Submucosal Gland Secretion Research Articles

Normalizing salt content by mixing native human airway mucus samples normalizes sample rheology.

Across the globe, millions of people are affected by muco-obstructive pulmonary diseases like cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In MOPDs, the airway mucus becomes hyperconcentrated, increasing viscoelasticity and impairing mucus clearance. Research focused on treatment of MOPDs requires relevant sources of airway mucus both as a control sample type and as a basis for manipulation to study the effects of additional hyperconcentration, inflammatory milieu, and biofilm growth on the biochemical and biophysical properties of mucus. Endotracheal tube mucus has been identified as a prospective source of native airway mucus given its several advantages over sputum and airway cell culture mucus such as ease of access and in vivo production that includes surface airway and submucosal gland secretions. Still, many ETT samples suffer from altered tonicity and composition from either dehydration, salivary dilution, or other contamination. Herein, the biochemical compositions of ETT mucus from healthy human subjects were determined. Samples were characterized in terms of tonicity, pooled, and restored to normal tonicity. Salt-normalized ETT mucus exhibited similar concentration-dependent rheologic properties as originally isotonic mucus. This rheology agreed across spatial scales and with previous reports of the biophysics of ETT mucus. This work affirms previous reports of the importance of salt concentration on mucus rheology and presents methodology to increase yield native airway mucus samples for laboratory use and manipulation.

Aquaporins in Respiratory System.

Aquaporins (AQPs) are water channel proteins facilitating fluid transport in alveolar space, airway humidification, pleural fluid absorption, and submucosal gland secretion. In this chapter, we mainly focus on the expression of four AQPs in the lungs, which include AQP1, AQP2, AQP4, and AQP5 in normal and disease status, and the experience of AQPs function from various model and transgenic mice were summarized in detail to improve our understanding of the role of AQPs in fluid balance of respiratory system. It has been suggested that AQPs play important roles in various physiology and pathophysiology conditions of different lung diseases. There still remains unclear the exact role of AQPs in lung diseases, and thus continuous efforts on elucidating the roles of AQPs in lung physiological and pathophysiological processes are warranted.

Mucus concentration-dependent biophysical abnormalities unify submucosal gland and superficial airway dysfunction in cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Lipopolysaccharides induce a RAGE-mediated sensitization\xa0of sensory neurons and fluid hypersecretion in the upper airways

Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

Protective Effect of Diminazene Aceturate in Acid‐Induced Airway Obstruction

Mild airway acidification occurs in several airway diseases, including cystic fibrosis and asthma. However, the role of airway acidification in airway pathology is controversial. Here, we studied mucus secretion in piglets forty‐eight hours following an intra‐airway acid challenge. Airway submucosal glands of acid‐challenged piglets exhibited faulty submucosal gland secretion, with a retainment of mucin 5B (MUC5B) in the submucosal gland and decreased concentrations of MUC5B in lung lavage fluid following cholinergic stimulation. Concomitantly, intrapulmonary airways were obstructed with glycoprotein rich material under both basal and methacholine‐stimulated conditions. Blocking detection of acid with the diminazene aceturate, a small molecule that inhibits the acid‐sensing ion channel (ASIC), partially restored submucosal gland function and mitigated acid‐induced airway obstruction. These findings suggest that transient airway acidification significantly impacts mucus secretion and highlight diminazene aceturate as an agent beneficial in alleviating some mucus defects.Support or Funding InformationHL119560, 1OT2OD023854

Mucus strands from submucosal glands initiate mucociliary transport of large particles.

Mucus produced by submucosal glands is a key component of respiratory mucociliary transport (MCT). When it emerges from submucosal gland ducts, mucus forms long strands on the airway surface. However, the function of those strands is uncertain. To test the hypothesis that mucus strands facilitate transport of large particles, we studied newborn pigs. In ex vivo experiments, interconnected mucus strands moved over the airway surface, attached to immobile spheres, and initiated their movement by pulling them. Stimulating submucosal gland secretion with methacholine increased the percentage of spheres that moved and shortened the delay until mucus strands began moving spheres. To disrupt mucus strands, we applied reducing agents tris-(2-carboxyethyl)phosphine and dithiothreitol. They decreased the fraction of moving spheres and delayed initiation of movement for spheres that did move. We obtained similar in vivo results with CT-based tracking of microdisks in spontaneously breathing pigs. Methacholine increased the percentage of microdisks moving and reduced the delay until they were propelled up airways. Aerosolized tris-(2-carboxyethyl)phosphine prevented those effects. Once particles started moving, reducing agents did not alter their speed either ex vivo or in vivo. These findings indicate that submucosal glands produce mucus in the form of strands and that the strands initiate movement of large particles, facilitating their removal from airways.

