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(3)(-) secretion than on liquid secretion. Although preserved relative to liquid secretion, net HCO(3)(-) 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(3)(-). The pattern of responses to secretion agonists and antagonists suggests that the cystic fibrosis transmembrane conductance regulator mediates this process.

In porcine bronchi, inhibition of both Cl− and[Formula: see text] transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [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]. In this previous study, a combination of three potential [Formula: see text] transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl−/[Formula: see text]exchange, and Na+/H+exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced[Formula: see text] secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl−- and DMA-sensitive [Formula: see text] transport.

To model the airway glandular defect in cystic fibrosis (CF), the effect of anion secretion blockers on submucosal gland mucus secretion was investigated. Porcine distal bronchi were isolated, pretreated with a Cl- secretion blocker (bumetanide) and/or a combination of blockers to inhibit HCO3- secretion (dimethylamiloride, acetazolamide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), and then treated with acetylcholine (ACh), a glandular liquid and mucus secretagogue. Bronchi were then fixed, sectioned, and stained for mucins. Each gland duct was ranked for mucin content from zero (no mucin) to five (duct completely occluded with mucin). Untreated bronchi, bronchi treated only with ACh, and ACh-treated bronchi that received either bumetanide or the HCO3- secretion blockers all exhibited low gland duct mucin content (1.18 +/- 0.34, 0.59 +/- 0.07, 0.65 +/- 0.03, and 0.83 +/- 0.11, respectively). However, pretreatment with both Cl- and HCO3- secretion blockers before ACh addition resulted in substantial and significant ductal mucus accumulation (3.57 +/- 0.22). In situ videomicroscopy studies of intact airways confirmed these results. Thus inhibition of the anion (and presumably liquid) secretion response to ACh leads to mucus obstruction of submucosal gland ducts that resembles the early pathological changes observed in CF.

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Airway submucosal glands secrete both macromolecules and liquid, yet the mechanisms by which these substances are secreted are not well understood. In this study, a video microscope was used to directly visualize the submucosal glands in isolated porcine distal bronchi and to observe their responses to acetylcholine (ACh), a glandular secretagogue. Submucosal glands were classified as either "antral," "linear," or "convoluted" glands based on the morphology of their terminal collecting ducts. Because antral duct glands were most easily visualized, the response to ACh was studied in detail in this gland type. Within 5-10 s after addition of 10 microM ACh, the cross-sectional area of the gland duct openings to the airway surface increased severalfold but returned to pre-ACh dimensions within 1 min. Between 30 s and 10 min after ACh addition, spherical particles (1-10 microm) entered the antral ducts from distal acini and exited through the duct openings to the airway surface. Some of the particles were retained within the antral duct where they were kept in constant motion by the action of cilia present within the antral duct. The particles, which are likely to contain the macromolecular secretory products of mucous and/or serous cells, maintained their spherical shape within the gland duct, suggesting that the secretion product was membrane bound. To our knowledge, these studies provide the first description of airway submucosal gland secretion as viewed in situ.

To evaluate the possible relationship between submucosal glands and transepithelial Cl- secretion, we compared the bioelectric properties of two distal airway regions: bronchioles, which contain few submucosal glands, and small bronchi, which contain numerous glands. Intact distal bronchi were dissected from the lungs of 4-8 wk old pigs and cannulated with micropipettes and perfused. Transepithelial potential difference (PD), short-circuit current (Isc), and barrier resistance (Rm) in distal bronchi, determined by cable analysis, were -3.0 +/- 0.4 mV, 50.7 +/- 5.9 microA/cm2, and 59.6 +/- 5.9 omega.cm2, respectively (means +/- SE). Bumetanide (10 microM), which blocks Cl- secretion through inhibition of Na(+)-K(+)-2Cl- cotransport, reduced Isc in distal bronchi by 30.0 +/- 5.5 microA/cm2 (59.0% of the total Isc). By comparison, a previous study of porcine distal airways [S.T. Ballard and A.E. Taylor, Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L79-L84, 1994] determined that bumetamide-sensitive Isc in bronchioles was 2.6 +/- 1.4 microA/cm2 (only 9.0% of the total Isc). Submucosal gland duct openings to the luminal surface were identified microscopically and counted in representative fields. In eight bronchioles, 6.8 +/- 4.4 gland duct openings/cm2 of airway surface were observed, whereas seven distal bronchi contained 916.8 +/- 84.0 gland duct openings/cm2, over a 100-fold difference. These data suggest that a direct relationship may exist between the magnitude of active transepithelial Cl- secretion and the presence of submucosal glands in normal distal airways.

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