Bovine Tracheal Epithelial Cells Research Articles

Bovine rhinitis B virus is highly prevalent in acute bovine respiratory disease and causes upper respiratory tract infection in calves.

Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log10 TCID50 BRBV. Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.

Xanthoxyletin Inhibits Proliferation of Human Oral Squamous Carcinoma Cells and Induces Apoptosis, Autophagy, and Cell Cycle Arrest by Modulation of the MEK/ERK Signaling Pathway.

BackgroundThis study aimed to investigate the effects of xanthoxyletin, a plant-derived coumarin, on human oral squamous cancer cells in vitro and in mouse xenografts in vivo.Materia/MethodsThe study included SCC-1 human oral cancer cells and EBTr normal embryonic bovine tracheal epithelial cells, which were treated with 0 μM, 5 μM, 10 μM, and 20 μM of xanthoxyletin for 24 hours. The MTT assay assessed cell viability, and autophagy was detected by electron microscopy. Cell apoptosis was investigated using 4′,6-diamidino-2-phenylindole (DAPI), annexin V, and propidium iodide (PI) fluorescence flow cytometry, which was also used to investigate the cell cycle. Protein expression was measured by Western blot. Mouse xenografts were used for the in vivo evaluation of the effects of xanthoxyletin.ResultsXanthoxyletin significantly inhibited the proliferation of oral cancer cells (IC50, 10–30 μM) with lower cytotoxicity for normal cells. Xanthoxyletin treatment was associated with G2/M arrest of the cell cycle and with increased apoptosis and autophagy of SCC-1 cells. Apoptosis and autophagy induced by xanthoxyletin were also associated with changes in expression of the apoptosis-associated proteins, Bax and Bcl-2, and the autophagy-associated proteins, LC3I, LC3II, Beclin 1, p62, and VSp34. Xanthoxyletin inhibited the expression of components of the signaling cascade of the MEK/ERK pathway in the SCC-1 oral cancer cells. The in vivo effects of xanthoxyletin showed inhibition of growth of mouse xenografts.ConclusionsXanthoxyletin inhibited the proliferation of human oral squamous carcinoma cells and induced apoptosis, autophagy, and cell cycle arrest by modulation of the MEK/ERK signaling pathway.

Benzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). β-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of β-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 μM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 μM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP—a toxic pollutant found in crude oil—on this respiratory innate immune response.

Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

β-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle.

Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

Biochemical Characterization of Native Usher Protein Complexes from a Vesicular Subfraction of Tracheal Epithelial Cells

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells

The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor κB (NF-κB) pathway can be involved in its regulation.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant ( p < 0.01), and these effects were significantly ( p < 0.02, p < 0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant ( p < 0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Effect of Corticosteroids and Neuropeptides on the Expression of Defensins in Bovine Tracheal Epithelial Cells

Susceptibility to bacterial pneumonia in cattle is enhanced by stressors such as transportation, weaning, and commingling, which trigger a physiologic stress response resulting in elevated levels of endogenous corticosteroids and catecholamines. To determine the effect of neuroendocrine mediators on the expression of innate defense peptides in the lung, bovine tracheal epithelial cells were exposed to dexamethasone, catecholamines, acetylcholine, or substance P, and then beta-defensin expression was quantified using real-time reverse transcription-PCR. Basal expression of tracheal antimicrobial peptide (TAP) mRNA was not affected by any of the mediators tested. However, induction of TAP expression by lipopolysaccharide was significantly inhibited by pretreatment with dexamethasone. Bronchial biopsy specimens from dexamethasone-treated calves had significantly lower expression of TAP and lingual antimicrobial peptide (LAP) mRNA than saline-treated controls following 48 h of treatment. Lipopolysaccharide-elicited neutrophil recruitment was enhanced in the lungs of dexamethasone-treated calves compared to saline-treated controls. These findings indicate that modulation of epithelial antimicrobial peptide expression is one mechanism through which corticosteroids and stress may impair innate pulmonary defenses.

Inhibition of β-Defensin Gene Expression in Airway Epithelial Cells by Low Doses of Residual Oil Fly Ash is Mediated by Vanadium

Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM(2.5)) on one antimicrobial mechanism of host defense in the airway, beta-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1beta. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0-20 microg/cm(2) residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1beta. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 microg/cm(2) of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO(2) and TiO(2) were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1beta-induced human beta-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V(2)O(5), and VOSO(4) inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens.

Suppression of NF-κB-mediated β-defensin gene expression in the mammalian airway by the Bordetella type III secretion system

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.

One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis.

The iron-binding protein lactoferrin is a ubiquitous and abundant constituent of human exocrine secretions. Lactoferrin inhibits bacterial growth by sequestering essential iron and also exhibits non-iron-dependent antibacterial, antifungal, antiviral, antitumor, anti-inflammatory, and immunoregulatory activities. All of these non-iron-dependent activities are mediated by the highly charged N terminus of lactoferrin. In this study we characterized a Lys/Arg polymorphism at position 29 in the N-terminal region of human lactoferrin that results from a single nucleotide polymorphism in exon 1 of the human lactoferrin gene. We expressed cDNAs encoding both lactoferrin variants in insect cells and purified the two proteins by ion exchange chromatography. The two lactoferrin variants exhibited nearly identical iron-binding and iron-releasing activities and equivalent bactericidal activities against a strain of the gram-negative bacterium Actinobacillus actinomycetemcomitans. When tested against the gram-positive species Streptococcus mutans and Streptococcus mitis, however, lactoferrin containing Lys at position 29 exhibited significantly greater bactericidal activity than did lactoferrin containing Arg. In addition, the Lys-containing lactoferrin stimulated bovine tracheal epithelial cells to synthesize much higher levels of tracheal antimicrobial peptide mRNA than did the Arg-containing variant. A genotyping assay that distinguished between the two alleles based on a polymorphic EarI restriction site showed that the Lys and Arg alleles had frequencies of 24% and 76%, respectively, among 17 healthy human subjects, and 72% and 28%, respectively, among nine patients with localized juvenile periodontitis. Our findings suggest that these two lactoferrin variants are functionally different and that these differences may contribute to the pathogenesis of localized juvenile periodontitis.

