Acetylcholine-induced liquid secretion by bronchial epithelium: role of Clminus and HCOminus 3 transport

Laura Trout1, John T. Gatzy2, and Stephen T. Ballard1

1 Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; and 2 Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Inhibitors of Cl- and HCO-3 secretion reduce acetylcholine-induced liquid, but not mucin, secretion by bronchial submucosal glands [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L372-L377, 1997]. The present study quantified contributions of Cl- and HCO-3 transport to volume and composition of acetylcholine-induced liquid secretion by airway epithelium. When distal bronchi were excised from 33 pigs and treated with 10 µM acetylcholine, the airways secreted 13.4 ± 0.7 µl · cm-2 · h-1. Bumetanide (10 µM) pretreatment reduced acetylcholine-induced liquid and Cl- secretion rates by ~70%, but HCO-3 secretion fell by only 40%. Dimethylamiloride (DMA; 100 µM) pretreatment reduced Cl- secretion rates by ~15%, but HCO-3 secretion fell 47%. DMA alone had little effect on liquid secretion. When airways were pretreated with both bumetanide and DMA, acetylcholine-induced liquid secretion was nearly abolished. We conclude that about three-fourths of acetylcholine-induced liquid secretion in distal bronchi is dependent on Cl- secretion. Most of the remaining response is driven by HCO-3 secretion. We speculate that the principal source of this liquid is submucosal glands. Crossover inhibition of bumetanide on HCO-3 secretion and DMA on Cl- secretion implies modulation of anion secretion secondary to changes in cell electrolyte composition.

cystic fibrosis; dimethylamiloride; bumetanide; bronchi; submucosal glands; bicarbonate; chloride

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE LUMINAL SURFACE of the pulmonary airways is lined with a thin layer of liquid that normally approximates the depth of the cilia of airway surface epithelial cells. Islands of mucus rest atop this layer of liquid and are propelled by the cilia from distal airways toward the pharynx. It has been assumed that regulation of the depth of airway surface liquid (ASL) is critical to normal ciliary clearance functions. That is, if the depth of the ASL is too great, the tips of the cilia cannot contact the mucus and mucociliary transport is impeded. On the other hand, if the ASL depth is too shallow, the cilia become entangled in the mucus and transport efficiency is reduced. Therefore, adequate removal of inhaled and endogenous debris depends on regulation of ASL depth.

The dynamics of ASL are poorly understood. Whereas basal liquid absorption by airway surface epithelia is driven by active Na+ transport (4), the mechanism and site of liquid secretion in the airways remain controversial. Specifically, surface epithelial cells have the capacity to secrete Cl- and liquid, but in most species, this process is best demonstrated after inhibition of active Na+ absorption (5). In contrast, submucosal glands in nasal, tracheal, and bronchial airways of higher mammals secrete copious quantities of mucus and liquid in response to cholinergic or adrenergic stimulation (16, 22). Consequently, submucosal glands could serve as a major source of ASL.

In cystic fibrosis (CF), mutations in the CF transmembrane conductance regulator protein (CFTR), a cAMP-regulated Cl- channel (1), disable Cl- secretion by airway epithelia (17). Airway obstruction and increased susceptibility to bacterial pulmonary infections that typify this disease have long been thought, albeit without definitive evidence, to result from impaired Cl--dependent secretion of ASL. CFTR is normally localized at high density to the serous cells of the submucosal glands (8), suggesting that physiologically important secretion of Cl- occurs at this site. A previous study (11) showed that mucins accumulate in the gland ducts of acetylcholine-treated porcine airways in the presence, but not in the absence, of a combination of Cl- and HCO-3 secretion inhibitors. Inhibitor pretreatment also substantially reduces the volume of mucus liquid produced in response to acetylcholine and increases the solids content of mucus threefold (21). These findings support the notion that acetylcholine-induced glandular liquid secretion is driven by both Cl- and HCO-3 secretion and that inhibition of this process uncouples liquid from mucin secretion by glands. Interpretation of these studies depended on the putative selectivity of the inhibitors because the ion composition of the secreted liquid was not measured.

