Stimulation Of Airway Epithelial Cells Research Articles

CALHM1-Mediated ATP Release and Ciliary Beat Frequency Modulation in Nasal Epithelial Cells

Mechanical stimulation of airway epithelial cells causes apical release of ATP, which increases ciliary beat frequency (CBF) and speeds up mucociliary clearance. The mechanisms responsible for this ATP release are poorly understood. CALHM1, a transmembrane protein with shared structural features to connexins and pannexins, has been implicated in ATP release from taste buds, but it has not been evaluated for a functional role in the airway. In the present study, Calhm1 knockout, Panx1 knockout, and wild-type mouse nasal septal epithelial cells were grown at an air-liquid interface (ALI) and subjected to light mechanical stimulation from an air puff. Apical ATP release was attenuated in Calhm1 knockout cultures following mechanical stimulation at a pressure of 55 mmHg for 50 milliseconds (p < 0.05). Addition of carbenoxolone, a PANX1 channel blocker, completely abolished ATP release in Calhm1 knockout cultures but not in wild type or Panx1 knockout cultures. An increase in CBF was observed in wild-type ALIs following mechanical stimulation, and this increase was significantly lower (p < 0.01) in Calhm1 knockout cultures. These results demonstrate that CALHM1 plays a newly defined role, complementary to PANX1, in ATP release and downstream CBF modulation following a mechanical stimulus in airway epithelial cells.

Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells.

The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK) 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA). However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs), cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs) to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG) but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE) or myeloperoxidase (MPO) attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC), nonsevere asthma (NSA), and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

Cell-contact dependent inhibition of monocytes by airway epithelial cells and reversion by infection with Respiratory Syncytial Virus

Airway epithelial cells (AEC) are the first line of defense against airborne infectious microbes and play an important role in regulating the local immune response. However, the interplay of epithelial cells and professional immune cells during both homeostasis and infection has only been partially studied. The present study was performed to determine how bronchial epithelial cells affect the activation of monocytes. Under healthy conditions, AECs were shown to inhibit reactivity of monocytes. We hypothesized that upon infection, monocytes might be released from inhibition by AECs. We report that direct contact of monocytes with unstimulated BEAS2B epithelial cells results in inhibition of TNF secretion by activated monocytes. In addition to the known soluble modulators, we show that cell contacts between epithelial cells and monocytes or macrophages also contribute to homeostatic inhibitory actions. We find AECs to express the inhibitory molecule PD-L1 and blockade of PD-L1 results in increased secretion of pro-inflammatory cytokines from monocytes. Contrary to the inhibitory activities during homeostasis, epithelial cells infected with Respiratory Syncitial Virus (RSV) induce a significant release of inhibition. However, release of inhibition was not due to modulation of PD-L1 expression in AECs. We conclude that airway epithelial cells control the reactivity of monocytes through direct and indirect interactions; however tonic inhibition can be reverted upon stimulation of AECs with RSV and thereof derived molecular patterns. The study confirms the important role of airway epithelial cells for local immune reactions.

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.

Role of defensins in the pathogenesis of chronic lung allograft rejection

Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n=28), BOS− (n=26) and normal healthy controls (n=24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1–3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p=0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP–AAT complexes in BAL (p=0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2-fold (p=0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx.

99 : IL-33 splice variants are critical to its regulated secretion from airway epithelial cells

IL-33 is a member of the IL-1 family of cytokines. It plays a critical role in allergic disease as an upstream regulator of Th2 type inflammation, and multiple genome wide association studies have implicated this cytokine in the pathogenesis of asthma. IL-33 localizes in the airway to the nuclei of airway epithelial basal cells. To date, the mechanisms of its release from epithelial cells have been elusive. Furthermore, its precise role and regulation in the asthmatic airway remains unclear. Recently, we discovered that IL-33 splice variants transcripts are present in human airway epithelial cells. Furthermore, we find that splice variants vary in their localization to the nucleus or cytoplasm. We have gone onto show that following stimulation of airway epithelial cells with secretogogues, cytoplasmic IL-33 variants are released extracellularly while nuclear forms are not. Finally, we find by immunoblot that IL-33 present in the airway secretions of asthmatics is a truncated form of the protein (not full length) and contains both the N and C terminus, arguing against proteolytic degradation of the IL-33 protein in vivo. These studies provide new insights to the mechanisms that airway epithelial cells use to regulate the secretion of this important Th2 cytokine and help to inform the development of anti-IL-33 therapeutics.

