Mechanism of substance P-induced liquid secretion across bronchial epithelium

Laura Trout1, Michel R. Corboz2, and Stephen T. Ballard1

1 Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688; and 2 Schering-Plough Research Institute, Kenilworth, New Jersey 07033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was undertaken to identify and determine the mechanism of noncholinergic pathways for the induction of liquid secretion across airway epithelium. Excised porcine bronchi secreted substantial and significant quantities of liquid when exposed to acetylcholine, substance P, or forskolin but not to isoproterenol, norepinephrine, or phenylephrine. Bumetanide, an inhibitor of Na+-K+-2Cl- cotransport, reduced the liquid secretion response to substance P by 69%. Approximately two-thirds of bumetanide-insensitive liquid secretion was blocked by dimethylamiloride (DMA), a Na+/H+ exchange inhibitor. Substance P responses were preserved in airways after surface epithelium removal, suggesting that secreted liquid originated from submucosal glands. The anion channel blockers diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited >90% of substance P-induced liquid secretion, whereas DIDS had no effect. DMA, DPC, and NPPB had greater inhibitory effects on net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion than on liquid secretion. Although preserved relative to liquid secretion, net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion was reduced by 39% in the presence of bumetanide. We conclude that substance P induces liquid secretion from bronchial submucosal glands of pigs through active transport of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The pattern of responses to secretion agonists and antagonists suggests that the cystic fibrosis transmembrane conductance regulator mediates this process.

cystic fibrosis transmembrane conductance regulator; bicarbonate; bumetanide; dimethylamiloride; cystic fibrosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRACHEOBRONCHIAL SUBMUCOSAL GLANDS secrete numerous substances such as mucins, which facilitate mucociliary clearance of inhaled debris, and various antibacterial proteins including lysozyme, lactoferrin, and beta -defensins that are critical to normal lung function (3, 20, 44). Normally, these proteinaceous substances are flushed from gland ducts by cosecretion of liquid that is thought to originate from serous cells that populate the distal ducts and demilunes of the glands. Close coupling of liquid and macromolecule secretion ensures that mucins are adequately hydrated for optimal ciliary transport by surface epithelial cells and that antibacterial substances are delivered to the airway surface in quantities sufficient to prevent pathogen colonization.

Secretion of liquid from airway glands is principally under neural control. Immunohistochemical and radioligand binding studies demonstrated that M3 muscarinic receptors (4), alpha 1-adrenergic receptors (5), beta 2-adrenergic receptors (29), and substance P receptors (6) are present on airway submucosal gland cells. Muscarinic or alpha -adrenergic receptor agonists substantially enhance volume secretion from feline tracheal submucosal glands in vivo (36, 47). Intravenous administration of substance P also greatly stimulates tracheal gland liquid secretion in domestic pigs, suggesting that sensory afferents play a role in regulating submucosal gland function (14). In cats, beta -adrenergic receptor stimulation produces a smaller liquid secretion response that varies from mild to scant (26, 36).

Recent studies (2, 16, 45) provide significant insight into the mechanisms responsible for liquid secretion in airway submucosal glands. Intact distal bronchi of pigs, when stimulated with acetylcholine, secrete liquid through the active transport of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. This secreted liquid originates in large part from submucosal glands and is probably mediated by the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein (2). Similarly, Calu-3 cells, a human airway cell line believed to be of serous cell origin, have been shown to secrete both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by a CFTR-dependent mechanism (9, 25). Involvement of CFTR in this process implies that derangement of liquid secretion from airway submucosal glands and the resultant uncoupling of mucin and liquid secretion could be of paramount importance to the development of lung disease in CF, a genetic disorder caused by deleterious mutations in the CFTR. Indeed, the earliest pathological sign in the lungs of CF patients is obstruction of submucosal gland ducts with mucinous material (32, 52), a finding that can be reproduced in vitro by treating porcine airways with Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion inhibitors (15, 17).