The present study was designed to 1) measure the secretion rate and composition of liquid that accumulated in lumens of excised distal airways pretreated with acetylcholine and 2) evaluate the effects of inhibitors of active Cl- and HCO-3 secretion on these properties.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Airway excision. Young pigs (10-15 kg) were obtained from a local vendor. Animals were sedated with an intramuscular injection of xylazine (80 mg) and ketamine (4 mg) and killed with an intravenous overdose of pentobarbital sodium. The chest was rapidly opened, and portions of both lungs were excised and placed in Krebs-Ringer bicarbonate (KRB) solution at room temperature. Distal bronchi (external diameter 2-3 mm, length 25-35 mm) were immediately dissected free from the surrounding tissue. Airway branches were tightly ligated with sutures close to the airway trunk to prevent leakage of liquid into the luminal space. The isolated bronchi were then warmed slowly (~0.1°C/min) from room temperature to 37°C.

Treatment with inhibitors of ion secretion. The role of Cl- and HCO-3 transport in acetylcholine-induced liquid secretion was evaluated by exposing both the luminal and submucosal surfaces of the bronchi to either 10 µM bumetanide, an Na+-K+-2Cl- cotransport inhibitor, or Cl--free KRB to block Cl- secretion; or dimethylamiloride (DMA; 100 µM), an Na+/H+ exchange inhibitor, to block HCO-3 secretion. The action of DMA is thought to result from inhibition of proton extrusion across the basolateral membrane, which reduces the capacity of the epithelial cells to generate cytoplasmic HCO-3 (19). We also exposed bronchi to bilateral Na+-free KRB to inhibit both Na+-K+-2Cl- cotransport and Na+/H+ exchange. Bilateral ouabain (1 mM), an Na+-K+-ATPase inhibitor, was used to inhibit primary Na+ active transport and any secondary transport that depends on Na+ and K+ gradients across the cell membrane. In each experiment, treated tissues were paired with control (vehicle-treated) bronchi from the same animal.

Collection of airway liquid. Bronchi were pretreated with inhibitors of ion secretion for 45 min. Paired control airways were pretreated with an equal volume of the vehicle dimethyl sulfoxide (DMSO) for the same period of time. Tissues exposed to Cl-- or Na+-free KRB solution were incubated in these solutions before being warmed and were not pretreated for an additional 45 min. During the pretreatment period, the solutions bathed both the luminal and adventitial surfaces of each bronchus. At the end of pretreatment, the airway was removed from the bath and the lumen was cleared of all liquid and mucus. Both ends of the airway were cannulated with polyethylene tubing, and the preparation was returned to the pretreatment bath. Then, acetylcholine (10 µM) was added to the bath solution to stimulate liquid secretion. After 2 h, the bronchi were removed from the bath and cut lengthwise. Mucus liquid in the airway lumens was collected with a 2-mm-diameter glass rod and forceps. The non-Newtonian properties of mucus allow secretions to be picked up and manipulated as a semisolid. Residual liquid, if present in the lumens, was collected with a pipette. When liquid was present in the cannulas, the liquid was gently cleared from the tubes with forced air. Collected liquid was transferred to tared microcentrifuge tubes. The sealed specimens were weighed (Mettler H20 balance) and frozen for analysis. Liquid secretion rate (JV) was calculated from total secretion volume and normalized to the estimated surface area. Luminal surface area (SA) was calculated from airway length (L) and outer diameter (D) by the following relationship: SA = 0.682Dpi L. Histological sections of six different distal bronchi showed that the cross-sectional inner-surface circumference was 68.2 ± 2.6% of the outer-surface circumference (Trout, unpublished observations). This relationship between inner and outer circumference is unaffected by constriction of airway smooth muscle due to folding of the airway mucosa (Trout, unpublished observations). Collected liquid volume (in ml) was considered to be equivalent to wet weight (in g).

Ion analysis. Frozen specimens were thawed and diluted with 1 ml of 0.2 N HNO3 and 1 ml of distilled, deionized water. Standards and blanks were similarly prepared. The diluted, acidified specimens were divided into two 1-ml samples for flame photometric analysis and amperometric Cl- titration. Specimens, standards, and blanks were directly assayed for Na+ and K+ with a Coleman model 51 flame photometer. To measure Cl-, 1-ml samples were combined with 1 ml of 0.1 N HNO3, 2 ml of acid reagent, 0.2 ml of glacial acetic acid, and 2 drops of indicator gelatin solution. Then, the samples were amperometrically titrated with a Labconco digital chloridimeter.