Mucociliary Transport in Healthy and Cystic Fibrosis Pig Airways.

Cystic fibrosis (CF) lung disease is the major cause of morbidity and mortality in people with CF. Abnormal mucociliary transport has been the leading hypothesis for the underlying pathogenesis of CF airway disease. However, this has been difficult to investigate at very early time points. A porcine CF model, which recapitulates many features of CF disease in humans, enables studies to be performed in non-CF and CF pigs on the day that they are born. In newborn CF pigs, we found that under basal conditions, mucociliary transport rates in non-CF and CF pigs are similar. However, after cholinergic stimulation, which stimulates submucosal gland secretion, particles become stuck in the CF airways owing to a failure of mucus strands to release from submucosal glands. In this review, we summarize these recent discoveries and also discuss the morphology, composition, and function of mucins in the porcine lung.

Tubotympanic system functioning

The Eustachian (auditory) tube and tympanomastoid cavities form an anatomic and functional whole that cannot easily be divided, and is therefore known as the “tubotympanic system”. The system has been the focus of several studies, with complex and sometimes contradictory results, making an overview of its functioning difficult to obtain. The objective of the present article is to review the current state of knowledge, as an indispensable preliminary to understanding tubotympanic system dysfunction, and notably the development of chronic otitis. The system as a whole is covered by mucosa, which provides continuity, although with certain particularities from one area to another, and plays a primordial role. Thus, under physiological conditions, gas diffusion across the tympanomastoid mucosa largely ensures the equilibrium of pressure between the middle ear and outside environment, the tube orifice being very little involved. Under large rapid change in atmospheric pressure, the aeration function of the Eustachian tube comes into play, governed by a reflex mechanism. The system also has other functions that are essential to good middle-ear functioning: protection against nasopharyngeal secretions and pathogens and against certain physiological noises; middle-ear cavity clearance by mucociliary transport of pathogens, partly related to submucosal gland secretion; and immune defense.

Aquaporins in Respiratory System.

Aquaporins (AQPs ) are water channel proteins supposed to facilitating fluid transport in alveolar space, airway humidification, pleural fluid absorption, and submucosal gland secretion . In this chapter, we mainly focus on the expression of 4 AQPs in the lungs which include AQP1, AQP2 , AQP4 and AQP5 in normal and disease status, and the experience of AQPs function from various model and transgenic mice were summarized in detail to improve our understanding of the role of AQPs in fluid balance of respiratory system. It has been suggested that AQPs play important roles in various physiology and pathophysiology conditions of different lung diseases. There still remains unclear the exact role of AQPs in lung diseases, and thus continuous efforts on elucidating the roles of AQPs in lung physiological and pathophysilogical processes are warranted.

Secretory IgA from submucosal glands does not compensate for its airway surface deficiency in chronic obstructive pulmonary disease

Secretory immunoglobulin A (SIgA) reaches the airway lumen by local transcytosis across airway epithelial cells or with tracheobronchial submucosal gland secretions. In chronic obstructive pulmonary disease (COPD), deficiency of SIgA on the airway surface has been reported. However, reduction of SIgA levels in sputum and bronchoalveolar lavage (BAL) fluid has not been consistently observed. To explain this discrepancy, we analyzed BAL fluid and lung tissue from patients with COPD and control subjects. Immunohistochemical analysis of large and small airways of COPD patients showed that MUC5AC is the predominant mucin expressed by airway epithelial cells, whereas MUC5B is expressed in submucosal glands of large airways. Dual immunostaining with anti-IgA and anti-MUC5B antibodies showed reduction of IgA on the airway surface as well as accumulation of IgA within MUC5B-positive luminal mucus plugs, suggesting that luminal SIgA originates from submucosal glands in COPD patients. We found that the concentration of SIgA in BAL is inversely correlated with forced expiratory volume in 1s (FEV1) in COPD, but that the ratio of SIgA/MUC5B is a better predictor of FEV1, particularly in patients with moderate COPD. Together, these findings suggest that SIgA production by submucosal glands, which are expanded in COPD, is insufficient to compensate for reduced SIgA transcytosis by airway epithelial cells. Localized SIgA deficiency on the surface of small airways is associated with COPD progression and represents a potential new therapeutic target in COPD.

