Cigarette smoke and calcium conspire to impair CFTR function in airway epithelia

To maintain health and function in response to inhaled environmental irritants and toxins, the lungs and airways depend upon an innate defense system that involves the secretion of mucus (i.e., mucin, salts, and water) by airway epithelium onto the apical surface to trap foreign particles. Airway mucus is then transported in an oral direction via ciliary beating and coughing, which helps to keep the airways clear. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated Cl^- channel in the apical membrane of epithelium that contributes to salt and water secretion onto the luminal surface of airways, thereby ensuring that secreted mucus is sufficiently hydrated for movement along the epithelial surface. Dehydration of airway mucus, as occurs in cystic fibrosis, results in a more viscous, less mobile secretion that compromises the lung’s innate defense system by facilitating a build-up of foreign particles and bacterial growth. Related to this situation is chronic obstructive pulmonary disease (COPD), which is a leading cause of death globally. A major cause of COPD is cigarette smoking, which has been reported to decrease the cellular levels of CFTR in airway epithelia. In their recent article, Rasmussen and coworkers now report that exposure to cigarette smoke elevates cytosolic free Ca²⁺ in airway epithelium, leading to decreased surface localization and cellular expression of CFTR and reduced levels of secreted airway surface liquid. Blocking this increase in cytosolic Ca²⁺ largely prevented CFTR loss in airway epithelium and surprisingly, cellular lysosomes appear to be a major source for smoke-induced Ca²⁺ elevation.     

Cigarette Smoke-induced Ca2+ Release Leads to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Dysfunction

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca²⁺ that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca²⁺, but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca²⁺ release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca²⁺ and CFTR internalization. We hypothesize that CS-induced Ca²⁺ release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Ca signaling and fluid secretion by secretory cells of the airway epithelium

Cytoplasmic Ca²⁺ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca²⁺ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca²⁺. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca²⁺ as a second messenger. Changes in intracellular Ca²⁺ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca²⁺-activated K⁺ channels and Cl⁻ channels. We also review evidence of interactions of Ca²⁺ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca²⁺ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.     

CFTR-Adenylyl Cyclase I Association Responsible for UTP Activation of CFTR in Well-Differentiated Primary Human Bronchial Cell Cultures

Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca²⁺-activated chloride channel (CaCC) is activated by Ca²⁺ agonists like UTP. We found that most chloride current elicited by Ca²⁺ agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca²⁺ activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca²⁺ agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca²⁺ agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca²⁺/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1–CFTR association is responsible for Ca²⁺/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca²⁺ agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.     

Innate immune response in CF airway epithelia: hyperinflammatory?

The lack of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the apical membranes of CF airway epithelial cells abolishes cAMP-stimulated anion transport, and bacteria, eventually including Pseudomonas aeruginosa, bind to and accumulate in the mucus. Flagellin released from P. aeruginosa triggers airway epithelial Toll-like receptor 5 and subsequent NF-B signaling and production and release of proinflammatory cytokines that recruit neutrophils to the infected region. This response has been termed hyperinflammatory because so many neutrophils accumulate; a response that damages CF lung tissue. We first review the contradictory data both for and against the idea that epithelial cells exhibit larger-than-normal proinflammatory signaling in CF compared with non-CF cells and then review proposals that might explain how reduced CFTR function could activate such proinflammatory signaling. It is concluded that apparent exaggerated innate immune response of CF airway epithelial cells may have resulted not from direct effects of CFTR on cellular signaling or inflammatory mediator production but from indirect effects resulting from the absence of CFTRs apical membrane channel function. Thus, loss of Cl^–, HCO₃^–, and glutathione secretion may lead to reduced volume and increased acidification and oxidation of the airway surface liquid. These changes concentrate proinflammatory mediators, reduce mucociliary clearance of bacteria and subsequently activate cellular signaling. Loss of apical CFTR will also hyperpolarize basolateral membrane potentials, potentially leading to increases in cytosolic [Ca²⁺], intracellular Ca²⁺, and NF-B signaling. This hyperinflammatory effect of CF on intracellular Ca²⁺ and NF-B signaling would be most prominently expressed during exposure to both P. aeruginosa and also endocrine, paracrine, or nervous agonists that activate Ca²⁺ signaling in the airway epithelia. Pseudomonas aeruginosa; Toll-like receptor; NF-B; oxidative stress; acidic airway surface liquid; calcium     

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.     

