Measurement of fluid secretion from intact airway submucosal glands.

Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

Properties of substance P-stimulated mucus secretion from porcine tracheal submucosal glands.

Human and pig airway submucosal glands secrete mucus in response to substance P (SubP), but in pig tracheal glands the response to SubP is >10-fold greater than in humans and shares features with cholinergically produced secretion. CFTR-deficient pigs provide a model for human cystic fibrosis (CF), and in newborn CF pigs the response of tracheal glands to SubP is significantly reduced (Joo et al. J Clin Invest 120: 3161-3166, 2010). To further define features of SubP-mediated gland secretion, we optically measured secretion rates from individual adult porcine glands in isolated tracheal tissues in response to mucosal capsaicin and serosal SubP. Mucosal capsaicin (EC(50) = 19 µM) stimulated low rates of secretion that were partially inhibited by tetrodotoxin and by inhibitors for muscarinic, VIP, and SubP receptors, suggesting reflex stimulation of secretion by multiple transmitters. Secretion in response to mucosal capsaicin was inhibited by CFTR(inh)-172, but not by niflumic acid. Serosal SubP (EC(50) = 230 nM) stimulated 10-fold more secretion than mucosal capsaicin, with a V(max) similar to that of carbachol. Secretion rates peaked within 5 min and then declined to a lower sustained rate. SubP-stimulated secretion was inhibited 75% by bumetanide, 53% by removal of HCO(3)(-), and 85% by bumetanide + removal of HCO(3)(-); it was not inhibited by atropine but was inhibited by niflumic acid, clotrimazole, BAPTA-AM, nominally Ca(2+)-free bath solution, and the adenylate cyclase inhibitor MDL-12330A. Ratiometric measurements of fura 2 fluorescence in dissociated gland cells showed that SubP and carbachol increased intracellular Ca(2+) concentration by similar amounts. SubP produced rapid volume loss by serous and mucous cells, expansion of gland lumina, mucus flow, and exocytosis but little or no contraction of myoepithelial cells. These and prior results suggest that SubP stimulates pig gland secretion via CFTR- and Ca(2+)-activated Cl(-) channels.

Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs.

Cystic fibrosis (CF) results from mutations that disrupt CF transmembrane conductance regulator (CFTR), an anion channel found mainly in apical membranes of epithelial cells. CF leads to chronic infection of the airways with normally innocuous bacteria and fungi. Hypotheses to explain the pathophysiology of CF airways have been difficult to test because mouse models of CF do not develop human-like airway disease. The recent production of pigs lacking CFTR and pigs expressing the most common CF-causing CFTR mutant, DeltaF508, provide another model that might help clarify the pathophysiology of CF airway disease. Here, we studied individual submucosal glands from 1-day-old piglets in situ in explanted tracheas, using optical methods to monitor mucus secretion rates from multiple glands in parallel. Secretion rates from control piglets (WT and CFTR+/-) and piglets with CF-like disease (CFTR-/- and CFTR-/DeltaF508) were measured under 5 conditions: unstimulated (to determine basal secretion), stimulated with forskolin, stimulated with carbachol, stimulated with substance P, and, as a test for synergy, stimulated with forskolin and a low concentration of carbachol. Glands from piglets with CF-like disease responded qualitatively to all agonists like glands from human patients with CF, producing virtually no fluid in response to stimulation with forskolin and substantially less in response to all other agonists except carbachol. These data are a step toward determining whether gland secretory defects contribute to CF airway disease.

Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process.

Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.