Airway Submucosal Gland Cells Research Articles

205 Airway submucosal gland cell biology, physiology, and pathophysiology in cystic fibrosis

205 Airway submucosal gland cell biology, physiology, and pathophysiology in cystic fibrosis

IRBIT as a Regulator of Bicarbonate Transport in the Small Intestine

BackgroundSmall intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5‐trisphosphate (IP3) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown.AimTo examine duodenal expression and function of IRBIT in regulating bicarbonate transport.MethodsDuodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical‐in or flipped apical‐out enteroids. mRNA expression was evaluated using qPCR and re‐analysis of a duodenal sc‐RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back‐titration.ResultsIRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5‐fold higher expression than Calu‐3 airway submucosal gland cells or HEK293 kidney‐derived cells (n=1 each). Analysis of duodenal sc‐RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt‐villus axis, primarily localizing in E‐cadherin positive epithelial cells, but also present in the lamina propria. In patient‐derived duodenal enteroids, IRBIT was expressed in both crypt‐like (undifferentiated) and villus‐like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc‐RNAseq analysis, AHCYL1 and SLC26A3 were co‐expressed in 38% of all villus cells, compared to only 5% co‐expressing AHCYL1 and SLC26A6. CFTR‐independent duodenal bicarbonate secretion stimulated by linaclotide (10‐7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10‐6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh‐A250, 10‐5 M; 68±8%, n=10). Linaclotide stimulation of apical‐out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking.Summary and ConclusionsIRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition.

S-allylmercapto-l-cysteine modulates MUC5AC and AQP5 secretions in a COPD model via NF-кB signaling pathway

Garlic has shown versatile medicinal activities in the prevention and treatment of diseases such as chronic obstructive pulmonary disease (COPD). However, no individual garlic bioactive components have yet been determined in the COPD treatment effects. In this work, S-allylmercapto-l-cysteine (SAMC) identified in the aged garlic was selected as a model compound to determine its COPD therapeutic potential. The COPD model was established by using lipopolysaccharides (LPS) to stimulate the human airway submucosal gland cell line SPC-A1. Previous studies show that both MUC5AC up-regulation and AQP5 down-regulation play an important role in viscous COPD mucus secretions. The modulation effects of SAMC on LPS-induced MUC5AC and AQP5 productions in SPC-A1 cells were then evaluated. Pretreatment of the SPC-A1 cells with SAMC attenuated MUC5AC secretion and increased AQP5 expression in a dose-dependent manner in the non-cytotoxic concentration range of 20 to 100μM. Mechanistic studies suggested that SAMC could suppress the accumulation of MUC5AC mRNA and inhibit IкBα degradation and NF-кB p65 translocation. These results suggest that SAMC could be a promising candidate in the prevention and treatment of MUC5AC-associated disorders such as COPD.

Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non‐cystic fibrosis epithelia

The lantibiotic duramycin (Moli1901, Lancovutide) has been suggested as a drug of choice in the treatment for cystic fibrosis (CF). It has been proposed that duramycin may stimulate chloride secretion through Ca²(+) -activated Cl⁻ channels (CaCC). We investigated whether duramycin exhibited any effect on Cl⁻ efflux and intracellular Ca²(+) concentration ([Ca²(+)](i)) in CF and non-CF epithelial cells. Duramycin did stimulate Cl⁻ efflux from CF bronchial epithelial cells (CFBE) in a narrow concentration range (around 1 μM). However, 100 and 250 μM of duramycin inhibited Cl⁻ efflux from CFBE cells. An inhibitor of the CF transmembrane conductance regulator (CFTR(inh)₋₁₇₂) and a blocker of the capacitative Ca²(+) entry, gadolinium chloride, inhibited the duramycin-induced Cl⁻ efflux. No effect on Cl⁻ efflux was observed in non-CF human bronchial epithelial cells (16HBE), human airway submucosal gland cell line, human pancreatic epithelial cells, CF airway submucosal gland epithelial cells, and CF pancreatic cells. The [Ca²(+)](i) was increased by 3 μM duramycin in 16HBE cells, but decreased after 1, and 3 μM of duramycin in CFBE cells. The results suggest that the mechanism responsible for the stimulation of Cl⁻ efflux by duramycin is mainly related to unspecific changes of the cell membrane or its components rather than to effects on CaCC.

