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A small molecule CFTR inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways¹

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California, USA

²Correspondence: 1246 Health Sciences, East Tower, Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu

SPECIFIC AIMS

It has been difficult to define the role of CFTR in airway submucosal gland fluid secretion because of a lack of suitable CFTR inhibitors and concerns in interpreting experiments on cystic fibrosis (CF) human airways due to severe gland pathology. The purpose of this study was to investigate the role of CFTR on rate, composition, and physical properties of fluid secreted from airway submucosal glands in the absence of secondary factors such as airway infection, inflammation, and gland hypertrophy. We have used a recently identified thiazolidine CFTR inhibitor (CFTR_inh-172) that reversibly and selectively inhibits CFTR Cl^– conductance with 300 nM affinity. The tested hypothesis was whether acute CFTR inhibition would alter secreted gland fluid volume, composition, and viscosity.

PRINCIPAL FINDINGS

1. CFTR_inh-172 inhibits gland fluid secretion rate by multiple stimuli in pig and human airways
Fluid droplets on the mucosal surface of freshly obtained bronchi were observed by light microscopy after removing serosal tissue, mounting in a 37°C / 5% CO₂ incubation chamber, and covering the mucosa with oil. By image analysis of expanding fluid droplets, single gland fluid secretion rates in pig bronchi were (in nL/min): 1.1 ± 0.1 (basal), 7.3 ± 1.0 (pilocarpine-stimulated), and 2.2 ± 0.3 (forskolin-stimulated). CFTR_inh-172 significantly reduced secretion stimulated by both cholinergic and cAMP agonists, but did not significantly reduce basal secretion (Fig. 1 A). Secretion in response to pilocarpine was also reduced in HCO₃^– free solutions. Figure 1B shows a series of micrographs of expanding gland fluid droplets after stimulation with pilocarpine, without and after application of CFTR_inh-172. In human airways, as shown in Fig. 1C , CFTR_inh-172 also inhibited pilocarpine-stimulated gland fluid secretion. Significant though lesser reduction in fluid secretion was seen in airways from CF subjects, which may be related to chronic glandular hypertrophy in CF. CFTR_inh-172 did not affect fluid secretion in CF airways.

View larger version (31K): [in this window] [in a new window]   Figure 1. Fluid secretion rate from submucosal glands of pig and human airways.A) Averaged fluid secretion rates from pig airways (SE, n=4–18 airways, >10 droplets measured per airway) under basal conditions and after addition of pilocarpine (50 µM), IBMX (100 µM), forskolin (20 µM), cocktail (forskolin 20 µM, IBMX 100 µM, apigenin 20 µM) in the absence and presence of 20 µM CFTRinh-172. *P < 0.01 compared with corresponding agonist without inhibitor (one way ANOVA with Tukey-Kramer post hoc test). B) Bright-field micrographs of expanding fluid droplets secreted from submucosal glands after pilocarpine (50 µM) stimulation. Scale bar, 50 µm. C) Fluid secretion from human airways (SE, n=2–3 airways, >6 droplets measured per airway) under basal conditions and after pilocarpine (50 µM). *P < 0.01 compared with stimulated control airway.

2. CFTR inhibition reduces gland fluid pH but does change [Na⁺] and [Cl^–]
Gland fluid ionic content was determined by ratio imaging of Na⁺, Cl^–, and pH-sensitive fluorescent dyes microinjected into gland fluid droplets. pH in freshly secreted gland fluid droplets was measured by excitation ratio imaging using the pH-sensitive fluorescent probe BCECF-dextran. pH was determined in secreted fluid droplets under basal and pilocarpine-stimulated conditions, without and with CFTR inhibition. Gland fluid pH under basal conditions was 6.9 ± 0.06, and was mildly elevated after stimulation with pilocarpine (7.1±0.07). Application of CFTR_inh-172 did not change pH under basal conditions, but significantly reduced pH to 6.7 ± 0.09 after stimulation. Secreted fluid [Na⁺] and [Cl^–] measured by fluorescence ratio imaging after microinjection with fluorescent indicators were 101 ± 1 mM and 116 ± 2 mM, respectively. There was no significant effect of stimulation on [Na⁺] and [Cl^–] or a significant effect of CFTR inhibition.

