by selectively targeting Na+-K+-2Cl- cotransport and Na+/H+ exchange (22). If Cl- and HCO-3 both flow across the apical membrane of glandular epithelial cells through CFTR, we expect that equivalent inhibition of liquid secretion would occur with blockers of this channel. The results of the present paper confirm this hypothesis, with DPC and NPPB producing equivalent if not greater inhibition of liquid secretion than bumetanide+DMA. We expect that these same Cl--channel blockers would be useful for modeling the acute pathophysiology of CF disease in surrogate species such as the pig, in which airway gland expression resembles that of humans.

If blockade of the CFTR by DPC or NPPB acutely mimics the CF condition, the pH of airway surface liquid in CF patients might be relatively more acidic than normal. In the present study, control airway surface liquid contains 20-25 mM HCO-3, whereas liquid from NPPB-treated airways contains only 8.1 ± 3.2 mM HCO-3. With the assumption that all solutions were equilibrated with 5% CO2, the Henderson-Hasselback relationship predicts that the pH of airway surface liquid would be ~7.33-7.45 in control airways and as low as 6.95 in inhibitor-treated airways. It is possible that this approximately threefold higher H+ concentration is relevant to the development of CF lung pathology. However, findings that the pH of cholinergically induced airway surface liquid is normally ~7.0 in ferrets (18), a species that exhibits no unusual propensity toward airway disease, argues against this notion.

Because we expected the surface epithelium to provide a significant barrier to passive liquid movement, we were surprised to observe that the rate of liquid flux into the lumen of epithelium-denuded and epithelium-intact airways was very similar. Cannulated airways were submerged vertically in Krebs buffer; therefore, interstitial fluid pressure ranged from 0 cmH2O to as much as +5 cmH2O relative to the air-filled lumen in some tissues. This hydrostatic pressure gradient should have favored liquid flow into the airway lumen. From our data, it is clear that little passive liquid flow into the lumen occurred when the epithelium was removed. We speculate that passive liquid movement through the interstitial space was restricted by the gellike properties of the interstitial matrix and perhaps by the uniform layer of smooth muscle cells that lies within the airway wall.

In summary, we show that airway submucosal glands are the principal site of ACh-induced liquid secretion from porcine airways. We also provide evidence that CFTR is present in submucosal glands and supports not only Cl- and liquid secretion but HCO-3 secretion as well. By demonstrating a physiological function for the CFTR in glandular transepithelial anion and liquid secretion, we are hopeful that these findings provide a crucial link toward our understanding of the pulmonary pathogenesis of CF.


    ACKNOWLEDGEMENTS

This work was funded by the National Institutes of Health Grants HL-48622 and SCOR DK-53090 and Cystic Fibrosis Foundation RDP Grant R464.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. T. Ballard, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688 (E-mail: sballard{at}usamail.usouthal.edu).

Received 2 April 1999; accepted in final form 2 June 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, M. P., R. J. Gregory, S. Thompson, D. W. Sousa, S. Paul, R. C. Mulligan, A. E. Smith, and M. J. Welsh. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253: 202-205, 1991[Medline].

2.   Anderson, M. P., D. N. Sheppard, H. A. Berger, and M. J. Welsh. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L1-L14, 1992[Abstract/Free Full Text].

3.   Clarke, L. L., and M. C. Harline. Dual role of CFTR in cAMP-stimulated HCO-3 secretion across murine duodenum. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G718-G726, 1998[Abstract/Free Full Text].

4.   Engelhardt, J. F., J. R. Yankaskas, S. T. Ernst, Y. Yang, C. R. Marino, R. C. Boucher, J. A. Cohn, and J. M. Wilson. Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat. Genet. 2: 240-248, 1992[Medline].

5.   Gray, M. A., S. Plant, and B. E. Argent. cAMP-regulated whole cell chloride currents in pancreatic duct cells. Am. J. Physiol. 264 (Cell Physiol. 33): C591-C602, 1993[Abstract/Free Full Text].