Aquaporin 5 expression inhibited by LPS via p38/JNK signaling pathways in SPC-A1 cells

Proper H(2)O to mucin ratio of airway mucus is important for mucociliary clearance. Recent studies suggest that decreased aquaporin 5 (AQP5) is correlated with increased staining of MUC5AC in submucosal glands of COPD patients. Lipopolysaccharide (LPS) is one of the major insults in airway mucin secretion in COPD. In this study, changes in both AQP5 and MUC5AC expression levels in SPC-A1, a human airway submucosal gland cell line, were quantified after exposure of the cells to LPS. AQP5 transcription and protein expression were decreased while MUC5AC expression was increased by LPS exposure in SPC-A1 cells. Further studies revealed that AQP5 expression was down-regulated via the p38/JNK signaling pathway, while MUC5AC was up-regulated through the EGFR-p38/JNK pathway. Therefore, p38 and JNK may become promising targets to preserve AQP5 expression and prevent MUC5AC over-expression to restore proper H(2)O to mucin ratio of the airway mucus, which may be beneficial to the clinical management of COPD patients.

PKA Enhances the Sensitivity of IP3 Receptors in Swine Airway Submucosal Gland Cells.

Increased sensitivity of airway submucosal gland cells (SMGC) to ACh by PKA activation results in increased transepithelial ion movement. The mechanism of the increased sensitivity induced by PKA is unclear. Since ACh‐induced transepithelial ion movement is dependent on the IP3/Ca2+ signaling pathway, we hypothesized that PKA enhances sensitivity of IP3 receptors in airway SMGC, resulting in increased [Ca2+]i. Patch clamp of swine airway SMGC was performed in these studies. We showed that forskolin decreased the EC50 for IP3 about 4‐fold measured as the peak KCa current induced upon whole cell formation with IP3 in the pipette. VIP (10 nM), a co‐neurotransmitter present in cholinergic airway nerve fibers, enhanced the response to 0.3 µM IP3. Neither VIP nor IP3 (0.3 µM) induced KCa current when applied alone. VIP induced a significant KCa current in SMGC only when the internal solution contained IP3. Removing external Ca2+ did not abolish the VIP‐induced sensitization of the peak IP3 response although the plateau response was lost. This suggests that Ca2+ release is sensitized as well as Ca2+ influx. Elevating external Ca2+ to 5 mM revealed a KCa current induced only in the presence of IP3 (0.3 µM). Thus Ca2+ entry may also be increased by IP3 (0.3 µM). These results suggest that PKA increases the sensitivity of IP3 receptors by phosphorylation, resulting in the increased intracellular Ca2+ release and Ca2+ entry.

Moli1901 (duramycin) increases chloride transport in cystic fibrosis airway epithelial and pancreatic cell lines

The lantibiotic Moli1901 is a polycyclic peptide derived from Streptomyces cinnamoneum, which has been reported to stimulate chloride (Cl−) efflux from airway epithelial cells, and therefore it has been suggested as a potential drug for the treatment of cystic fibrosis (CF). In the present study, we investigated the effect of Moli1901 on Cl− efflux from normal human bronchial epithelial (16HBE) cells, a CF airway epithelial (CFBE) cell line, a normal pancreatic epithelial cell line (PANC-1), a CF pancreatic cell line (CFPAC-1), normal human airway submucosal gland cells (Calu-3), CF airway submucosal gland cells (CFSME), and untransfected baby hamster kidney fibroblasts (BHK-wt). Cl− efflux was determined with the help of a fluorescent probe, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE). It was found that Moli1901 dissolved in Standard Ringer’s solution without Cl− ions (SR0) significantly stimulated Cl− efflux from CFBE cells (at a concentration of 1mM), from CFPAC cells (at 3mM) and from Calu-3 cells (at 1 and 10mM). In these cells, stimulated Cl− efflux was significantly increased over basal Cl− efflux, and a stimulation corresponding to 30−40% of wild-type CFTR activity was obtained. Further experiments in the presence of inhibitors of CFTR (inhibitor-172), of Ca2+activated Cl− channels (gadolinium), and of the calcium ionophore A23187 were performed. These results show that Moli1901 causes a CFTR-independent Cl− efflux, which may explain the beneficial action of Moli1901 on FEV1, recently documented in a short-term clinical trial (Chest 131:4161; 2007). Supported by: Swedish Association for Cystic Fibrosis, the Swedish Heart-Lung Foundation and the Swedish Science Research Council.