A spatial model of fluid recycling in the airways of the lung

The genetic disease cystic fibrosis (CF) is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and results in viscous mucus and impaired mucociliary clearance leading to chronic recurring pulmonary infections. Although extensive experimental research has been conducted over the last few decades, CF lung pathophysiology remains controversial. There are two competing explanations for the observed depletion of periciliary liquid (PCL) in CF lungs. The low volume hypothesis assumes fluid hyperabsorption through surface epithelia due to an over-active epithelial Na+ channel (ENaC), and the low secretion hypothesis assumes inspissated mucins secreted from glands due to lack of serous fluid secreted from gland acini.We present a spatial mathematical model that reflects in vivo fluid recycling via submucosal gland (SMG) secretion, and absorption through surface epithelia. We then test the model in CF conditions by increasing ENaC open probability and decreasing SMG flux while simultaneously reducing CFTR open probability. Increasing ENaC activity only results in increased fluid absorption across surface epithelia, as seen in in vitro experiments. However, combining potential CF mechanisms results in markedly less fluid absorbed while providing the largest reduction in PCL volume, suggesting that a compromise in gland fluid secretion dominates over increased ENaC activity to decrease the amount of fluid transported transcellularly in CF lungs in vivo. Model results also indicate that a spatial model is necessary for an accurate calculation of total fluid transport, as the effects of spatial gradients can be severe, particularly in close proximity to the SMGs.

Neural control of submucosal gland and apical membrane secretions in airways.

The mechanisms that lay behind the low-level secretions from airway submucosal glands and the surface epithelium in the absence of external innervation have been investigated in small areas (1.0–1.5 cm2) of mucosa from sheep tracheas, freshly collected from a local abattoir. Glandular secretion was measured by an optical method while short circuit current was used as a measure of surface secretion. Activation of neurones in the intrinsic nerve net by veratrine alkaloids caused an immediate increase in both glandular secretion and short circuit current, both effects being blocked by the addition of tetrodotoxin. However, agents known to be acting directly on the glands, such as muscarinic agonists (e.g., carbachol) or adenylate cyclase activators (e.g., forskolin) were not influenced by tetrodotoxin. The toxin alone had no discernable effect on the low-level basal secretion shown by unstimulated glands. Calu-3 cell monolayers, generally agreed to be a surrogate for the secretory cells of submucosal glands, showed no sensitivity to veratrine alkaloids, strengthening the view that the veratrine-like drugs acted exclusively on the intrinsic nerve net. The data are discussed in relation way in which transplanted lungs can maintain mucociliary clearance and hence a sterile environment in the absence of external innervation, as in transplanted lungs.

Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea.

In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends on gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho HJ, Joo NS, Wine JJ. Am J Physiol Lung Cell Mol Physiol 299: L124-L136, 2010). Stimulated MCC velocities (mm/min, means ± SE for 10- to 35-min period poststimulation) were as follows: 1 μM carbachol: 19.1 ± 3.3 > 10 μM phenylephrine: 15.3 ± 2.4 ≈ 10 μM isoproterenol: 15.0 ± 1.9 ≈ 10 μM forskolin: 14.6 ± 3.1 > 1 μM vasoactive intestinal peptide (VIP): 10.2 ± 2.2 >> basal (t15): 1.8 ± 0.3; n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70% and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3 (-)-free solutions did not significantly inhibit MCC to either intracellular Ca(2+) concentration or intracellular cAMP concentration ([cAMP]i)-elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion, and bumetanide almost completely inhibited [cAMP]i-stimulated MCC while it had a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this.