HCO-3 in another set of frozen specimens was measured by the method of Van Slyke as modified by Bittner and Hall (2). Each specimen was thawed and diluted to 100 µl with distilled, deionized water. Standards and blanks were prepared similarly. Each was combined with 900 µl of a bromcresol green indicator solution and titrated to the indicator end point with 0.005 N NaOH dispensed by an Oxford titrator (model 301).

Solutions and drugs. KRB solution was composed of (in mM) 112 NaCl, 4.7 KCl, 2.5 CaCl2, 2.4 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.6 glucose. Cl- was replaced by equimolar gluconate in Cl--free KRB. Na+ was replaced by equimolar choline in Na+-free KRB solution. Solution pH was maintained in solutions by constant gassing with 95% O2-5% CO2. All drugs were purchased from Sigma. Stock solutions of bumetanide, DMA, and ouabain were prepared with DMSO. Stock solutions of acetylcholine were prepared with deionized water.

Statistics. Data are expressed as means ± SE. Paired comparisons were made with either dependent or independent t-tests. Bonferroni corrections were made for multiple comparisons. Differences were considered to be significant when P < 0.05. The number of observations (animals) in each group is given by n.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Liquid volume secretion. In the absence of stimulation, bronchi secreted very little liquid (0.4 ± 0.2 µl · cm-2 · h-1; n = 6). When treated with 10 µM acetylcholine, however, bronchi secreted 13.4 ± 0.7 µl · cm-2 · h-1 (n = 33). Liquid secretion was not transient but continuous, as indicated by the relatively constant rate of liquid accumulation in the cannulas during the 2-h incubation. The acetylcholine-induced JV was significantly reduced by 70% when airways were exposed to 10 µM bumetanide (Table 1). Pretreatment with 100 µM DMA alone did not alter JV (Table 1). The JV of tissues pretreated with both bumetanide and DMA decreased by 89% compared with control bronchi, an effect that was significantly greater than bumetanide pretreatment alone (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of anion secretion inhibitors on acetylcholine-induced liquid secretion

When bronchi were bathed in Na+-free KRB before acetylcholine, JV was virtually abolished compared with airways bathed in Na+-replete KRB (Table 1). A similar change from the acetylcholine-stimulated control bronchi was induced by exposing airways to 100 µM ouabain (Table 1). Compared with airways bathed in KRB, Cl--free KRB inhibited the acetylcholine-induced JV by 78% (Table 1), similar to the magnitude of inhibition induced by bumetanide pretreatment.

Ion analysis. Table 2 shows the ion composition of the luminal liquid that accumulated in response to acetylcholine. The concentrations of ions in luminal liquid from control airways resemble those of plasma except that the K+ concentration is about threefold higher. When airways were pretreated with DMA, the HCO-3 concentration in the liquid was significantly reduced. Reduction in the HCO-3 concentration tended to be compensated for by an increase in the Cl- concentration, but the rise was not significant. Pretreatment of airways with bumetanide to block Cl- secretion resulted in a significant 229% increase in HCO-3 concentration. The Cl- concentration tended to fall during exposure to bumetanide, but this difference was not significant. We noted no appreciable change in Na+ or K+ concentration with either pretreatment. Although osmolality was not measured in the present study, summation of solution ions in Table 2 suggests that the acetylcholine-induced secretion product is nearly isosmotic to normal plasma (vehicle pretreatment = 303 µeq/l; DMA pretreatment = 310.1 µeq/l; and bumetanide pretreatment = 336.6 µeq/l).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Ion composition of acetylcholine-induced airway liquid

Net secretory flows of ions were calculated from the ion concentrations in Table 2 and the JV for the same tissues. In this subset of tissues, DMA pretreatment decreased JV to 91% of control levels, whereas bumetanide pretreatment reduced JV to 30% of control levels (Table 3). Reductions in net flux of Na+, K+, and Cl- with DMA or bumetanide pretreatment parallel fractional reductions in liquid volume secretion. However, DMA pretreatment caused a fractionally smaller HCO-3 secretion than that predicted (about twice the decrease) from the change in JV, whereas bumetanide pretreatment resulted in a greater HCO-3 flux (about twice) than that predicted by JV.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Acetylcholine-induced ion and liquid flux