Prostaglandin E2Enhances Acetylcholine-Induced, Ca2+-Dependent Ionic Currents in Swine Tracheal Mucous Gland Cells

Airway submucosal gland cell (SMGC) secretions are under the control of various neurotransmitters and hormones. Interactions between different pathways, such as those mediated by cAMP and Ca(2+), in controlling mucus or electrolyte secretions are not well understood. Prostaglandin E(2) (PGE(2)) or forskolin has been shown to enhance acetylcholine (ACh)-induced short circuit current (I(SC)) in SMGC mucous cell monolayers. We show that PGE(2), by activating cAMP-dependent protein kinase A (PKA), enhanced ACh-induced, Ca(2+)-mediated current and changes in [Ca(2+)](i) in mucous cells. PGE(2) pretreatment sensitized ACh-induced I(SC) (DeltaI(SC)) by activating endoprostanoid (EP(2)) receptors. PKA inhibitors 14-22 amide PKI (PKI) and Rp-diastereomer (Rp) of cAMPs prevented the effect of PGE(2). Removing external Ca(2+) or pretreatment with the Ca(2+) entry blocker, SKF96365 [1-[beta-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole], shifted the concentration-response relationships for ACh to the right but did not abolish PGE(2)-induced sensitization of the ACh response. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist and Ca(2+) entry blocker, 2-aminoethoxydiphenyl borate, abolished the ACh-induced response. Charybdotoxin, but not iberiotoxin (IbTX), inhibited the ACh-induced DeltaI(SC). Clotrimazole, but not IbTX, inhibited the ACh-induced serosal K(+) current. Under whole-cell patch clamp, ACh-induced K(+) and Cl(-) currents were coincident with increases in [Ca(2+)](i) in single mucous cells. PGE(2) or forskolin pretreatment did not induce current or [Ca(2+)](i) changes but enhanced ACh-induced currents, membrane hyperpolarization, and [Ca(2+)](i) changes. Intra-cellular dialysis with the PKA-catalytic subunit enhanced ACh-induced whole-cell current as well. These findings demonstrate that PGE(2), via EP(2) receptors and the cAMP/PKA pathway, activates Ca(2+) entry-independent mechanisms, possibly by increasing IP(3)-mediated Ca(2+) release, resulting in the sensitization of ACh-induced currents.

PGE2 enhances Ca2+‐dependent ionic currents in the airway mucus gland cells

Airway submucosal gland cell (SMGC) secretions are under the control of various secretagogues. Mechanisms of interactions between PGE2 and ACh in activating mucus cell ion current responses are examined in this study. PGE2 pretreatment sensitized ACh-induced short-circuit current (ISC) that were mediated by EP2 receptors and mimicked by forskolin. PKA inhibitors, 14–22 amide PKI (PKI) and Rp-cAMPs, prevented the effect of PGE2. Treatment with 0 [Ca2+]o or Ca2+ entry blocker, SKF96365, shifted the ACh concentration-response relationships to the right, but did not abolish PGE2-induced sensitization of the ACh response. An IP3 receptor antagonist and the Ca2+ entry blocker, 2-APB, abolished the ACh-induced response. Under whole-cell patch clamp, IP3, dialyzed in the cytosol via patch pipette upon whole-cell formation, induced immediate increases in both KCa and CaCC currents that were prolonged by simultaneous inclusion of cAMP in the internal solution. The plateau phases of both currents were related to Ca2+ entry, abolishable by treatments with 0 [Ca2+]o, Gd3+, 2-APB, or SKF96365, and were also enhanced by membrane hyperpolarization. Abundant mRNA expressions for TRPC channels (TRPC-1>>3≈6>4) were detected in the mucus cells using real-time RT-PCR. Thus, PGE2, via EP2 receptors and the cAMP/PKA pathway, enhances ACh-induced, Ca2+-mediated ionic currents in mucus cells through activating Ca2+-entry independent mechanisms that are primarily responsible for sensitization of the ACh response. ACh-induced Ca2+ entry via non-selective cation channels was also enhanced by cAMP treatment, important for sustaining the ACh-induced response [Supported by a grant from the American Heart Association to J.M.F.].