6.   Hogan, D. L., D. L. Crombie, J. I. Isenberg, P. Svendsen, O. B. Schaffalitsky de Muckadell, and M. A. Ainsworth. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO-3 secretion. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G872-G878, 1997[Abstract/Free Full Text].

7.   Inglis, S. K., M. R. Corboz, and S. T. Ballard. Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L762-L766, 1998[Abstract/Free Full Text].

8.   Inglis, S. K., M. R. Corboz, A. E. Taylor, and S. T. Ballard. Effect of anion transport inhibition on mucus secretion by airway submucosal glands. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997[Abstract/Free Full Text].

9.   Inglis, S. K., M. R. Corboz, A. E. Taylor, and S. T. Ballard. Regulation of ion transport across porcine distal bronchi. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L289-L297, 1996[Abstract/Free Full Text].

10.   Jacob, T. J. C., and M. M. Civan. Role of ion channels in aqueous humor formation. Am. J. Physiol. 271 (Cell Physiol. 40): C703-C720, 1996[Abstract/Free Full Text].

11.   Jacquot, J. E., E. Puchelle, J. Hinnrasky, C. Fuchey, C. Bettinger, C. Spilmont, N. Bonnet, A. Dieterle, D. Dreyer, A. Pavirani, and W. Dalemans. Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands. Eur. Respir. J. 6: 169-176, 1993[Abstract].

12.   Jiang, C., W. E. Finkbeiner, J. H. Widdicombe, and S. S. Miller. Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis. J. Physiol. (Lond.) 501: 637-647, 1997[Abstract].

13.   Lee, M. C., C. M. Penland, J. H. Widdicombe, and J. J. Wine. Evidence that Calu-3 human airway cells secrete bicarbonate. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L450-L453, 1998[Abstract/Free Full Text].

14.   Moon, S., M. Singh, M. Krouse, and J. J. Wine. Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L1208-L1219, 1997[Abstract/Free Full Text].

15.   Oppenheimer, E. H., and J. R. Esterly. Pathology of cystic fibrosis: review of the literature and comparison with 146 autopsied cases. In: Perspectives in Pediatric Pathology, edited by H. S. Dosenberg, and R. P. Bolarde. Chicago: Yearbook Medical Publications, 1975, vol. 2, p. 241-278.

16.   Peng, S., M. Sommerfelt, J. Logan, Z. Huang, T. Jilling, K. Kirk, E. Hunter, and E. J. Sorscher. One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system. Protein Expr. Purif. 4: 95-100, 1993[Medline].

17.   Poulsen, J. H., H. Fischer, B. Illek, and T. E. Machen. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. USA 91: 5340-5344, 1994[Abstract].

18.   Robinson, N. P., H. Kyle, S. E. Webber, and J. G. Widdicombe. Electrolyte and other chemical concentrations in tracheal airway surface liquid and mucus. J. Appl. Physiol. 66: 2129-2135, 1989[Abstract/Free Full Text].

19.   Sehgal, A., A. Presente, and J. F. Engelhardt. Developmental expression patterns of CFTR in ferret tracheal surface airway and submucosal gland epithelia. Am. J. Respir. Cell Mol. Biol. 15: 122-131, 1996[Abstract].

20.   Sheppard, D. N., and M. J. Welsh. Effect of ATP-sensitive K+-channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents. J. Gen. Physiol. 100: 573-591, 1992[Abstract].

21.   Smith, J. J., and M. J. Welsh. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J. Clin. Invest. 89: 1148-1153, 1992[Medline].

22.   Trout, L., J. T. Gatzy, and S. T. Ballard. Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl- and HCO-3 transport. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1095-L1099, 1998[Abstract/Free Full Text].

23.   Trout, L., M. King, W. Feng, S. K. Inglis, and S. T. Ballard. Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L258-L263, 1998[Abstract/Free Full Text].

24.   Vandorpe, D., N. Kizer, F. Ciampollilo, B. Moyer, K. Karlson, W. B. Guggino, and B. A. Stanton. CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells. Am. J. Physiol. 269 (Cell Physiol. 38): C683-C689, 1995[Abstract].


Am J Physiol Lung Cell Mol Physiol 277(4):L694-L699
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