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study, either bumetanide pretreatment or Cl--free buffer blocked ~70% of the liquid secretion response to acetylcholine. These results suggest that Cl- transport is responsible for most of the liquid that is secreted in response to acetylcholine and is consistent with the well-established model for Cl- secretion first described by Silva et al. (18). According to this model, Cl- enters across the basolateral membrane of epithelial cells by Na+-K+-2Cl- cotransport, which uses the electrochemical gradient for Na+ to drive cell Cl- above its equilibrium. Given a sufficient electrochemical driving force, Cl- exits the cells through apical membrane Cl- channels. A lumen negative transepithelial potential difference (PD) draws cations, predominantly Na+, through the paracellular path. This model has been widely accepted for airway epithelia (4, 17). Secretion of Cl- can be raised either by opening apical membrane Cl- channels or by opening basolateral K+ channels, which increases the electrical force that drives Cl- efflux. Recent studies (13, 14) with airway gland cells provide evidence that cholinergic agonists open basolateral Ca2+-activated K+ channels and induce secretion through the CFTR in the luminal membrane.

Most of the acetylcholine-induced liquid secretion that is bumetanide insensitive is blocked by pretreatment with DMA. Because DMA inhibits HCO-3 secretion by this preparation, we think HCO-3 secretion drives the bumetanide-insensitive liquid secretion. These data are consistent with the model for HCO-3 secretion proposed by Smith and Welsh (19). According to this model, carbonic anhydrase catalyzes the intracellular formation of HCO-3 and H+ from CO2 and H2O. Subsequent removal of H+ from the intracellular space by basolateral Na+/H+ exchange leaves HCO-3 to exit in exchange for another anion or coupled to the flow of a cation in the same direction. Further validation of this model is drawn from our observations that Na+-free solution (which removes substrate for both Na+-K+-2Cl- and Na+/H+) or ouabain (which disrupts transmembrane Na+ and K+ gradients) abolishes acetylcholine-induced liquid secretion. The path(s) by which HCO-3 exits bronchial epithelial cells in our preparation cannot be determined from our data, but it has been suggested that HCO-3 passes through the CFTR in the apical membrane of respiratory epithelia (19). However, the permeability of the CFTR to HCO-3 is only about one-fourth that of Cl- (15). The outwardly rectifying Cl- channel is less selective than the CFTR (20) but may not be capable of aggregate open times that would support an HCO-3 flux equal to roughly one-third of the Cl- flow through all channels.

It is not possible to conclude from these data the contribution of HCO-3 secretion to acetylcholine-induced liquid secretion when Cl- secretion is intact. In Table 3, DMA inhibits ~9% of the liquid secretion rate compared with control bronchi, but this difference is not significant. It is possible that DMA alone inhibits a fraction of liquid secretion, but the response could be masked by the variance between experimental and control responses. On the other hand, HCO-3 secretion could be induced under conditions where Cl- secretion is inhibited. Evidence for this notion arises from a study of salivary glands (7) where furosemide decreases liquid secretion by only 60% but increases HCO-3 concentration in the secreted liquid approximately fivefold. In the present study with porcine bronchi, however, inhibition of Cl- secretion with bumetanide is associated with a decrease rather than an increase in HCO-3 net flow (Table 3).

If HCO-3 and Cl- flow through the same channel(s), selective inhibition of a process that ordinarily maintains cellular chemical activity of one species would be expected not to change flow of the unaffected species through the channel. Alternatively, flow of the unaffected species could increase if the anions "compete" for channel entry. Independent paths for HCO-3 and Cl- flow across the luminal membrane also project no change in unaffected anion flow. None of the inhibitors we tested increased or preserved secretion of an anion species, so our findings suggest a more complex process(es). Bumetanide inhibited the acetylcholine-induced secretion of Na+, K+, and Cl- by 68-74% but reduced HCO-3 secretion by only 40% (Table 3). Consequently, the fall in Cl- secretion was partially compensated for by the persistence of HCO-3 secretion. DMA reduced HCO-3 secretion by 47% and Na+ and K+ secretion by 20-26% but Cl- secretion by only 16%. Even though the changes were smaller with DMA, there is a tendency for Cl- secretion to compensate for the loss of HCO-3 secretion. However, each inhibitor reduced secretion of all ion species. This could result from secondary effects of an inhibitor on channel activity in the same cell (e.g., change in intracellular pH, Na+, or K+). Consequently, our results are compatible with HCO-3 and Cl- secretion through the same cell but cannot distinguish HCO-3 flow through Cl- channels from flow through an independent path. Observations by Inglis et al. (11) and Trout et al. (21) that liquid secretion can be uncoupled from mucus secretion imply that secretion by the serous cell was the principal target of the inhibitors we tested. Modification of secreted solution by another acinar cell type or a complex series of processes in different gland regions (e.g., secretion by the acinus and selective absorption/secretion by ducts) seems, with our current limited knowledge, to be unnecessarily complicated. There are, however, difficulties with the same-cell hypothesis. For example, inhibition of basolateral Na+ entry through the cotransporter by bumetanide would be expected to decrease the intracellular Na+ concentration, promote Na+/H+ exchange, raise the cell HCO-3 concentration, and increase rather than decrease HCO-3 secretion. Effects of injection of ion channel inhibitors into duct and acinar lumens would help resolve modes of gland secretion, but this approach has not been developed.