TSG-6 Potentiates the Antitissue Kallikrein Activity of Inter–α-inhibitor through Bikunin Release

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-alpha-inhibitor and pre-alpha-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6.HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin.HC complexes (byproducts of TSG-6.HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-alpha and IL-1beta, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-alpha-inhibitor) were also induced by TNF-alpha in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Regulation of MUC5AC mucin secretion by depletion of AQP5 in SPC-A1 cells

Airway mucus is regulated by many inflammatory mediators such as ILs, TNF-α, EGF, PGF2α, LT, and so on. Recently, the relationship between membrane ion channel and mucus production has been under investigation. The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1). A recombinant plasmid ( pShAQP5) containing small hairpin RNA expression cassette targeting AQP5 sequence was constructed. In pShAQP5 transiently transfected cells, ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3%, respectively, on day 5 after pShAQP5 transfection. While in five stably transfected clones (shAQP5-G1, G2, G3, A2, and A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123%, and 38%, respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59–156% and 33–166%, respectively. This is the first reliable investigation of the regulation of MUC5AC mucin secretion by silencing AQP5. Further study of the regulatory mechanism between AQPs and mucins may provide new strategies for development of novel antihypersecretory drugs in airway diseases.

Datura stramonium in asthma treatment and possible effects on prenatal development

Southern Africa has a variety of medicinal plants, used as remedies; however, little information is available regarding the cytotoxic potential, particularly when used during pregnancy. One such plant is Datura stramonium (DS) (Solanaceae), used frequently as an anti-asmatic treatment. DS contains a variety of alkaloids including atropine and scopolamine that can cause anticholinergic poisoning if taken in large doses. Atropine and scopolamine act on the muscarinic receptors by blocking them (particularly the M 2 receptors) on airway smooth muscle and submucosal gland cells. However, this will cause a continuous release in acetylcholine (Ach). Ach also act on nicotinic receptors; however, it is known that “over exposure” of nicotinic receptors may cause desensitization. We suggest that exposure of the foetus to DS when a mother uses it for asthma, will cause a continuous release of Ach, resulting in the desensitizing of nicotinic receptors, this could ultimately result in permanent damage to the foetus. Therefore we conclude that this African herbal remedy should be used with caution during pregnancy.

Downregulation of aquaporin 5 induced by vector-based short hairpin RNA and its effect on MUC5AC gene expression in human airway submucosal gland cells

The aquaporins (AQPs) are a family of homologous water channels expressed in many epithelial and endothelial cells, however no reliable and non-toxic inhibitors of AQPs have been reported yet. Our researchers have analyzed the changes of AQP5 expression induced by vector-based short hairpin RNA (shRNA) in the human airway submucosal gland cell line (SPC-A1) and observed its regulation on the expression of MUC5AC gene. Localizations of AQP5 and MUC5AC in SPC-A1cells were detected by Immunofluorescence. AQP5 mRNA was significantly reduced by 75.1% one day after transfection with specific shRNA, named shAQP5. However, the significant suppression of AQP5 protein did not appear until day 5 after transfection. MUC5AC mRNA was remarkably increased by 119.9% On day 3 after shAQP5 transfection, while comparable MUC5AC protein changes were not found in SPC-A1 cells with flow cytometry analysis. These results indicate that vector-based shRNA could be used as a potential tool to inhibit the expression of AQP5. This is the first investigation providing evidence demonstrating the regulation of the mucin gene by AQP5 gene silencing.

Prostanoids Secreted by Alveolar Macrophages Enhance Ionic Currents in Swine Tracheal Submucosal Gland Cells