The cytokines interleukin-1β and tumor necrosis factor-α stimulate CFTR-mediated fluid secretion by swine airway submucosal glands

The airway is kept sterile by an efficient innate defense mechanism. The cornerstone of airway defense is mucus containing diverse antimicrobial factors that kill or inactivate pathogens. Most of the mucus in the upper airways is secreted by airway submucosal glands. In patients with cystic fibrosis (CF), airway defense fails and the lungs are colonized by bacteria, usually Pseudomonas aeruginosa. Accumulating evidence suggests that airway submucosal glands contribute to CF pathogenesis by failing to respond appropriately to inhalation of bacteria. However, the regulation of submucosal glands by the innate immune system remains poorly understood. We studied the response of submucosal glands to the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α. These are released into the airway submucosa in response to infection with the bacterium P. aeruginosa and are elevated in CF airways. Stimulation with IL-1β and TNF-α increased submucosal gland secretion in a concentration-dependent manner with a maximal secretion rate of 240 ± 20 and 190 ± 40 pl/min, respectively. The half maximal effective concentrations were 11 and 20 ng/ml, respectively. The cytokine effect was dependent on cAMP but was independent of cGMP, nitric oxide, Ca(2+), or p38 MAP kinase. Most importantly, IL-1β- and TNF-α-stimulated secretion was blocked by the CF transmembrane conductance regulator (CFTR) blocker, CFTRinh172 (100 μmol/l) but was not affected by the Ca(2+)-activated Cl(-) channel blocker, niflumic acid (1 μmol/l). The data suggest, that during bacterial infections and resulting release of proinflammatory cytokines, the glands are stimulated to secrete fluid, and this response is mediated by cAMP-activated CFTR, a process that would fail in patients with CF.

Protease-activated receptor 2–dependent fluid secretion from airway submucosal glands by house dust mite extract

The submucosal gland (SMG) is important in the control of airway surface fluid. Protease-activated receptor (PAR) 2 contributes to the pathophysiology of allergies in response to nonspecific allergens bearing proteases and anion secretion. House dust mites (HDMs) have abundant proteases that can activate PAR2, but little is known about the direct effect of HDM on SMG secretion. The aim of this study was to investigate the effect of HDMs on glandular secretion and its mechanism in allergic patients, patients with chronic rhinosinusitis (CRS), or both. Inferior nasal turbinates were harvested from 55 patients and classified into 4 groups (the control, allergic rhinitis [AR], CRS, and AR+CRS groups). Amicroscope attached to a digital camera was used to quantify mucus bubbles from individual SMGs while stimulated with HDM extract, PAR2-activating peptide, and carbachol. PAR2 expression in the SMGwas determined by means of immunostaining with anti-PAR2 mAb. HDM induced a significantly higher secretion rate and number of responding glands in the AR and AR+CRS groups than in the control group. Interestingly, patients in the CRS group, who had no HDM-specific IgE antibody, showed a higher response than the control group, and its response was suppressed by a PAR2-selective antagonist. The responses to PAR2-activating peptide were similar to those to HDM, and their secretion rates positively correlated with HDM responses. PAR2 was highly expressed in all 3 disease groups with immunostaining. HDM allergens can induce glandular secretion in patients with AR, CRS, or both, and PAR2 represents a possible mechanism for nonspecific hyperreactivity in inflammatory airway diseases.

Small‐molecule activators of TMEM16A, a calcium‐activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction

TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca(2+). By patch-clamp, N-aroylaminothiazole "activators" (E(act)) strongly increased Cl(-) current at 0 Ca(2+), whereas tetrazolylbenzamide "potentiators" (F(act)) were not active at 0 Ca(2+) but reduced the EC(50) for Ca(2+)-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (E(act)) and potentiator (F(act)) produced large and more sustained CaCC Cl(-) currents than general agonists of Ca(2+) signaling, with EC(50) 3-6 μM and Cl(-) conductance comparable to that induced transiently by Ca(2+)-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl(-) conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.