Surface epithelial cells could also affect the composition and volume of liquid secreted in response to acetylcholine. We cannot dismiss this possibility, but several lines of evidence suggest that this liquid arises largely, if not entirely, from the submucosal glands. First, cholinergic agonists have been shown to be efficacious stimulants of gland secretion in intact airways (3, 16, 22). Second, pretreatment of bronchi with inhibitors of Cl- and HCO-3 secretion causes mucin to accumulate in gland ducts after application of acetylcholine (9, 11). These results suggested indirectly that inhibitors of Cl- and HCO-3 secretion blocked glandular liquid secretion and thereby uncoupled liquid and mucin secretion in glands. Third, cultured airway gland cells have been shown to secrete liquid in response to cholinergic stimulation (13). Fourth, acetylcholine stimulates secretory fluxes of Na+ and Cl- across excised canine airways (6), which contain glands, but exerts no effect on fluxes of these ions across excised rabbit trachea (12), which is aglandular.

Because inhibitors of Cl- and HCO-3 secretion also inhibit acetylcholine-induced isotonic liquid secretion, we conclude that both anions are capable of driving liquid and net counterion flows. In the absence of inhibitors, we estimate that distal bronchi secrete ~13.4 µl · cm-2 · h-1 in response to acetylcholine. A previous study (10) showed that acetylcholine causes a 15 µA/cm2 increase in equivalent short-circuit current (Isc) across distal bronchi. Assuming that the increase in Isc represents a sustained active anion secretion that drives secretion of isosmotic liquid, electrogenic transport accounts for only ~3.9 µl · cm-2 · h-1 or ~29% of the measured liquid secretion. Because these tissues were not clamped to zero PD, JV projected from Isc would be expected to be reduced by ~20% under open-circuit conditions (change in PD with acetylcholine ~5 mV). These data indicate that a substantial fraction of the liquid secretion response to acetylcholine is electrically silent. Boucher and Gatzy (6) also reported that acetylcholine-induced increases in Na+ and Cl- secretion across canine airways were not accompanied by a large bioelectric response. They concluded that acetylcholine-induced secretion across intact airways was electrically silent and originated from glands.

The results of the present study imply that the mechanism of secretion by porcine bronchi is similar to that of rabbit mandibular salivary glands (7). About 60% of acetylcholine-stimulated liquid secretion from salivary glands was blocked by furosemide, which, like bumetanide, inhibits Cl- secretion by blocking Na+-K+-2Cl- cotransport. Residual salivary liquid secretion required the presence of HCO-3. The authors also observed that furosemide caused HCO-3 concentrations in salivary liquid to increase from 13 to 55-80 mM. Unlike the present study, however, furosemide induced an increase rather than a decrease in HCO-3 secretion, an observation more in keeping with expectations for anion flow through the same channels. The study of rabbit salivary glands also reported that carbonic anhydrase inhibitors block only about one-half of the HCO-3-dependent salivary liquid flow. This finding is intriguing because Inglis et al. (9) previously showed that gland ducts are occluded by exposure to the combination of bumetanide and DMA but not to the combination of acetazolamide and bumetanide.