We considered the possibility that noncholinergic pathways might induce gland liquid secretion by CFTR-independent mechanisms. The existence of a CFTR-independent secretion pathway could be important therapeutically in that it could be manipulated pharmacologically to bypass the CFTR-dependent defect in gland liquid secretion that likely occurs in CF. To identify these possible pathways, we screened numerous secretagogues as potential stimulators of gland liquid secretion in porcine bronchi. We found that liquid was secreted in response to both muscarinic and substance P receptor agonists but that alpha - and beta -adrenergic receptor agonists produced no measurable effect. We further report evidence that the liquid secretion response to substance P closely mirrors that of acetylcholine in that it is driven by the transepithelial secretion of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and likely requires the CFTR.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental protocol. Young domestic pigs (~10-15 kg) were sedated with intramuscular xylazine (4 mg) and ketamine (80 mg) and euthanized with an intravenous overdose of pentobarbital sodium. The lungs were removed and placed in Krebs-Ringer bicarbonate (KRB) at room temperature. Distal bronchi were dissected from the surrounding tissue, and the side branches of the bronchi were ligated with sutures. Each bronchus was warmed slowly to 37°C (~0.1°C/min) in a KRB bath.

At the end of the warming period, the airways were removed from their bath solutions, and the lumens were cleared of all fluid and mucus. The air-filled bronchi were then cannulated with polyethylene tubing and returned to their respective baths. Either 100 µM acetylcholine (muscarinic receptor agonist), 1 µM substance P (neurokinin receptor agonist), 10 µM norepinephrine (agonist for alpha - and beta 1-adrenergic receptors), 10 µM phenylephrine (alpha -adrenergic agonist), 10 µM isoproterenol (beta -adrenergic receptor agonist), or 10 µM forskolin (direct stimulator of adenylyl cyclase) was then added to the bath to stimulate secretion. In all studies in which substance P was used, 1 µM phosphoramidon, a protease inhibitor needed to preserve substance P activity, was added 15 min before cannulation.

To determine the mechanism of substance P-induced liquid secretion, the following transport inhibitors were used. Atropine (10 µM), a muscarinic receptor antagonist, was used to ensure that responses to substance P were not due to secondary release of acetylcholine. Bumetanide (10 µM), a loop diuretic that blocks Na+-K+-2Cl- cotransport, was used to block transepithelial Cl- secretion. Dimethylamiloride (DMA; 100 µM), an inhibitor of Na+/H+ exchange, was used to block HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (45). Three anion channel blockers were used: 100 µM diphenylamine-2-carboxylate (DPC), a relatively nonselective anion channel inhibitor (38); 1 mM DIDS, an inhibitor that blocks Ca2+-activated anion channels, outwardly rectifying anion channels, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, and Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport but not the CFTR (1, 34, 38); and 100-300 µM 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB), an anion channel inhibitor that reportedly exhibits some specificity for the CFTR (21). The transport inhibitors were added 30 min before the addition of phosphoramidon for a total pretreatment period of 45 min. All inhibitor-pretreated tissues were paired with control tissues, which received only the inhibitor vehicle.

To localize liquid secretion to submucosal glands, the surface epithelium of some airways was removed with a wooden ream. Selected airways were viewed under saline with a Zeiss ACM microscope equipped with a ×20 water-immersion objective to confirm that this technique adequately removed the surface epithelium while leaving the submucosal structures intact.

Collection of airway liquid. After a 2-h incubation with the secretion agonists, the bronchi were removed from their cannulas and sectioned lengthwise, and all luminal mucus liquid was recovered. Mucus liquid was placed into tared tubes that were sealed and then weighed to determine secretion volume. Liquid samples were frozen (-70°C) for later analysis. Airway lengths and outer diameters were measured and used to estimate luminal surface areas as previously described (45). Net liquid secretion rate (Jv) was calculated based on the total secretion volume, the luminal surface area, and the time of agonist exposure.

Bicarbonate analysis. Frozen samples were thawed to room temperature, and 1.4-10.0 µl of each sample were added to 1,000 µl of Sigma INFINITY CO2 reagent (Sigma). The solutions were mixed with a vortex mixer for 15-20 s, and the absorbance of each sample was measured at 380 nm with a Beckman DU-65 spectrophotometer. Blank samples containing reagent and distilled H2O were assayed at the beginning and end of the sample readings to allow correction for temporal variance in the enzymatic reaction. Because the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in liquid from bumetanide-treated tissues was substantially higher than that in the control tissues, these samples were diluted so that the concentrations would fall within the linear range of the assay. The rate of net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> flux (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) was calculated from the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]) in the secreted liquid and the Jv.