House Dust Mite Nonspecifically Induces Fluid Secretion from Nasal Glands

Objective: Investigate the effects of the house dust mite (HDM) on fluid secretion in submucosal glands in nasal tissue and to compare its effects in control and diseased groups. In addition, we looked to see if PAR2 was involved in the HDM-induced fluid response. Method: Inferior nasal turbinate tissue was harvested from 26 patients with either allergic rhinitis (with HDM-specific IgE) and/or chronic rhinosinusitis, and eight controls. We used a microscope attached to a camera to quantify mucus bubbles secreted from individual submucosal glands in response to HDM extract and PAR2-AP (activating peptide). Results: HDM stimulated fluid secretion, and the diseased group showed a higher secretion rate than did the controls. Interestingly, there was no significant difference between the three diseased groups, and CRS patients without HDM-specific IgE were also stimulated by HDM. The nonspecific response induced by HDM was hypothesized to be through PAR2. The expression of PAR2 was predominant on the basolateral side of gland acini. HDM-induced secretion rates positively correlated with PAR2-AP-induced secretion rates. Carbachol responses and gland density were not different among the three groups. Conclusion: These findings suggest that HDM induces an IgE-independent nonspecific response from nasal submucosal glands, generating fluid secretion in allergic or infective nasal tissue, and PAR2 is at least partially involved in this process.

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.

Lubiprostone targets prostanoid EP4 receptors in ovine airways

Lubiprostone, a prostaglandin E₁ derivative, is reported to activate ClC-2 chloride channels located in the apical membranes of a number of transporting epithelia. Lack of functioning CFTR chloride channels in epithelia is responsible for the genetic disease cystic fibrosis, therefore, surrogate channels that can operate independently of CFTR are of interest. This study explores the target receptor(s) for lubiprostone in airway epithelium. All experiments were performed on the ventral tracheal epithelium of sheep. Epithelia were used to measure anion secretion from the apical surface as short circuit current or as fluid secretion from individual airway submucosal glands, using an optical method. The EP₄ antagonists L-161982 and GW627368 inhibited short circuit current responses to lubiprostone, while EP₁(,)₂(&)₃ receptor antagonists were without effect. Similarly, lubiprostone induced secretion in airway submucosal glands was inhibited by L-161982. L-161982 effectively competed with lubiprostone with a K(d) value of 0.058 µM, close to its value for binding to human EP₄ receptors (0.024 µM). The selective EP₄ agonist L-902688 and lubiprostone behaved similarly with respect to EP₄ receptor antagonists. Results of experiments with H89, a protein kinase A inhibitor, were consistent with lubiprostone acting through a G(s) -protein coupled EP₄ receptor/cAMP cascade. Lubiprostone-induced short-circuit currents and submucosal gland secretions were inhibited by selective EP₄ receptor antagonists. The results suggest EP₄ receptor activation by lubiprostone triggers cAMP production necessary for CFTR activation and the secretory responses, a possibility precluded in CF tissues.

CAMP-activated Ca2+ signaling is required for CFTR-mediated serous cell fluid secretion in porcine and human airways

Cystic fibrosis (CF), which is caused by mutations in CFTR, affects many tissues, including the lung. Submucosal gland serous acinar cells are primary sites of fluid secretion and CFTR expression in the lung. Absence of CFTR in these cells may contribute to CF lung pathogenesis by disrupting fluid secretion. Here, we have isolated primary serous acinar cells from wild-type and CFTR-/- pigs and humans without CF to investigate the cellular mechanisms and regulation of fluid secretion by optical imaging. Porcine and human serous cells secrete fluid in response to vasoactive intestinal polypeptide (VIP) and other agents that raise intracellular cAMP levels; here, we have demonstrated that this requires CFTR and a cAMP-dependent rise in intracellular Ca2+ concentration ([Ca2+]i). Importantly, cAMP induced the release of Ca2+ from InsP3-sensitive Ca2+ stores also responsive to cAMP-independent agonists such as cholinergic, histaminergic, and purinergic agonists that stimulate CFTR-independent fluid secretion. This provides two types of synergism that strongly potentiated cAMP-mediated fluid secretion but differed in their CFTR dependencies. First, CFTR-dependent secretion was strongly potentiated by low VIP and carbachol concentrations that individually were unable to stimulate secretion. Second, higher VIP concentrations more strongly potentiated the [Ca2+]i responses, enabling ineffectual levels of cholinergic stimulation to strongly activate CFTR-independent fluid secretion. These results identify important molecular mechanisms of cAMP-dependent secretion, including a requirement for Ca2+ signaling, and suggest new therapeutic approaches to correct defective submucosal gland secretion in CF.