Mucus secretion by single tracheal submucosal glands from normal and cystic fibrosis transmembrane conductance regulator knockout mice

Abstract

Submucosal glands line the cartilaginous airways and produce most of the antimicrobial mucus that keeps the airways sterile. The glands are defective in cystic fibrosis (CF), but how this impacts airway health remains uncertain. Although most CF mouse strains exhibit mild airway defects, those with the C57Bl/6 genetic background have increased airway pathology and susceptibility to Pseudomonas. Thus, they offer the possibility of studying whether, and if so how, abnormal submucosal gland function contributes to CF airway disease. We used optical methods to study fluid secretion by individual glands in tracheas from normal, wild-type (WT) mice and from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice (Cftr(m1UNC)/Cftr(m1UNC); CF mice). Glands from WT mice qualitatively resembled those in humans by responding to carbachol and vasoactive intestinal peptide (VIP), although the relative rates of VIP- and forskolin-stimulated secretion were much lower in mice than in large mammals. The pharmacology of mouse gland secretion was also similar to that in humans; adding bumetanide or replacement of HCO(3)(-) by Hepes reduced the carbachol response by approximately 50%, and this inhibition increased to 80% when both manoeuvres were performed simultaneously. It is important to note that glands from CFTR knockout mice responded to carbachol but did not secrete when exposed to VIP or forskolin, as has been shown previously for glands from CF patients. Tracheal glands from WT and CF mice both had robust secretory responses to electrical field stimulation that were blocked by tetrodotoxin. It is interesting that local irritation of the mucosa using chili pepper oil elicited secretion from WT glands but did not stimulate glands from CF mice. These results clarify the mechanisms of murine submucosal gland secretion and reveal a novel defect in local regulation of glands lacking CFTR which may also compromise airway defence in CF patients.

Figure 1. Fluid secretion by mouse airway submucosal glands

Digital image of mucus droplets that form under paraffin oil following stimulation of isolated mouse trachea with carbachol. Mouse glands are smaller than those in humans but there are more per unit area in this region of the trachea.

Figure 2. Cumulative secreted volumes versus time from single glands

Carbachol (1 μm) was added at time 0. Each curve indicates the fluid secreted by one gland. A, wild-type mice (n = 7 glands from four tracheas). B, CFTR−/− mice (n = 7 glands from three tracheas). Insets show the calculated secretion rates.

Figure 3. Mean concentration dependence of carbachol stimulation in wild-type (WT) and CFTR−/− knockout mouse glands

Data from WT (○) and CFTR−/− (•) submucosal glands were fitted with a three-parameter logistic curve using non-linear regression (n = 6 glands for each point). The concentration giving half-maximal stimulation (EC₅₀) was 1.7 μm for WT mice and 1.8 μm for CFTR−/− mice. Maximum secretion rates (V_max) were also similar: 1.58 ± 0.16 nl min⁻¹ (n = 55) and 1.66 ± 0.25 (n = 36), respectively.

Figure 4. Effect of bumetanide, ouabain and niflumic acid on carbachol stimulated fluid secretion by wild-type (WT) mouse submucosal glands

A, effect of 50 nm carbachol on submucosal glands (n = 11 glands from five tracheas). B, effect of 10 μm bumetanide on fluid secretion (n = 5 glands from three tracheas). C, effect of 1 mm ouabain on fluid secretion (n = 5 glands from two tracheas). D, effect of 10 μm niflumic acid on fluid secretion by mouse submucosal glands (n = 8 glands from from tracheas). E, effect of bumetanide, ouabain and niflumic acid on carbachol-induced fluid secretion rate (mean + s.e.m.). The secretion rate during the time interval 15–35 min after addition of blocker was compared with that measured after 35–55 min stimulation with carbachol. *Significant differences (Student's t test, P < 0.05) between the control and experimental groups.

Figure 5. Effect of 40 μm bumetanide, replacement of bicarbonate by Hepes, and both interventions simultaneously on carbachol-induced secretion

Data were fitted to a three-parameter logistic curve using non-linear regression. Control carbachol responses (•) yielded EC₅₀ of 0.51 ± 0.03 μm and maximal secretion rate (V_max) of 1.84 ± 0.02 nl min⁻¹ (68 glands from five tracheas). V_max was decreased by 40% when glands were exposed to 40 μm bumetanide (○), by 55% by replacement of bicarbonate-containing Krebs-Ringer solution by Hepes-buffered solution lacking HCO3⁻ and gassed with O₂ (▵), and by 80% with the combined treatment (▿). EC₅₀ values were not affected significantly by the treatments.

Figure 6. Effect of VIP on cumulative fluid volumes and secretion rates

VIP (100 μm) was added to glands at time 0. A, Wild-type (n = 6 glands from three tracheas) and B, CFTR−/− (n = 12 glands from five tracheas) mice. Carbachol (1 μm) was added to the glands from CFTR−/− mice shown in B to confirm their viability. The insets show the secretion rates.

Figure 7. Effect of forskolin (10 μm) on submucosal gland secretion

A, wild-type (n = 6 glands from seven tracheas) and B, CFTR−/− (n = 7 glands from three tracheas) mice. Carbachol (1 μm) was added to the CFTR−/− knockout mouse glands as a positive control for viability. The insets show the secretion rates.

Figure 8. Effect of chili pepper oil (capsaicinoids) on fluid secretion by submucosal glands from wild-type mice

A, secreted volume (32 glands from eight tracheas) before and after addition of chili pepper oil. Note the break in the ordinate. B, secretion rates calculated under control conditions and after exposure to chili pepper oil (n = 8 tracheas). C, chili pepper oil response measured in the presence of CFTRinh172 (nominally 100 μm; n = 53 glands from seven tracheas). Carbachol (1 μm) was added to the CFTRinh172-treated glands as a positive control for viability.

Figure 9. Effect of tetrodotoxin (TTX) on responses to chili pepper oil, VIP (100 μm) and carbachol (1 μm)

The first time point shown is at 40 min; however, TTX (1 μm) was added at t = 0, then glands were sequentially exposed to chili pepper oil, VIP and carbachol (n = 24 glands from three tracheas).

Figure 10. Effect of electrical field stimulation (EFS) on fluid secretion by submucosal glands from wild-type mice

A, cumulative fluid secretion by individual glands before and after EFS (duration indicated by hatched vertical bar). B, mean secretion rates calculated immediately before and after EFS. *Significant difference between the control and experimental groups (12 glands from two tracheas).

Figure 11. Effect of chili pepper oil and electrical field stimulation (EFS) on fluid secretion by submucosal glands from CFTR−/− knockout mice

Note insensitivity to capsaicinoids but strong regulation by EFS to mimic central nervous system stimulation (12 glands from four tracheas). Most glands were non-secreting until stimulated by EFS (duration indicated by hatched vertical bar), despite the presence of chili pepper oil.