Solution composition and drugs. KRB contained 112.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 2.4 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, and 11.6 mM glucose. The pH of all KRB solutions was maintained at 7.4 by constant gassing with 95% O2-5% CO2. DPC (as N-phenylanthranilic acid) was purchased from Aldrich, and NPPB was obtained from Calbiochem. All other drugs were purchased from Sigma. Stock solutions of substance P and phosphoramidon were prepared in 0.9% saline. Stock solutions of norepinephrine, phenylephrine, and isoproterenol were prepared in deionized H2O, and stock solutions of all inhibitors and forskolin were prepared in DMSO. Equal volumes of the vehicle were added to all control tissues.

Statistics. The data are expressed as means ± SE. Statistical comparisons were made with paired t-tests, unpaired t-tests, or ANOVA, with either Dunnett's or Tukey's test for multiple comparisons when appropriate. Differences were considered significant when P < 0.05. The number of observations is indicated by n (animals).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Untreated porcine bronchi secreted little liquid (1.9 ± 0.3 µl · cm-2 · h-1; n = 37; Fig. 1). However, substantial liquid secretion occurred when the airways were exposed to 100 µM acetylcholine (16.9 ± 2.7 µl · cm-2 · h-1; n = 6), 1 µM substance P (18.0 ± 1.0 µl · cm-2 · h-1; n = 46), and 10 µM forskolin (8.5 ± 1.9 µl · cm-2 · h-1; n = 6). No significant increase in Jv was observed after treatment with either alpha - or beta -adrenergic agonists. Norepinephrine (10 µM), phenylephrine (10 µM), and isoproterenol (10 µM) induced secretions of only 2.5 ± 0.4 µl · cm-2 · h-1 (n = 4), 0.9 ± 0.4 µl · cm-2 · h-1 (n = 5), and 0.7 ± 0.5 µl · cm-2 · h-1 (n = 4), respectively. In a separate group of paired tissues, the Jv response to substance P (16.5 ± 2.8 µl · cm-2 · h-1; n = 4) was unaffected by 10 µM atropine (19.4 ± 4.1 µl · cm-2 · h-1; n = 4), indicating that the stimulatory effect was not caused by secondary release of acetylcholine.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of candidate secretagogues on rate of liquid secretion (Jv) by porcine bronchi. Agonists are 100 µM acetylcholine (ACh), 1 µM substance P (SP) in the presence of 1 µM phosphoramidon, 10 µM norepinephrine (NE), 10 µM phenylephrine (PE), 10 µM isoproterenol (Iso), and 10 µM forskolin (For). NT, no treatment. Nos. in parentheses, no. of observations. *Significant difference from NT group (P < 0.05).

When substance P-stimulated airways were pretreated with 100 µM DMA to block HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, an insignificant 9% decrease in Jv occurred (Table 1). However, when airways were pretreated with 10 µM bumetanide to block Cl- secretion, substance P-induced liquid secretion was significantly reduced by 69%. In the presence of both bumetanide and DMA, substance P-induced liquid secretion fell by 89%, an inhibitory effect that was significantly greater than that of bumetanide pretreatment alone. DPC (100 µM) pretreatment, which was intended to block apical membrane anion channels, nearly abolished the Jv response as did pretreatment with 300 µM NPPB. Pretreatment with 1 mM DIDS, which should have blocked Ca2+-activated anion channels, outwardly rectifying anion channels, some volume-sensitive anion channels, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, and Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport but not CFTR, had no effect on Jv. Removal of the surface epithelium from the bronchi did not significantly affect the rate of substance P-induced secretion nor the inhibitory effect of bumetanide and DMA on the Jv (Fig. 2), suggesting that the principal source of the liquid was the submucosal glands.

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



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of surface epithelium removal on substance P-induced liquid secretion. DMA, dimethylamiloride. Nos. in parentheses, no. of observations; +, presence; -, absence. Responses in epithelium-denuded airways were not significantly different from responses in epithelium-intact airways. *Significant difference from airways treated with substance P alone (P < 0.05).