We examined the effect of substances released by swine alveolar macrophages (AMs) on ionic currents in airway submucosal gland cells (SGCs). AMs obtained by lavage were activated by 24-h zymosan exposure (0.1 mg/ml). Supernatant was collected and used to stimulate short-circuit current changes (DeltaI(SC)) in SGC monolayers in Ussing chambers. Dexamethasone (1 microM) or indomethacin (5 muM) during zymosan exposure of AMs reduced or abolished the supernatant-induced DeltaI(SC). Zymosan exposure induced a 5-fold increase in cyclooxygenase (COX)-2 but not COX-1 protein levels in AMs. Prostaglandin E(2) (PGE(2)) concentration in the supernatant from zymosan-activated AMs was 550 +/- 10 nM (n = 3) compared with 28 +/- 3 nM for unstimulated AMs (n = 3). PGE(2), applied serosally, induced DeltaI(SC) with an EC(50) of 15.5 +/- 1.3 nM (n = 4) and 3.6 +/- 1.8 microM (n = 3) when applied apically. Four types of endoprostanoid receptors (EP(1-4)) were detected in SGCs using Western blot. PGE(2)-induced DeltaI(SC) were inhibited by AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) but not by SC19220 (8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide), suggesting that endoprostanoid (EP)(2) but not EP(1) receptors were activated by PGE(2). Pretreatment of SGCs with supernatant from zymosan-activated AMs, PGE(2), or forskolin enhanced the sensitivity to acetylcholine (ACh)-induced DeltaI(SC). PGE(2)-induced DeltaI(SC) were blocked by charybdotoxin (ChTX), chromanol 293B, or glibenclamide. ACh-induced DeltaI(SC) were only blocked by ChTX or glibenclamide. None of these blockers altered PGE(2) pretreatment-induced sensitization of ACh-induced DeltaI(SC). These results demonstrate that prostanoids released from activated AMs directly increase cystic fibrosis transmembrane conductance regulator and K(+) channel activity. ACh-induced DeltaI(SC) are also enhanced due to enhanced activation of Ca(2+)-activated K(+) channels (K(Ca)).

Characterization of Novel Airway Submucosal Gland Cell Models for Cystic Fibrosis

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitrostudies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl^- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting.

CFTR and lysozyme secretion in human airway epithelial cells

Lysozyme is secreted in large quantities in human airways (10-20 mg/day), where it helps to defend against bacterial and fungal infection. Lysozyme expression is restricted to the serous cells of the submucosal glands, which also express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. It is often assumed that mucus secretion in human airways is coupled to anion secretion through CFTR Cl(-) channels located in the apical membrane. Therefore, a defect in CFTR function could cause abnormal mucus secretion leading to persistent bacterial infection and inflammation of the airways. In this study we measured simultaneous secretion of lysozyme and Cl(-) from human airway epithelial serous cells. Secretion of lysozyme was measured by a turbidimetric assay that relies on the ability of lysozyme to disrupt the wall of the bacterium Micrococcus lysodeikticus, thus causing a fall in the optical density of the sample. Secretion of Cl(-) was measured as short-circuit current in a modified Ussing chamber. Activation of Cl(-) secretion by stimulation of cAMP- or Ca(2+)-dependent pathways caused comparable increases in lysozyme secretion. Similarly, blockers of Cl(-) secretion, such as diphenylamine-2-carboxylate (DPC), also reduced lysozyme secretion. However, while treatment of airway submucosal gland cells with antisense oligonucleotides directed against CFTR reduced Cl(-) secretion, it had no significant effect on the total amount of lysozyme secretion. These results suggest a role for functional CFTR in regulation of lysozyme secretion in human airways.

Surfactant protein A2 gene expression by human airway submucosal gland cells.

To determine whether human airway submucosal glands produce and secrete surfactant proteins, we examined their protein and gene expression in submucosal glands from trachea and bronchi obtained from operated and autopsied lungs within 4 h of death. Using a monoclonal antibody (PE-10) against surfactant protein A (SP-A), a positive immunoperoxidase stain was observed over serous cells of submucosal glands in histologic sections of airway walls. Measurement of SP-A in culture medium samples using single-step enzyme-linked immunosorbent assay showed a significant secretion of SP-A by isolated submucosal glands (1.2 +/- 0.08 ng/ml/h, SEM, n = 40). In gene expression experiments by reverse transciption-polymerase chain reaction, the SP-A complementary DNA (cDNA) segment was amplified from isolated submucosal glands, indicating the presence of SP-A messenger RNA (mRNA) in airway submucosal glands. Bronchial superficial epithelial cells failed to show the presence of SP-A mRNA. No cDNA segment of SP-B, SP-C, or SP-D cDNA was amplified from isolated submucosal glands or superficial epithelial cells, whereas all were amplified from alveolar tissue. Furthermore, in contrast to the control alveolar tissue, which expressed both SP-A1 and SP-A2 genes, SP-A2 gene transcript alone was detected in isolated submucosal glands by Southern analysis that included the digestion of the amplified SP-A cDNA fragment with the restriction enzyme Apa I. These findings indicate that human airway submucosal gland cells can transcribe the SP-A2 gene and produce SP-A protein in a manner different from peripheral airways and alveoli, playing a role in the airway defense mechanism.