3. CFTR inhibition increases secreted fluid viscosity
Linear (or "zeroth order") viscosity was determined from translational diffusive mobility of a noninteracting fluorescent probe (FITC-dextran, 10 kDa) as measured by fluorescence recovery after photobleaching. Freshly secreted gland fluid droplets were microinjected with FITC-dextran, and fluorescence recovery was measured in individual fluid droplets after bleaching a cylindrical region through the droplet by a brief, intense laser pulse. Figure 2 A (left) shows recovery of fluorescence following bleaching in saline (top), and gland fluid under control conditions and after CFTR inhibition. Fluorescence recovery was slowed 1.3-fold in pilocarpine-stimulated gland fluid compared with saline, and was further slowed by 3.3-fold after CFTR inhibition. Figure 2A (right) summarizes recovery rates and relative fluid viscosities measured in gland fluid under basal and pilocarpine-stimulated conditions, without and after CFTR inhibition. Fluid viscosity increased remarkably after CFTR inhibition and stimulation by pilocarpine. An aspect of nonlinear viscosity with direct relevance to bacterial adhesion and clearance mechanisms (adhesivity) was estimated from maximum length of secreted gland fluid strands pulled with glass needles as illustrated by micrographs in Fig. 2B (top). Figure 2B (bottom) shows significantly greater adhesivity in pilocarpine-stimulated secreted fluid after CFTR inhibition.

View larger version (40K): [in this window] [in a new window]   Figure 2. Elevated viscosity and protein concentration of freshly-secreted gland fluid after CFTR inhibition. A) Left: fluorescence recovery after photobleaching of 10 kDa FITC-dextran diffusion in saline (top), and in basal and pilocarpine-stimulated fluid secreted from pig submucosal glands (without and with CFTR inhibition) as indicated. Right: averaged fluorescence recovery rates (inversely proportional to fluid viscosity) (SE, n=6 airways, 4–6 fluid droplets studied per airway). *P < 0.001 comparing with vs. without inhibition. B) Left: non-linear viscous properties of submucosal gland fluid secretions. Adhesivity/spinability measured as length of fluid strands drawn from gland secretion. Top: micrographs showing fluid strands (arrows) on airway mucosal surface. Glass micropipettes shown and location of fluid droplet indicated by red arrow. Bottom: averaged length of strands before breaking in pilocarpine-stimulated gland secretions (SE, n=5 airways). C) Left: total protein content in pig submucosal gland fluid secretions under basal and pilocarpine-stimulated condition with and without CFTRinh-172 (SE, n=4 airways). Fluid was collected at 15 min after pilocarpine. Right: representative SDS-PAGE of secreted fluid. b,basal; p,pilocarpine.

Protein content of gland fluid secretions was assayed in fluid collected from many individual glands using glass micropipettes. Figure 2C (left) shows that total protein content of secreted gland fluid was significantly elevated by 2-fold after CFTR inhibition. SDS-PAGE analysis in Fig. 2C (right) confirms differences in protein concentration, but shows similar band patterns suggesting similar relative protein composition.

CONCLUSIONS AND SIGNIFICANCE

Airway disease is the major cause of morbidity and mortality in CF. Disease progression is characterized by recurrent bouts of airway infection and inflammation, airway obstruction, and deteriorating lung function. Several lines of evidence have suggested that a primary factor in CF airway disease may be altered airway fluid / mucus properties leading to decreased mucociliary clearance and consequent airway obstruction. Submucosal glands are a major contributor of airway fluid / mucus, and glandular secretions in CF patients are abnormal. Airways from CF subjects, especially at the time of lung transplantation, are usually associated with extensive infection, inflammation, airway remodeling, and gland hypertrophy. Abnormal gland secretions may therefore be attributed to factors associated with airway disease progression and secondary to loss of CFTR function. Principal findings here are a remarkably reduced rate of fluid secretion after CFTR inhibition, with corresponding increased protein concentration and viscosity. CFTR inhibition also produced a mild reduction in gland fluid pH, but no significant changes in [Na⁺] or [Cl^–].