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism.     

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.     

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Background

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods

Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results

We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions

CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.     

Upregulation of TMEM16A Protein in Bronchial Epithelial Cells by Bacterial Pyocyanin

Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca²⁺-activated Cl^- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca²⁺-dependent Cl^- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways.     

The TMEM16A chloride channel as an alternative therapeutic target in cystic fibrosis

Cystic fibrosis (CF), a multiorgan genetic disease, is caused by loss of function of CFTR, a cAMP-regulated anion channel. In CF airway epithelia, defective Cl⁻ and bicarbonate secretion impairs mucociliary clearance and other innate defense mechanisms, favoring the colonization of the lungs by highly virulent bacteria. The airway epithelium expresses TMEM16A, a second type of Cl⁻ channel that is activated by cytosolic Ca²⁺. TMEM16A is particularly expressed in goblet cells. This specific localization could be important in the release and hydration of mucins. Activation of TMEM16A with pharmacological agents could circumvent the primary defect in CF. This strategy needs to be carefully designed and tested to avoid possible undesired effects due to the expression of TMEM16A in other cell types such as bronchial smooth muscle cells.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca²⁺ ([Ca²⁺]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca²⁺ regulatory proteins leading to increased store operated Ca²⁺ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca²⁺]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca²⁺]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca²⁺ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.     

Acinar origin of CFTR-dependent airway submucosal gland fluid secretion

Cystic fibrosis (CF) airway disease arises from defective innate defenses, especially defective mucus clearance of microorganisms. Airway submucosal glands secrete most airway mucus, and CF airway glands do not secrete in response to VIP or forskolin. CFTR, the protein that is defective in CF, is expressed in glands, but immunocytochemistry finds the highest expression of CFTR in either the ciliated ducts or in the acini, depending on the antibodies used. CFTR is absolutely required for forskolin-mediated gland secretion; we used this finding to localize the origin of forskolin-stimulated, CFTR-dependent gland fluid secretion. We tested the hypothesis that secretion to forskolin might originate from the gland duct rather than or in addition to the acini. We ligated gland ducts at various points, stimulated the glands with forskolin, and monitored the regions of the glands that swelled. The results supported an acinar rather than ductal origin of secretion. We tracked particles in the mucus using Nomarski time-lapse imaging; particles originated in the acini and traveled toward the duct orifice. Estimated bulk flow accelerated in the acini and mucus tubules, consistent with fluid secretion in those regions, but was constant in the unbranched duct, consistent with a lack of fluid secretion or absorption by the ductal epithelium. We conclude that CFTR-dependent gland fluid secretion originates in the serous acini. The failure to observe either secretion or absorption from the CFTR and epithelial Na(+) channel (ENaC)-rich ciliated ducts is unexplained, but may indicate that this epithelium alters the composition rather than the volume of gland mucus.     

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.     

Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl(-) channel. Its dysfunction limits Cl(-) secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl(-) channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca(2+) activate the epithelial CaCCs, which provides an alternative Cl(-) secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl(-) secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl(-) secretion in CF via a pathway independent of CFTR.     

Airway epithelial cells-Functional links between CFTR and anoctamin dependent Cl(-) secretion

Airways consist of a heterogeneous population of cells, comprising ciliated cells, Clara cells and goblet cells. Electrolyte secretion by the airways is necessary to produce the airway surface liquid that allows for mucociliary clearance of the lungs. Secretion is driven by opening of Cl(-) selective ion channels in the apical membrane of airway epithelial cells, through either receptor mediated increase in intracellular cAMP or cytosolic Ca(2+). Traditionally cAMP-dependent and Ca(2+)-dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1) and with detailed knowledge of the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR), it has become possible to look more closely into this relationship.