In a subset of airways, the [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in the secreted liquid was determined, and JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was calculated. When airways were pretreated with DMA, both [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] and JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were significantly reduced (Table 2). The reduction in JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (to 57.1% of control value) was far greater than the reduction in Jv (to 91.2% of control value) for these tissues, suggesting that DMA preferentially inhibited secretion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Bumetanide pretreatment, intended to selectively inhibit Cl- secretion, increased [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in the secreted liquid twofold. This increased [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] apparently reflects the greater inhibitory effect of bumetanide on the Jv than on the JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Even though JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was preserved relative to Jv, bumetanide actually inhibited JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by 38.9% compared with levels in control airways. The combination of DMA and bumetanide nearly abolished both JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Jv. Because a greater inhibition was seen in JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> than in Jv, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in secreted liquid was significantly reduced with the combination of these agents.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of anion transport inhibitors on substance P-induced bicarbonate secretion

DPC pretreatment also nearly abolished JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, causing [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in secreted fluid to fall to <5 meq/l (Table 2). Because 300 mM NPPB reduced secretion volumes to levels that were too low to accurately measure HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, a lesser concentration of NPPB, 100 mM, was used to assess its effects on JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]. At this lower concentration, NPPB substantially reduced JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]. For both DPC and NPPB, residual JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (as a percentage of control value) was approximately one-third of the residual Jv. DIDS had no significant effect on Jv, JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, or [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These studies show that substance P is an effective stimulator of liquid secretion in porcine bronchi. Approximately 70% of the Jv response to substance P was inhibited by bumetanide, suggesting that active secretion of Cl- comprises the major driving force for liquid secretion. Most of the bumetanide-insensitive Jv was blocked with DMA, apparently by inhibiting active HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Because removal of the surface epithelium did not significantly affect the rate of substance P-induced liquid secretion or the inhibitory responses to the secretion blockers, the source of the secreted liquid is most likely the submucosal glands. The pattern of inhibitory responses to several potential anion channel blockers and mimicry of the secretory response by forskolin are traits consistent with participation of the CFTR in the secretion response to substance P.

Previous studies demonstrate that substance P evokes liquid secretion from tracheal submucosal glands (14) and that afferent C fiber stimulation induces liquid secretion via an efferent vagal response (8). Together with a study (31) showing localization of substance P-containing neurons in the airways, these findings suggest the presence of 1) sensory afferent neurons that are capable of direct release of tachykinins at the site of stimulation within the airways and 2) efferent neurons passing through the vagus that release cholinergic neurotransmitters within airway tissues. The existence of this dual-excitation pathway ensures that a vigorous glandular secretion response will result from an appropriate mechanical or chemical stimulus of the airway mucosa. Copious secretion of both liquid and mucin from glands should aid mucociliary transport by increasing the volume of mucus liquid at the airway surface. Endogenous release of either acetylcholine or substance P in the vicinity of the surface epithelial cells should also increase ciliary beat frequency (49, 50), further accelerating mucociliary transport and facilitating removal of mucosal irritants.

A role for CFTR in secretion of airway gland liquid has been suspected since the finding that serous cells of submucosal glands were a major site of CFTR expression in human airways (11). Similar distribution patterns occur in cows (19), ferrets (39), and pigs (2). Reports (9, 40) that the Calu-3 cell, an airway serous cell line of human origin, secretes Cl- by a CFTR-dependent mechanism further support this notion. Moon and colleagues (30) concluded that CFTR is the exclusive Cl- conductive pathway expressed in the apical membrane of Calu-3 cells, suggesting that anion secretion in this cell type is critically dependent on the function of this channel. In the present study, we conclude that CFTR mediates the liquid secretion responses to substance P for the following reasons. First, substance P-induced liquid secretion was abolished by both DPC and NPPB, which are known to inhibit CFTR by blocking the ion pore from the cytoplasmic side of the channel (51). However, inhibition of secretion by DPC and NPPB does not alone confirm CFTR involvement in this process because these agents are known to exert nonselective effects that could complicate interpretation of their actions. For instance, DPC has been shown to inhibit cyclooxygenase (43), and NPPB has been shown to uncouple oxidative phosphorylation in nonepithelial cells by acting as a protonophore (28). Additionally, these agents block other anion channels besides the CFTR (1, 18). The second reason to suspect CFTR involvement is that the secretion response to substance P is insensitive to DIDS, which does not inhibit the CFTR but does inhibit both Ca2+-activated Cl- channels and outwardly rectifying Cl- channels, the most likely non-CFTR anion channels that would support transepithelial anion secretion in postnatal airway epithelia (1). The third piece of evidence implicating CFTR in gland secretion is the fluid secretion response to acetylcholine and substance P that is partially mimicked by forskolin, which increases intracellular cAMP by direct stimulation of adenylyl cyclase. cAMP is a well-known activator of CFTR (35). Finally, a role for CFTR in gland liquid secretion is implied from studies of early CF disease (32, 52) where mucoid obstruction of submucosal gland ducts is one of the earliest signs of pathology in the lung. Similar mucin obstruction of gland ducts can be reproduced in pig airways after inhibition of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion with bumetanide and DMA, respectively (15). This mucin obstruction of both CF and porcine airway gland ducts after Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion inhibition apparently reflects continued glandular mucin secretion in the face of impaired liquid secretion. We therefore believe that the data collectively support a role for CFTR in the liquid secretion response to substance P.