Reduced tonsillar expression of human β-defensin 1, 2 and 3 in allergic rhinitis

Airway infections are known to cause exacerbations of allergy and asthma. Tonsils constitute a primary site for microbial recognition and triggering of the immune system in the airways. Human β-defensins (HBDs) are antimicrobial peptides with an important role in this defense. Our aim was to investigate HBD1-3 in tonsillar tissue and their potential role in allergic rhinitis (AR). Tonsils, obtained from patients with AR and non-allergic controls, and isolated tonsillar CD4(+), CD8(+) and CD19(+) lymphocytes were analyzed for HBD1-3 expression using real-time RT-PCR and/or immunohistochemistry. Tonsillar tissue, mixed tonsillar lymphocytes and airway epithelial cells (AECs) were cultured with or without IL-4, IL-5, IL-13 or histamine followed by measurements of HBD1-3 release using ELISA. HBD1-3 were present in tonsillar tissue, including epithelial, CD4(+), CD8(+) and CD19(+) cells. The expression was reduced in allergic compared to healthy tonsils. Stimulation of AECs with IL-4, IL-5 and histamine down-regulated the HBD release, whereas no effects were seen in cultured tonsils or lymphocytes. This study demonstrates presence of HBD1-3 in tonsils and that the levels are reduced in patients with AR. Together with the down-regulation of HBDs in epithelial cells in the presence of allergic mediators suggest that AR patients have an impaired antimicrobial defense that might make them more susceptible to respiratory tract infections.

Timothy grass pollen extract‐induced gene expression and signalling pathways in airway epithelial cells

Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment.

Leptin up-regulates MUC5B expression in human airway epithelial cells via mitogen-activated protein kinase pathway

ABSTRACTLeptin, an adipocyte-secreted hormone that regulates food intake and metabolic response, has been recently reported to increase in the serum during inflammatory airway disease associated with mucus-hypersecretion. We investigated the effects of leptin on mucin expression in human airway epithelial cells and the signaling pathways. The expression of the leptin receptor was evaluated in human nasal mucosa and NCI-H292 cells. Leptin-induced expression of major respiratory mucins in NCI-H292 cells was analyzed. Mutant leptin, which acts as a receptor antagonist, and specific inhibitors of extracellular signal-regulated kinase (ERK1/2), p38 and Janus kinase-2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) were used. Leptin receptors were expressed in the nasal mucosa and NCI-H292 cells. Treatment with leptin significantly increased the expression of MUC5AC and MUC5B in NCI-H292 cells; these effects were blocked by mutant leptin. The cells activated by leptin showed increased ERK1/2, p38, and STAT3 phosphorylation. Leptin-induced MUC5B expression was blocked by the ERK1/2 and p38 pathway inhibitors, but not by the JAK2/STAT3 pathway inhibitor. Leptin might significantly contribute to the production of major gel-forming mucins by direct stimulation of airway epithelial cells and the activation of leptin receptors coupled with the activation of ERK1/2 or p38, but not the JAK2/STAT3 pathway.

Epidermal Growth Factor Receptor Transactivation by ATP-mediated Purinoceptor Stimulation of Airway Epithelial Cells is Mediated by the NADPH Oxidase DUOX1

Epidermal Growth Factor Receptor Transactivation by ATP-mediated Purinoceptor Stimulation of Airway Epithelial Cells is Mediated by the NADPH Oxidase DUOX1

IL-17 primes airway epithelial cells lacking functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) to increase NOD1 responses

In Cystic Fibrosis (CF), the absence of functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is key to better therapies for CF. In this manuscript, we have found that the presence of IL-17 in the airways of CF patients not only exacerbates inflammation through the recruitment of neutrophils via secretion of CXCL8, but also by priming airway epithelial cells lacking functional CFTR to increase response to the bacterial sensor NOD1. IL-17 stimulation of airway epithelial cells (AECs) lacking functional CFTR increased the expression of NOD1, NOD2, TLR4 and its own receptors IL-17RA and IL-17RC. Moreover, prior stimulation of AECs expressing the CFTRΔF508 mutant with IL-17 showed much greater CXCL8 secretion in response to a NOD1 agonist and Pseudomonas aeruginosa diffusible material. Taken together our results show that IL-17 primes AECs expressing CFTRΔF508 to increase host defence response to bacteria through the up-regulation of PRRs, and in particular of NOD1, and identifies another mechanism of action through which the CFTRΔF508 mutation leads to increase inflammation in response to bacterial ligands. Therefore preventing IL-17 function in CF may prove an important strategy in decreasing lung inflammation due to both direct and indirect effects.

CARMA3 Mediates Lysophosphatidic Acid–Stimulated Cytokine Secretion by Bronchial Epithelial Cells

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.

Cigarette Smoke Condensate Enhances Respiratory Syncytial Virus–Induced Chemokine Release by Modulating NF-kappa B and Interferon Regulatory Factor Activation

Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-kappaB), as shown by increased NF-kappaB DNA binding to its specific site of the IL-8 promoter and increased NF-kappaB-driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.

Lipid body mobilization in the ExoU-induced release of inflammatory mediators by airway epithelial cells

This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE 2 that was reduced by the bacterial treatment with MAFP, a cPLA 2 inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE 2. Also, in PA103-infected cells, PGE 2 was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE 2 and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.

(R)-albuterol elicits antiinflammatory effects in human airway epithelial cells via iNOS.

