Airway Surface Liquid Height Research Articles

Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways.

In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Lipoxin A4 prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells by Pseudomonas aeruginosa.

The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance.

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

Mucociliary dysfunction in HIV and smoked substance abuse.

Impaired mucociliary clearance (MCC) is a hallmark of acquired chronic airway diseases like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD) and asthma. This manifests as microbial colonization of the lung consequently leading to recurrent respiratory infections. People living with HIV demonstrate increased incidence of these chronic airway diseases. Bacterial pneumonia continues to be an important comorbidity in people living with HIV even though anti-retroviral therapy has succeeded in restoring CD4+ cell counts. People living with HIV demonstrate increased microbial colonization of the lower airways. The microbial flora is similar to that observed in diseases like cystic fibrosis and COPD suggesting that mucociliary dysfunction could be a contributing factor to the increased incidence of chronic airway diseases in people living with HIV. The three principal components of the MCC apparatus are, a mucus layer, ciliary beating, and a periciliary airway surface liquid (ASL) layer that facilitates ciliary beating. Cystic fibrosis transmembrane conductance regulator (CFTR) plays a pivotal role in regulating the periciliary ASL. HIV proteins can suppress all the components of the MCC apparatus by increasing mucus secretion and suppressing CFTR function. This can decrease ASL height leading to suppressed ciliary beating. The effects of HIV on MCC are exacerbated when combined with other aggravating factors like smoking or inhaled substance abuse, which by themselves can suppress one or more components of the MCC system. This review discusses the pathophysiological mechanisms that lead to MCC suppression in people living with HIV who also smoke tobacco or abuse illicit drugs.

Development of Confocal Reflection Microscopy for the Study of Airway Surface Liquid Dysregulation in Cystic Fibrosis

In cystic fibrosis(CF) patients and βENaC-overexpressing mice, impaired airway epithelial ion transport produces airway surface liquid(ASL) depletion, leading to mucociliary dysfunction, mucus obstruction, chronic airway inflammation and infection. ASL homeostasis can be studied by measuring ASL height in primary epithelial cultures from murine and human airways. The height can be determined using confocal fluorescence microscopy; however the current protocol requires fluorescently labelled liquid addition to the apical side of the epithelium resulting in nonphysiological conditions.

NOVOSTI V OBRAVNAVI PLJUČNE BOLEZNI PRI OTROCIH IN MLADOSTNIKIH S CISTIČNO FIBROZO

Cystic fibrosis (CF) is a multi-organ disease, affecting mostly lungs and gastrointestinal tract. Data from patient registries show that the survival of patients with CF has progressively improved over the past several decades, as a result of advances in antibiotic treatment, supplementation of pancreatic enzymes, better nutrition and a holistic approach to treatment in CF centres.The purpose of this review is to survey recent developments in the treatment of lung disease in children and adolescents with CF.We describe newborn screening for CF.When chronic respiratory insufficiency occurs, lung transplantation becomes a very important issue.Lung disease is the most common cause of morbidity and mortality in CF patients. Emerging new therapies are targeted at all points in the pathogenesis of lung disease, from drugs that treat infection and inflammation in the airways to gene transfer studies and to drugs that augment airway surface liquid height. A number of antibacterial agents formulated for inhalation are at various stages of study and there are several anti-inflammatory candidate drugs in clinical trials. The most important development in the recent years is modulation of the abnormal protein that causes CF, the cystic fibrosis transmembrane regulator (CFTR), where drugs are targeted at specific defects in the transcription, processing or functioning.When chronic respiratory insufficiency occurs, lung transplantation becomes a very important issue. The role of the CF nurse, who has responsibilities in educating and teaching clinical skills to patients and families, is described.

Na+ transport by small airway surface epithelia

BackgroundMaintaining an adequate level of Airway Surface Liquid (ASL) is crucial for the normal functioning of the immune system of the lungs. A balance between the secretion and the absorption of ions and fluid by the airways surface epithelia (ASE) ensures proper amount of ASL for mucociliar clearance. ASE expresses Epithelial Na+ Channel (ENaC) and the Cl‐ channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Currently, there are two opposing hypotheses to explain how the interplay between CFTR and ENaC activity regulate ASL height. One theory proposes that each surface epithelial cell expresses both CFTR and ENaC and is able to secrete or reabsorb fluid and ions. A recent hypothesis proposes that CFTR and ENaC are expressed in different groups of cells. Cells within the pleats of ASE express CFTR and are mainly secretory while the cells of the folds express ENaC and are principally absorptive.QuestionAre the cells of ASE able to transport ions in both directions?MethodsTo detect transepithelial Na+ flux we used Scanning Ion Selective Techniquethe, which measures ion concentration gradient generated by the epithelia using ion selective microelectrodes.We investigated the ion transport in the folds of the swine distal airways under normal Kreb's solution and in the presence of CFTR and ENaC modulators.ResultsASE showed spontaneous basal Na+ transport from the lumen into the tissue. Adding the sodium azide blocked the basal secretion. Stimulation with forskolin or thapsigargin triggered rapid and reversal Na+ transport and was blocked by treatment with the CFTR blocker.ConclusionsThe epithelial cells of the fold regions of ASE are able to secret and absorb Na+. Our results allow us to reject the hypothesis of the monodirectional Na+ transportation in the surface epithelia.