In the present study, virtually no liquid secretion resulted from administration of isoproterenol, a beta -adrenergic receptor agonist. Reports by others similarly suggest that beta -agonists are weak stimulators, at best, of gland liquid secretion. In studies with cat tracheae, Leikauf and coworkers (26) reported that isoproterenol induced about half the secretion response of acetylcholine, whereas Quinton (36) reported very little effect of this agent. These findings are somewhat surprising, however, in that beta -agonists have been shown to stimulate CFTR-dependent Cl- secretion in nasal epithelium, presumably through elevation of intracellular cAMP (12). Our results do show that liquid secretion is stimulated by forskolin, suggesting that the secretion response is sensitive to cAMP. Therefore, it is likely that beta -adrenoceptors are either sparse or absent on the gland cells that are responsible for liquid secretion. Indeed, Mak and coworkers (29) showed by radioligand binding that beta 2-adrenoceptors were present in airway submucosal gland cells but that receptor density was much less than in airway surface epithelium and alveolar walls.

We also saw no effect of alpha -adrenergic agonists on airway liquid secretion. This finding was unexpected because alpha -agonists have been shown to evoke vigorous liquid secretion from feline tracheal glands in vivo (36, 47). Similar to our studies, Joo and coworkers (24) showed in preliminary studies that phenylephrine is a poor agonist for liquid secretion by individual airway glands from sheep tracheae. We speculate that these findings reflect species differences in functional alpha -adrenergic receptor expression in airway gland cells.

The mechanism of substance P-induced Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion closely resembles that previously described for acetylcholine (2). The liquid secretion response to both agonists is inhibited ~70% by bumetanide, with most of the remaining secretion being inhibited by DMA. Both DPC and NPPB block >= 90% of the liquid secretion responses to both agonists, whereas DIDS has no effect. This pattern of responses is most consistent with the mechanism for Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion described by Smith and Welsh (41). According to this model, Cl- enters the cell across the basolateral membrane by Na+-K+-2Cl- cotransport and exits across the apical membrane through the CFTR. Both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+ are synthesized intracellularly from CO2 and H2O, either spontaneously or through the actions of carbonic anhydrase, with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> crossing the apical membrane through CFTR and H+ crossing the basolateral membrane through a Na+/H+ antiporter. Although we cannot discount the presence of basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, which could support transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion as described in Calu-3 cells (9, 25), the insensitivity of liquid secretion to DIDS in our preparation suggests that it plays little functional role in gland liquid secretion across porcine airways. We admit, however, that DIDS-insensitive isoforms of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport could exist in our preparation. In apparent contradiction to the Smith and Welsh (41) model, we observed that bumetanide alone inhibited ~40% of the net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secreted in response to substance P, a finding similar to that previously reported for acetylcholine-induced secretion (2, 45). We cannot account for this result with certainty, but several explanations are possible. One is that bumetanide partially blocks the CFTR, an action of this agent that has been previously documented (33). We believe that this explanation is unlikely, however, because HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is clearly preserved relative to Cl- in the presence of bumetanide, a finding that is inconsistent with conductance of both anions through a single channel. Another possibility is that the composition of the secreted liquid is modified by the surface epithelium. For instance, acid equivalents could be added to the luminal liquid by H+ transporters in the apical membrane during the 2-h incubation period. Indeed, preliminary studies by Coakley and coworkers (7) suggest that airway surface liquid is acidified with time through the actions of H+-K+-ATPases expressed in the apical membrane of the surface epithelium. Alternatively, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in the apical membrane of surface epithelium could replace HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the luminal liquid with Cl-. The actions of either transport process could also account for the relatively low [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] values in airway liquid when secretion volumes are greatly reduced, as seen with DPC and NPPB treatments. Finally, secreted liquid could contain very high [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] values when it exits the glands but become diluted with time as HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> diffuses out of and Cl- diffuses into the luminal liquid through the paracellular junctions of the surface epithelium.