Cocaine Inhibits Chloride Secretion by Swine Tracheal Submucosal Gland Cells Grown in Culture

Active ion transport by airway submucosal gland cells plays an important role in maintaining effective mucociliary clearance. Alterations in the clearance will affect normal airway functions. Nasal and airway mucosal surfaces are exposed to high concentrations of cocaine after snorting crystals or smoking the freebase form, causing respiratory complications. The actions of cocaine on airway gland cell ion transport were determined by measuring the basal electrical properties and ion transport activated by acetylcholine (ACh) and isoproterenol (ISO) in swine tracheal submucosal gland cells grown in culture. The cells were enzymatically isolated, purified on Percoll density gradients, and then grown on microporous inserts at an air interface. Chloride secretion was measured as short-circuit current (Isc) utilizing Ussing chamber methodology. Amiloride (10 μm) was added mucosally to block sodium reabsorption. The maximal changes in Isc (ΔIsc) induced by ACh and ISO were 42.0 ± 4.4 and 8.8 ± 0.8 μA/cm2. Cocaine decreased basal Isc and the increase in Isc induced by ACh and ISO. The concentration–response relationships for ACh and ISO were shifted to the right by cocaine (1–3 mm) when applied serosally. The maximal increase in Isc induced by ISO was inhibited by serosal cocaine, but, the maximal response to ACh was not. Thus, cocaine inhibits ACh-induced increases in Isc by competitively blocking the receptor and the response to ISO by noncompetitive inhibition. Mucosal cocaine reduced the maximal response to ACh and ISO and also caused a rightward shift. We conclude that cocaine directly alters submucosal gland cell function by reducing the basal and stimulated ability to transport ions and fluid.

Bradykinin regulation of airway submucosal gland secretion: role of bradykinin receptor subtype

To clarify the role of bradykinin receptor subtypes, we examined the effect of bradykinin on feline tracheal and human airway submucosal gland secretion using an isolated gland preparation. Bradykinin induced a significant increase in [3H]glycoconjugate secretion in a dose-dependent manner from isolated glands, which was significantly inhibited by D-Arg-(Hyp3, Thi5,8, D-Phe7)-bradykinin (the B2-receptor antagonist), whereas Des-Arg9-(Leu8)-bradykinin (B1-receptor antagonist) or indomethacin did not significantly alter it. Nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) caused a significant inhibition of bradykinin-induced glycoconjugate secretion, which was reversed by the addition of L-arginine. Bradykinin evoked bidirectional current responses, and an initial inward current (Cl- current) was followed by an outward current (K+ current) of the acinar cells in a whole cell configuration by patch-clamp technique. Bradykinin induced an immediate increase in intracellular calcium concentration ([Ca2+]i) of the acinar cells followed by a prolonged plateau, and Ca2+ removal resulted in an initial increase alone. [Ca2+]i rise was significantly inhibited by the B2-receptor antagonist, whereas the B1-receptor antagonist did not significantly alter it. These findings suggest that B2-receptor stimulation and the resultant [Ca2+]i rise induced both mucus glycoprotein and electrolyte secretions, involving NO formation in airway submucosal gland cells.

Cystic fibrosis sputum induces a secretory response from airway gland serous cells that can be prevented by neutrophil protease inhibitors.

High activities of the neutrophil proteases, elastase and cathepsin G, are found in the sputum of patients with cystic fibrosis (CF). Because both proteases have been shown to be potent secretagogues for airway submucosal glands, and because hypersecretion is a characteristic feature of CF, the objective of the present study was to examine whether there is secretagogue activity in CF sputum, and to determine the contribution of neutrophil proteases to the secretagogue activity. Confluent monolayers of cultured bovine tracheal serous cells were pulse-labelled with Na2(35)SO4, incubated with diluted CF sputum supernatants in the presence or absence of different protease inhibitors, and the subsequent release of the radio-labelled macromolecules was measured. CF sputum potently induced secretion concentration-dependently. Addition of the selective neutrophil elastase inhibitor ICI 200,355 inhibited the secretory response to CF sputum supernatant by 89%. Addition of a cathepsin G-inhibitor resulted in further inhibition of the secretory response. Addition of phosphoramidon, a drug known to inhibit Pseudomonas aeruginosa elastase, had no effect. We conclude that CF sputum potently stimulates airway submucosal gland cell secretion. These studies with protease inhibitors suggest that neutrophil proteases account substantially for the secretagogue activity present in CF sputum.