Roflumilast combined with adenosine increases mucosal hydration in human airway epithelial cultures after cigarette smoke exposure.

Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Recent studies have shown that cigarette smoke (CS) induces cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, which leads to airway-surface liquid (ASL) dehydration. This in turn contributes to the mucus dehydration and impaired mucociliary clearance that are seen in the chronic bronchitis form of COPD. Roflumilast is a phosphodiesterase 4 inhibitor that may improve lung function and reduce the frequency of exacerbations in patients with COPD. Although roflumilast can affect cAMP metabolism, little is known about the downstream pharmacological effects in the airways. We hypothesized that roflumilast would increase ASL rehydration in human bronchial epithelial cultures (HBECs) after chronic CS exposure. cAMP production was measured by Förster resonance energy transfer in HEK293T cells and by ELISA in HBECs. ASL height was measured by xz-confocal microscopy after air exposure or following HBEC exposure to freshly produced CS. Roflumilast had little effect on cAMP or ASL height when applied on its own; however, roflumilast significantly potentiated adenosine-induced increases in cAMP and ASL height in CS-exposed HBECs. Roflumilast increased the rate of ASL height recovery in cultures after CS exposure compared with controls. In contrast, the β2-adrenergic receptor agonists isoproterenol and salmeterol failed to increase ASL height after CS exposure. Our data suggest that roflumilast can increase ASL hydration in CS-exposed HBECs, which is predicted to be beneficial for the treatment of mucus dehydration/mucus stasis in patients with COPD chronic bronchitis.

Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition.

Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by ∼ 50% (0.052 μM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 μg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 μg/kg (i.t.) and 75 μg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.

Effects of airway surface liquid height on the kinetics of extracellular nucleotides in airway epithelia

Experimental techniques aimed at measuring the concentration of signaling molecules in the airway surface liquid (ASL) often require an unrealistically large ASL volume to facilitate sampling. This experimental limitation, prompted by the difficulty of pipetting liquid from a very shallow layer (~15μm), leads to dilution and the under-prediction of physiologic concentrations of signaling molecules that are vital to the regulation of mucociliary clearance. Here, we use a computational model to describe the effect of liquid height on the kinetics of extracellular nucleotides in the airway surface liquid coating respiratory epithelia. The model consists of a reaction–diffusion equation with boundary conditions that represent the enzymatic reactions occurring on the epithelial surface. The simulations reproduce successfully the kinetics of extracellular ATP following hypotonic challenge for ASL volumes ranging from 25μl to 500μl in a 12-mm diameter cell culture. The model reveals that [ATP] and [ADO] reach 1200nM and 2200nM at the epithelial surface, respectively, while their volumetric averages remain less than 200nM at all times in experiments with a large ASL volume (500μl). These findings imply that activation of P2Y2 and A2B receptors is robust after hypotonic challenge, in contrast to what could be concluded based on experimental measurements of volumetric concentrations in large ASL volumes. Finally, given the central role that ATP and ADO play in regulating mucociliary clearance, we investigated which enzymes, when inhibited, provide the greatest increase in ATP and ADO concentrations. Our findings suggest that inhibition of NTPDase1/highTNAP would cause the greatest increase in [ATP] after hypotonic challenge, while inhibition of the transporter CNT3 would provide the greatest increase in [ADO].

Physiological levels of lipoxin A4 inhibit ENaC and restore airway surface liquid height in cystic fibrosis bronchial epithelium.

In cystic fibrosis (CF), the airway surface liquid (ASL) is depleted. We previously demonstrated that lipoxin A4 (LXA4) can modulate ASL height (ASLh) through actions on Cl− transport. Here, we report novel effects of lipoxin on the epithelial Na+ channel ENaC in this response. ASL dynamics and ion transport were studied using live‐cell confocal microscopy and short‐circuit current measurements in CF (CuFi‐1) and non‐CF (NuLi‐1) cell cultures. Low physiological concentrations of LXA4 in the picomolar range produced an increase in ASLh which was dependent on inhibition of an amiloride‐sensitive Na+ current and stimulation of a bumetanide‐sensitive Cl− current. These ion transport and ASLh responses to LXA4 were blocked by Boc‐2 an inhibitor of the specific LXA4 receptor ALX/FPR2. LXA4 affected the subcellular localization of its receptor and enhanced the localization of ALX/FPR2 at the apical membrane of CF cells. Our results provide evidence for a novel effect of low physiological concentrations of LXA4 to inhibit airway epithelial Na+ absorption that results in an ASL height increase in CF airway epithelia.