To sustain the electrical driving force for anion efflux across the apical membrane, effective secretogogues must not only increase the apical anion conductance but also increase the basolateral conductance to K+ (42). When stimulated, both M3 muscarinic receptors, which are the predominant muscarinic receptor subtype in glands (4), and substance P receptors have been shown in multiple systems to induce phospholipase C-dependent inositol phospholipid hydrolysis and increase intracellular Ca2+ concentrations (10, 13, 22, 23, 48). We suspect that both of these agonists open Ca2+-activated K+ channels in the basolateral membrane to augment the electrical driving force for anion efflux across the apical membrane. Sasaki and coworkers (37) demonstrated that acetylcholine induces both Cl- and K+ currents in airway acinar gland cells by an inositol 1,4,5-trisphosphate-dependent process. Unfortunately, to date, we have been unable to identify, through the use of inhibitors, which specific subtype of K+ channel is involved in the liquid secretion response to either substance P or acetylcholine in porcine airways.

Our findings have particular relevance to the etiology of CF lung disease. It is likely that liquid secretion from airway bronchial glands induced by either substance P or acetylcholine, is dependent on the CFTR. When the secretion of gland liquid is blocked, as occurs when both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion are inhibited, gland ducts become obstructed with mucin, mimicking one of the earliest pathological signs of CF airway disease (15, 17). Additionally, the nonvolatile solids content of the secretion product increases threefold in inhibitor-treated airways (46), mirroring the increased dry weight that has been reported for CF mucus (27). This increased solids content of airway liquid significantly alters the rheological properties of this fluid in a manner that predicts reduced cough clearance (46). Failure to adequately clear this thickened mucus from airways could thereby predispose the lung to bacterial colonization, a hallmark of the disease. Consequently, disruption of gland liquid secretion could be the seminal event in the development of CF airway pathology. More study is clearly needed to determine if these events alone are sufficient to produce the cascade of pathological events that typify this disease.


    ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Angela Crews.


    FOOTNOTES

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

Address for reprint requests and other correspondence: L. Trout, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688 (E-mail: ltrout{at}bbl.usouthal.edu).

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. Section 1734 solely to indicate this fact.

Received 10 January 2001; accepted in final form 25 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, MP, Shepard DN, Berger HA, and Welsh MJ. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am J Physiol Lung Cell Mol Physiol 263: L1-L14, 1992[Abstract/Free Full Text].

2.   Ballard, ST, Trout L, Bebök Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 277: L694-L699, 1999[Abstract/Free Full Text].

3.   Bals, R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, and Wilson JM. Human beta -defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest 102: 874-880, 1998[Abstract/Free Full Text].

4.   Barnes, PJ. Muscarinic receptor subtypes in airways. Life Sci 52: 521-527, 1993[ISI][Medline].

5.   Barnes, PJ, Basbaum CB, Nadel JA, and Roberts JM. Pulmonary alpha-adrenoceptors: autoradiographic localization using [3H]prazosin. Eur J Pharmacol 88: 57-62, 1983[ISI][Medline].

6.   Carstairs, JR, and Barnes PJ. Autoradiographic mapping of substance P receptors in lung. Eur J Pharmacol 127: 295-296, 1986[ISI][Medline].

7.   Coakley, RD, Grubb BR, Gatzy JT, Chadburn JL, and Boucher RC. Differential airway surface liquid (ASL) pH, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and K+ homeostasis in cultured human and dog bronchial epithelium (Abstract). Pediatr Pulmonol Suppl 20: 194, 2000.