Catecholamines can suppress production of inflammatory mediators in different cell types, including airway epithelium, but downstream signaling mechanisms involved in regulation of these antiinflammatory effects are largely unknown. We theorized that acute beta2-adrenergic stimulation of airway epithelial cells with albuterol could suppress the production and release of inflammatory mediators, specifically granulocyte macrophage-colony stimulating factor (GM-CSF) via a pathway involving inducible nitric oxide synthase (iNOS). Normal human bronchial epithelial (NHBE) cells in primary culture were exposed to a cytokine mixture (10 ng/ml each IFN-gamma and IL-1beta) to induce iNOS expression. (R)- and (S)-enantiomers of albuterol, as well as racemic mixtures, were added with these cytokines, and effects on GM-CSF expression and production were assessed. Specific inhibitors and activators of protein kinases (PKs), beta2-adrenergic receptor antagonists, and small interfering RNAs against iNOS were used to delineate signaling pathways involved. iNOS message was significantly upregulated in a concentration-dependent manner by the active (R)-enantiomer of albuterol. (R)-albuterol also attenuated cytokine-induced increases in GM-CSF steady-state mRNA expression and protein release. The (S)-enantomer of albuterol had no effect on these parameters. PKC, specifically, the delta isoform, was required for iNOS message increase, but PKA and PKG were not involved in the pathway. Overall, this study identifies a novel pathway by which beta2-adrenergic agonists may exhibit antiinflammatory effects in airway epithelium and surrounding milieu.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea.

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V(te)) and amiloride-sensitive short-circuit current (I(sc-Amil)) in mouse trachea. Exposure to apical ATP or UTP (each 100 micromol/l) caused a large initial increase in lumen negative V(te) and I(sc), corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of I(sc-Amil) that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 micromol/l) or the Ca2+ ionophore ionomycin (1 micromol/l). Removal of extracellular Cl- or exposure to 200 microM DIDS reduced UTP-mediated inhibition of I(sc-Amil) substantially. The phospholipase inhibitor U73122 (10 micromol/l) and pertussis toxin (PTX; 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of I(sc-Amil). Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia.

Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.

PKC role in mechanically induced Ca2+ waves and ATP-induced Ca2+ oscillations in airway epithelial cells.

Mechanical stimulation of airway epithelial cells generates the Ca2+ mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase (PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a "Ca2+ wave"); the role that diacylglycerol-activated PKC might play in the response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12-O-tetradecanoylphorbol 13-acetate- or 1, 2-dioctanyl-sn-glycerol-induced activation of PKC slows the Ca2+ wave, decreases the amplitude of induced intracellular free Ca2+ concentration ([Ca2+]i) increases, and decreases the number of affected cells. The PKC inhibitors bisindolylmaleimide and Gö 6976 slowed the spread of the wave but did not change the number of affected cells. We show that ATP-induced [Ca2+]i increases and oscillations, responses independent of intercellular communication, were inhibited by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2+]i increases and blocked oscillations, suggesting that PKC has an initial positive effect on Ca2+ mobilization and then mediates feedback inhibition. PKC activators also reduced the [Ca2+]i increase that followed thapsigargin treatment, indicating a PKC effect associated with the Ca2+ release mechanism.

Stretch increases inositol 1,4,5-trisphosphate concentration in airway epithelial cells.

Mechanical stimulation of airway epithelial cells with a microprobe leads to an increase in cytoplasmic [Ca2+] that appears to be due, in part, to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores (Boitano et al., Science 258:292[1992]). To investigate whether intracellular IP3 concentration ([IP3]i) increases in response to mechanical stimulation, we grew confluent monolayers from rabbit tracheal mucosal explants on flexible substrates and measured [IP3]i after stretching the substrate. The effect of stretch on [IP3]i was measured in the presence of Li+, an inhibitor of IP3 degradation. In unstretched cells, IP3 measured approximately 5.1 pmol/10(6) cells, from which we estimated [IP3]i to be 1.8 microM. Addition of Li+ had no effect on resting [IP3]i. When the flexible cell support was stretched to increase its surface area by 13%, mean [IP3]i increased about 3-fold with a half-time of approximately 1 s. The increased [IP3]i was maintained in a plateau phase for approximately 8 s and then decayed to near the unstretched level over the next 10 s, despite the sustained application of stretch. A transient stretch (0.5 s) induced a similar rate of increase and peak [IP3]i; however, [IP3]i subsided without a plateau phase. The magnitude of the [IP3]i increase was proportional to stimulus intensity between 0 and 13% increase in substrate surface area. In addition, dissociated airway epithelial cells were exposed to hypotonic solution to induce cell swelling. [IP3]i increased about 4-fold above control levels after 10 s of exposure to hypotonic solution. Basal [IP3]i of dissociated cells in isotonic solution was estimated to be 0.7 microM. These results are consistent with mechanical stimulation leading to phospholipase C synthesis of IP3, which mediates intracellular and intercellular Ca2+ signaling.