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

BackgroundThe 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.MethodsExpression 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.ResultsWe 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.ConclusionsCFTR 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.

In vitro studies of a distillate of rectified essential oils on sinonasal components of mucociliary clearance.

Herbal remedies predate written history and continue to be used frequently for many common ailments. The essential oil mixture standardized is a phytopharmaceutical with a distillate of a mixture of rectified essential oils of eucalyptus, sweet orange, myrtle, and lemon as active ingredients used to treat respiratory diseases such as bronchitis and rhinosinusitis. We evaluated the pharmacologic effects of a distillate of rectified essential oils standardized on primary human upper respiratory epithelial cultures specifically addressing electrolyte transport, cilia beat frequency (CBF), airway surface liquid (ASL) hydration, and mucus transport velocity. Well-differentiated primary human sinonasal epithelial cultures grown at an air-liquid interface were treated on the apical or basolateral surface with varying concentrations of a distillate of rectified essential oils standardized. Changes in CBF were determined using the Sissons-Ammons Video Analysis system while changes in chloride flux were determined using the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. ASL hydration was quantified using Texas red dextran and mucociliary transport velocity was measured using fluorescent microspheres and time lapse photography. When applied to the basolateral surface, a distillate of rectified essential oils standardized activated chloride efflux and ciliary beat in a dose-dependent fashion, increasing ASL height and accelerating mucociliary transport velocity. The ancillary apical application of a distillate of rectified essential oils standardized had minimal effects on the CBF. Basolateral application of a distillate of rectified essential oils standardized stimulates both chloride efflux and cilia beat frequency resulting in a synergistic effect dramatically augmenting mucociliary transport velocity. These in vitro data support the clinical efficacy of this phytopharmaceutical in respiratory inflammatory disorders.

Activation of P2RY11 and ATP Release by Lipoxin A4 Restores the Airway Surface Liquid Layer and Epithelial Repair in Cystic Fibrosis

In cystic fibrosis (CF), the airway surface liquid (ASL) height is reduced as a result of impaired ion transport, which favors bacterial colonization and inflammation of the airway and leads to progressive lung destruction. Lipoxin (LX)A4, which promotes resolution of inflammation, is inadequately produced in the airways of patients with CF. We previously demonstrated that LXA4 stimulates an ASL height increase and epithelial repair. Here we report the molecular mechanisms involved in these processes. We found that LXA4 (1 nM) induced an apical ATP release from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and CF primary cultures. The ATP release induced by LXA4 was completely inhibited by antagonists of the ALX/FPR2 receptor and Pannexin-1 channels. LXA4 induced an increase in intracellular cAMP and calcium, which were abolished by the selective inhibition of the P2RY11 purinoreceptor. Pannexin-1 and ATP hydrolysis inhibition and P2RY11 purinoreceptor knockdown all abolished the increase of ASL height induced by LXA4. Inhibition of the A2b adenosine receptor did not affect the ASL height increase induced by LXA4, whereas the PKA inhibitor partially inhibited this response. The stimulation of NuLi-1 and CuFi-1 cell proliferation, migration, and wound repair by LXA4 was inhibited by the antagonists of Pannexin-1 channel and P2RY11 purinoreceptor. Taken together, our results provide evidence for a novel role of LXA4 in stimulating apical ATP secretion via Pannexin-1 channels and P2RY11 purinoreceptors activation leading to an ASL height increase and epithelial repair.

Non-Genomic Estrogen Regulation of Ion Transport and Airway Surface Liquid Dynamics in Cystic Fibrosis Bronchial Epithelium