8.   Davis, B, Roberts AM, Coleridge HM, and Coleridge JCG Reflex tracheal gland secretion evoked by stimulation of bronchial C-fibers in dogs. J Appl Physiol 53: 985-991, 1982[Abstract/Free Full Text].

9.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 742-760, 1999.

10.   Dickinson, KEJ, Frizzell RA, and Sekar MC. Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor. Eur J Pharmacol 225: 291-298, 1992[Medline].

11.   Engelhardt, JF, Yankaskas JR, Ernst ST, Yang Y, Marino CR, Boucher RC, Cohn JA, and Wilson JM. Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 2: 240-248, 1992[ISI][Medline].

12.   Grubb, B, Lazarowski E, Knowles M, and Boucher RC. Isobutylmethylxanthine fails to stimulate chloride secretion in cystic fibrosis airway epithelia. Am J Respir Cell Mol Biol 8: 454-460, 1993[ISI][Medline].

13.   Hanley, MR, Lee CM, Jones LM, and Michell RH. Similar effects of substance P and related peptides on salivation and on phosphatidylinositol turnover in rat salivary glands. Mol Pharmacol 18: 78-83, 1980[Abstract].

14.   Haxhui, MA, Haxhui-Poskurica B, Moracic V, Carlo WA, and Martin RJ. Reflex and chemical responses of tracheal submucosal glands in piglets. Respir Physiol 82: 267-278, 1990[ISI][Medline].

15.   Inglis, SK, Corboz MR, and Ballard ST. Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts. Am J Physiol Lung Cell Mol Physiol 274: L762-L766, 1998[Abstract/Free Full Text].

16.   Inglis, SK, Corboz MR, Taylor AE, and Ballard ST. Regulation of ion transport across porcine distal bronchi. Am J Physiol Lung Cell Mol Physiol 270: L289-L297, 1996[Abstract/Free Full Text].

17.   Inglis, SK, Corboz MR, Taylor AE, and Ballard ST. Effect of anion transport inhibition on mucus secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 272: L372-L377, 1997[Abstract/Free Full Text].

18.   Jacob, TJC, and Civan MM. Role of ion channels in aqueous humor formation. Am J Physiol Cell Physiol 271: C703-C720, 1996[Abstract/Free Full Text].

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

20.   Jeffrey, PK. The origins of secretions in the lower respiratory tract. Eur J Respir Dis 71, Suppl 153: 34-42, 1987.

21.   Jiang, C, Finkbeiner WE, Widdicombe JH, and Miller SS. Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis. J Physiol (Lond) 501: 637-647, 1997[Abstract].

22.   Jones, SVP, Barker JL, Goodman MB, and Brann MR. Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 cells. J Physiol (Lond) 421: 499-519, 1990[Abstract].

23.   Jones, SVP, Helman CJ, and Brann MR. Functional responses of cloned muscarinic receptors expressed in CHO-K1 cells. Mol Pharmacol 40: 242-247, 1991[Abstract].

24.   Joo, NS, Saenz Y, Krause ME, Wu J, and Wine JJ. Mucous secretion from individual tracheal submucosal glands from sheep (Abstract). Pediatr Pulmonol Suppl 20: 205, 2000.

25.   Lee, MC, Penland CM, Widdicombe JH, and Wine JJ. Evidence that Calu-3 human airway cells secrete bicarbonate. Am J Physiol Lung Cell Mol Physiol 274: L450-L453, 1998[Abstract/Free Full Text].

26.   Leikauf, GD, Ueki IF, and Nadel JA. Autonomic regulation of viscoelasticity of cat tracheal gland secretions. J Appl Physiol 56: 426-430, 1984[Abstract/Free Full Text].

27.   Lethem, MI, James SL, and Marriott C. The role of mucous glycoproteins in the rheological properties of cystic fibrosis sputum. Am Rev Respir Dis 142: 1053-1058, 1990[ISI][Medline].

28.   Lukacs, GL, Nanda A, Rotstein OD, and Grinstein S. The chloride channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells. FEBS Lett 288: 17-20, 1991[ISI][Medline].

29.   Mak, JC, Grandordy B, and Barnes PJ. High affinity [3H]formoterol binding sites in lung: characterization and autoradiographic mapping. Eur J Pharmacol 269: 35-41, 1994[Medline].