From our visual inspection of the airway lumen, we are confident that we collected a large fraction of the mucus and liquid from the air space. We admit, however, that some of the ASL, particularly periciliary liquid, is not collected with this technique. Recently, we modified our original technique by adding aspiration with a syringe and attached polyethylene tubing to remove as much residual liquid as possible from the airway surface. We found that the rates of liquid secretion measured with the "improved" collection technique (control: 17.3 ± 1.4 µl · cm-2 · h-1; bumetanide: 5.0 ± 0.7 µl · cm-2 · h-1; bumetanide + DMA: 1.6 ± 0.2 µl · cm-2 · h-1) are very similar to the rates presented in the present study (control: 13.5 ± 1.2 µl · cm-2 · h-1; bumetanide: 4.0 ± 0.5 µl · cm-2 · h-1; bumetanide + DMA: 1.9 ± 0.4 µl · cm-2 · h-1). From these data, we feel confident that our original collection technique was quantitatively adequate.

In conclusion, we found that both Cl- and HCO-3 can drive acetylcholine-induced liquid secretion by distal bronchi. We think that this liquid emanates from glands. Studies with inhibitors of anion transport show that all agents reduce liquid and electrolyte secretion but that inhibition of Cl- secretion tends to maintain HCO-3 secretion, whereas inhibition of HCO-3 secretion tends to preserve Cl- secretion. To the extent that gland secretion contributes to the formation of ASL, anion transport by gland epithelia would be expected to regulate ASL volume and composition.

    ACKNOWLEDGEMENTS

We thank Dr. Richard C. Boucher for useful comments and suggestions. We are particularly indebted to Dr. Michael R. Van Scott, who first raised our interest in the importance of HCO-3 transport by airway epithelia.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-48622.

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: S. T. Ballard, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688.

Received 15 May 1998; accepted in final form 4 September 1998.

    REFERENCES
Top
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.   Bittner, O., and S. G. Hall. The titration of bicarbonate in plasma---critical factors. Am. J. Clin. Pathol. 42: 522, 1964.

3.   Borson, D. B., R. A. Chin, B. Davis, and J. A. Nadel. Adrenergic and cholinergic nerves mediate fluid secretion from tracheal glands of ferrets. J. Appl. Physiol. 49: 1027-1031, 1980[Abstract/Free Full Text].

4.   Boucher, R. C. Human airway ion transport, part one. Am. J. Respir. Crit. Care Med. 150: 271-281, 1994[Medline].

5.   Boucher, R. C. Human airway ion transport, part two. Am. J. Respir. Crit. Care Med. 150: 581-593, 1994[Medline].

6.   Boucher, R. C., and J. T. Gatzy. Regional effects of autonomic agents on ion transport across excised canine airways. J. Appl. Physiol. 52: 893-901, 1982[Abstract/Free Full Text].

7.   Case, R. M., M. Hunter, I. Novak, and J. A. Young. The anionic basis of fluid secretion by the rabbit mandibular salivary gland. J. Physiol. (Lond.) 349: 619-630, 1984[Abstract].

8.   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].

9.   Inglis, S. T., 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].

10.   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].

11.   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].

12.   Jarnigan, F., J. D. Davis, P. A. Bromberg, J. T. Gatzy, and R. C. Boucher. Bioelectric properties and ion transport of excised rabbit trachea. J. Appl. Physiol. 55: 1884-1892, 1983[Abstract/Free Full Text].

13.   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].

14.   Moon, S., M. Singh, M. E. 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.   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].

16.   Quinton, P. M. Composition and control of secretions from tracheal bronchial submucosal glands. Nature 279: 551-552, 1979[Medline].

17.   Quinton, P. M. Cystic fibrosis: a disease in electrolyte transport. FASEB J. 4: 2709-2717, 1990[Abstract/Free Full Text].

18.   Silva, P., J. Stoff, M. Field, L. Fine, J. N. Forrest, and F. H. Epstein. Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am. J. Physiol. 233 (Renal Fluid Electrolyte Physiol. 2): F298-F306, 1977[Medline].

19.   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].

20.   Tabcharan, J. A., T. J. Jensen, J. R. Riordan, and J. W. Hanrahan. Bicarbonate permeability of the outwardly rectifying anion channel. J. Membr. Biol. 112: 109-122, 1989[Medline].

21.   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].

22.   Ueki, I., V. F. German, and J. A. Nadel. Micropipette measurement of airway submucosal gland secretion. Am. Rev. Respir. Dis. 121: 351-357, 1980[Medline].


Am J Physiol Lung Cell Mol Physiol 275(6):L1095-L1099
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society