Male cystic fibrosis (CF) patients survive longer than females and lung exacerbations in CF females vary during the estrous cycle. Estrogen has been reported to reduce the height of the airway surface liquid (ASL) in female CF bronchial epithelium. Here we investigated the effect of 17β-estradiol on the airway surface liquid height and ion transport in normal (NuLi-1) and CF (CuFi-1) bronchial epithelial monolayers. Live cell imaging using confocal microscopy revealed that airway surface liquid height was significantly higher in the non-CF cells compared to the CF cells. 17β-estradiol (0.1–10 nM) reduced the airway surface liquid height in non-CF and CF cells after 30 min treatment. Treatment with the nuclear-impeded Estrogen Dendrimer Conjugate mimicked the effect of free estrogen by reducing significantly the airway surface liquid height in CF and non-CF cells. Inhibition of chloride transport or basolateral potassium recycling decreased the airway surface liquid height and 17β-estradiol had no additive effect in the presence of these ion transporter inhibitors. 17β-estradiol decreased bumetanide-sensitive transepithelial short-circuit current in non-CF cells and prevented the forskolin-induced increase in ASL height. 17β-estradiol stimulated an amiloride-sensitive transepithelial current and increased ouabain-sensitive basolateral short-circuit current in CF cells. 17β-estradiol increased PKCδ activity in CF and non-CF cells. These results demonstrate that estrogen dehydrates CF and non-CF ASL, and these responses to 17β-estradiol are non-genomic rather than involving the classical nuclear estrogen receptor pathway. 17β-estradiol acts on the airway surface liquid by inhibiting cAMP-mediated chloride secretion in non-CF cells and increasing sodium absorption via the stimulation of PKCδ, ENaC and the Na+/K+ATPase in CF cells.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion

Mucociliary clearance (MCC) is the primary physical airway defense against inhaled pathogens and particulates. MCC depends on both proper fluid/mucus homeostasis and epithelial ciliary beating. Vasoactive intestinal peptide (VIP) is a neurotransmitter expressed in the sinonasal epithelium that is up-regulated in allergy. However, the effects of VIP on human sinonasal physiology are unknown, as are VIP's interactions with histamine, a major regulator of allergic disease. We imaged ciliary beat frequency, mucociliary transport, apical Cl(-) permeability, and airway surface liquid (ASL) height in primary human sinonasal air-liquid-interface cultures to investigate the effects of VIP and histamine. VIP stimulated an increase in ciliary beat frequency (EC50 0.5 μM; maximal increase ∼40% compared with control) and cystic fibrosis transmembrane conductance regulator (CFTR)-dependent and Na(+)K(+)2Cl(-) cotransporter-dependent fluid secretion, all requiring cAMP/PKA signaling. Histamine activated Ca(2+) signaling that increased ASL height but not ciliary beating. Low concentrations of VIP and histamine had synergistic effects on CFTR-dependent fluid secretion, revealed by increased ASL heights. An up-regulation of VIP in histamine-driven allergic rhinitis would likely enhance mucosal fluid secretion and contribute to allergic rhinorrhea. Conversely, a loss of VIP-activated secretion in patients with CF may impair mucociliary transport, contributing to increased incidences of sinonasal infections and rhinosinusitis.

Nerve growth factor reduces amiloride‐sensitive Na+ transport in human airway epithelial cells

Nerve growth factor (NGF) is over‐expressed in patients with lung inflammatory diseases including airway virus infections. Optimum airway surface liquid (ASL), controlled by epithelial cells, is essential for normal lung function. NGF has been shown to affect ion transport properties of neurons, but no information is available as to the effect NGF has on ion transport of airway epithelial cells. To investigate if NGF can alter epithelial ion transport, we established human primary cultured epithelial cells in air‐interface that demonstrates desirable in situ morphology and ion transport function. These cells were placed in an Ussing chamber to obtain transepithelial voltage (Vt), short‐circuit current (Isc), transepithelial resistance (Rt), and to measure responses to known ion transport inhibitors. Cells had an average Vt of −7.1 mV, Isc of 5.9 μA, and Rt of 750 μΩ×cm2. Amiloride (apical, 3.5×10−5 M) decreased total Isc by 52%. When cells were first treated with 1 ng/ml NGF prior to the addition of amiloride, amiloride reduced total Isc by 38%. The addition of NGF (apical, 1 ng/ml) to the bath reduced the total Isc by 4.5 %. These results indicate that NGF reduces Na+ transport in airway epithelial cells. This could result in an increase in ASL height. Support provided by NIOSH.

Retraction

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl− secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA4 is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA4 are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA4 produced a rapid and transient increase in intracellular Ca2+. We have investigated, the effect of LXA4 on Cl− secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA4 stimulated a rapid intracellular Ca2+ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA4 stimulated whole-cell Cl− currents which were inhibited by NPPB (calcium-activated Cl− channel inhibitor), BAPTA-AM (chelator of intracellular Ca2+) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA4 increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA4 effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl− secretion. The LXA4 stimulation of intracellular Ca2+, whole-cell Cl− currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA4 in the stimulation of intracellular Ca2+ signalling leading to Ca2+-activated Cl− secretion and enhanced ASL height in non-CF and CF bronchial epithelia.