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

31.   Nishi, Y, Kitamura N, Otani M, Hondo E, Taguchi K, and Yamada J. Distribution of capsaicin-sensitive substance P and calcitonin gene-related peptide-immunoreactive nerves in bovine respiratory tract. Ann Anat 182: 319-326, 2000[ISI].

32.   Oppenheimer, EH, and Esterly JR. Pathology of cystic fibrosis: review of the literature and comparison with 146 autopsied cases. In: Perspectives in Pediatric Pathology, edited by Dosenberg HS, and Bolarde RP.. Chicago, IL: Yearbook Medical, 1975, vol. 2, p. 241-278.

33.   Reddy, MM, and Quinton PM. Effects of bumetanide on chloride transport in human eccrine sweat ducts: implications for cystic fibrosis. Isr J Med Sci 23: 1210-1213, 1984.

34.   Romero, MF, Hediger MA, Boulpaep EL, and Boron WF. Expression cloning and characterization of a renal electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. Nature 387: 409-413, 1997[ISI][Medline].

35.   Rommens, JM, Dho S, Bear CE, Kartner N, Kennedy D, Riordan JR, Tsui LC, and Foskett JK. cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 88: 7500-7504, 1991[Abstract].

36.   Quinton, PM. Composition and control of secretions from tracheal bronchial submucosal glands. Nature 279: 551-552, 1979[ISI][Medline].

37.   Sasaki, T, Shimura S, Wakui W, Ohkawara Y, Takishima T, and Mikoshiba K. Apically localized IP3 receptors control chloride current in airway gland cells. Am J Physiol Lung Cell Mol Physiol 267: L152-L158, 1994[Abstract/Free Full Text].

38.   Schultz, BD, Singh AK, Devor DC, and Bridges RJ. Pharmacology of CFTR chloride channel activity. Physiol Rev 79: S109-S144, 1999[Medline].

39.   Sehgal, A, Presente A, and Engelhardt JF. 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].

40.   Shen, BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, and Widdicombe JH. Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am J Physiol Lung Cell Mol Physiol 266: L493-L501, 1994[Abstract/Free Full Text].

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

42.   Smith, PL, and Frizzell RA. Chloride secretion by canine tracheal epithelium. IV. Basolateral membrane K permeability parallels secretion rate. J Membr Biol 77: 187-199, 1984[ISI][Medline].

43.   Stutts, MJ, Henke DC, and Boucher RC. Diphenylamine-2-carboxylate (DPC) inhibits both Cl- conductance and cyclooxygenase of canine tracheal epithelium. Pflügers Arch 415: 611-616, 1990[ISI][Medline].

44.   Tom-Moy, M, Basbaum CB, and Nadel JA. Localization and release of lysozyme from ferret tracheae: effects of adrenergic and cholinergic drugs. Cell Tissue Res 228: 549-562, 1983[ISI][Medline].

45.   Trout, L, Gatzy JT, and Ballard ST. Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. Am J Physiol Lung Cell Mol Physiol 275: L1095-L1099, 1998[Abstract/Free Full Text].

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

47.   Ueki, I, German VF, and Nadel JA. Micropipette measurement of airway submucosal gland secretion: autonomic effects. Am Rev Respir Dis 121: 351-357, 1980[ISI][Medline].

48.   Weiss, SJ, McKinney JS, and Putney JW. Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells. Biochem J 206: 555-560, 1982[ISI][Medline].

49.   Wong, LB, Miller IF, and Yeates DB. Stimulation of ciliary beat frequency by autonomic agonists in vivo. J Appl Physiol 65: 971-981, 1988[Abstract/Free Full Text].

50.   Wong, LB, Miller IF, and Yeates DB. Pathways of substance P stimulation of canine tracheal ciliary beat frequency. J Appl Physiol 70: 267-273, 1991[Abstract/Free Full Text].

51.   Zhang, ZR, Zeltwanger S, and McCarty NA. Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes. J Membr Biol 175: 35-52, 2000[ISI][Medline].

52.   Zuelzer, WW, and Newton WA. The pathogenesis of fibrocystic disease of the pancreas. Pediatrics 4: 53-69, 1949[Abstract].


Am J Physiol Lung Cell Mol Physiol 281(3):L639-L645
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society