Reduced rate of gland fluid secretion after CFTR inhibition provides strong evidence for involvement of CFTR in glandular epithelial fluid transport. Impaired fluid secretion from submucosal glands is thus predicted early in cystic fibrosis (Fig. 3 ). The situation later in the disease becomes complicated by changes in gland structure due to chronic inflammation and infection, as well as possible changes in functioning of other proteins (such as ENaC) in CFTR deficiency. Data here indicate that CFTR is the rate-limiting step in submucosal gland fluid secretion when Cl^– conductance is reduced to near zero as in cystic fibrosis. Substantially greater stimulation of gland fluid secretion by cholinergic compared with cAMP agonists agrees with previous data indicating vagal muscarinic stimulation as the principal physiological stimulus of gland fluid secretion.

View larger version (19K): [in this window] [in a new window]   Figure 3. Proposed mechanism by which defective CFTR function and hyperviscous gland secretions in CF produce airway disease. Fluid secretion in normal glands involves CFTR mediated anion transport driving osmotic water transport through AQP5. Mucins and other proteins are also secreted. In CF, loss of functional CFTR results in reduced fluid transport with continued mucus production, producing reduced and viscous secretions. Viscous gland secretions and surface fluid hyperabsorption result in decreased mucociliary clearance, which together with mucus plugging initiates a cycle of inflammation, infection, and further obstruction.

While CFTR inhibition did not alter salt concentrations, gland fluid pH was significantly more acidic after inhibition, consistent with involvement of CFTR-dependent HCO₃^– transport in gland fluid secretion (suggested from studies showing increased HCO₃^– secretion across forskolin-stimulated Calu-3 cells). In agreement with previous findings, we found that gland fluid secretion after cholinergic stimulation in the absence of HCO₃^– was reduced by 60%. The possibility cannot be excluded that Cl^–/HCO₃^– transport in collecting ducts of submucosal glands might be involved in CFTR-dependent differences in glandular fluid pH. Measurement of acinar fluid pH by micropuncture or fluorescence methods will be needed to address this issue.

Increased protein concentration and consequent greater viscosity of fluid secreted by submucosal glands after CFTR inhibition suggests that salt and water secretion are impaired to a greater extent than protein secretion. Total secreted protein (product of protein concentration and fluid secretion rate) was reduced, but to a lesser extent. Greater protein concentration in secreted fluid after CFTR inhibition resulted in increased linear viscosity as observed by the 3-fold slowed diffusion of a noninteracting fluorescent probe (FITC-dextran), as well as increased gland fluid adhesivity. These results suggest that CFTR loss of function, rather than effects of chronic inflammation and infection was responsible for our previous observation of elevated gland fluid viscosity in CF airways

In summary, acute CFTR inhibition reduced fluid secretion by submucosal glands in pig and human bronchi without altering salt concentration. We propose that preferential impairment of salt/water secretion vs. protein secretion after CFTR inhibition results in increased protein concentration and viscosity in secreted fluid. Increased viscosity of gland fluid after loss of CFTR function may be important in initiation of small airway mucus plugging and decreased mucociliary clearance in cystic fibrosis airway disease (Fig. 3) . Since acute inhibition of CFTR mimics defective gland function seen in CF, it follows that CFTR_inh-172 or a related CFTR blocker may be used to pharmacologically create a model of CF lung disease in airways of large animals that more accurately reflects human lung physiology.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1248fje;

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