FK506 inhibits Cl − secretion in airway epithelium via calcineurin-independent mechanism

FK506 (tacrolimus)-binding protein (FKBP) is associated with intracellular Ca 2+ release channel and modulates its function. To elucidate the effect of FK506 on Ca 2+ dynamics and Ca 2+-mediated Cl − secretion in airway epithelium, we studied intracellular Ca 2+ ([Ca 2+] i) concentration and Cl −-dependent short-circuit current ( I sc), in cultured bovine tracheal epithelial cells. Addition of ATP induced an increase in [Ca 2+] i, and this response was dose dependently inhibited by FK506. Rapamycin, which binds FKBP with high affinity, likewise inhibited the [Ca 2+] i rise, but cyclosporin A, a specific calcineurin inhibitor, did not. In Cl − secretion studies using Ussing chamber, ATP increased Ca 2+-mediated I sc in amiloride-treated cells, an effect that was inhibited by FK506 and rapamycin but not by cyclosporin A. Therefore, FK506 inhibits Ca 2+ mobilization in airway epithelium via FKBP but not calcineurin-dependent mechanism, which may result in the suppression of Ca 2+-activated Cl − secretion.

Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH.

The concentration of salt in the thin layer of fluid at the surface of large airways, the airway-surface liquid (ASL), is believed to be of central importance in airway physiology and in the pathophysiology of cystic fibrosis. Invasive sampling methods have yielded a wide range of ASL [NaCl] from 40 to 180 mM. We have developed novel fluorescent probes and microscopy methods to measure ASL thickness, salt concentration, and pH quantitatively in cell-culture models and in the trachea in vivo. By rapid z-scanning confocal microscopy, ASL thickness was 21 +/- 4 microm in well-differentiated cultures of bovine tracheal epithelial cells grown on porous supports at an air-liquid interface. By ratio imaging fluorescence microscopy using sodium, chloride, and pH-sensitive fluorescent indicators, ASL [Na+] was 97 +/- 5 mM, [Cl-] was 118 +/- 3 mM, and pH was 6.94 +/- 0.03. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 45 +/- 5 microm, [Na+] was 115 +/- 4 mM, [Cl-] was 140 +/- 5 mM, and pH was 6.95 +/- 0.05. Similar ASL tonicity and pH were found in cystic fibrosis (CFTR-null) mice. In freshly harvested human bronchi, ASL thickness was 55 +/- 5 microm, [Na+] was 103 +/- 3 mM, [Cl-] was 92 +/- 4 mM, and pH was 6.78 +/- 0.2. These results establish by a noninvasive approach the key properties of the ASL and provide direct evidence that the ASL is approximately isotonic and not saltier in cystic fibrosis.

Mucin biosynthesis and secretion in tracheal epithelial cells in primary culture

Density-gradient centrifugation of bovine tracheal epithelial cell extracts revealed a 'high-density' (1.48 g/ml) sialic-acid-rich population as well as a 'low-density' (1.42 g/ml) one that reacted more strongly with a periodate-Schiff (PAS) assay. The sialic-acid-rich mucins were oligomeric molecules containing disulphide- bond-linked subunits and large glycosylated domains, whereas the PAS-reactive component seemed to be smaller and 'monomeric'. Only the 'high-density' population was secreted from cells cultured for 5 days on plastic or a collagen type 1, Matrigel or Vitrogen substrate. Release was less from cells grown on plastic than from those on a substrate and the amount was unaffected by increasing the thickness of the collagen layer. For cells grown on collagen, the amount of the sialic-acid-rich mucin increased over 10 days, whereas the PAS-reactive component was largely absent after 24 h, which was consistent with an initial release of stored PAS-reactive molecules and synthesis of the sialic-acid-rich mucins de novo. Both [(3)H]proline and [(35)S]sulphate were poorly incorporated into mucins detected with the chemical assays but molecules with a higher buoyant density than that of either of the previously identified species were labelled with [(35)S]sulphate. The [(35)S]sulphate-labelled material yielded large trypsin-resistant fragments and contained O-linked glycans but was not affected by digestion with chondroitin ABC lyase or heparan sulphate lyase, suggesting that it is a mucin rather than a proteoglycan. [(35)S]Sulphate is thus a poor marker for the major oligomeric mucins produced by bovine tracheal epithelial cells but the radiolabel is incorporated into a heavily labelled mucin-like component.

H 2O 2 induces apoptosis in bovine tracheal epithelial cells in vitro

Oxidants play an important role in the pathogenesis of various airway diseases. Oxidants have been shown to induce two distinct types of cell Death, i.e., apoptosis and necrosis. However, whether oxidants induce apoptosis in airway epithelial cells remains unclear. To address this question, we evaluated the effect of H 2O 2 exposure on bovine tracheal epithelial cells cultured under different conditions. When tracheal epithelial cells were isolated and exposed to H 2O 2 in suspension cultures, they underwent apoptosis as demonstrated by characteristic ultrastructural changes and DNA fragmentation. Interestingly, apoptosis occurred in single cells but not in aggregated cells. In addition, apoptosis was seen in many ciliated and in fewer mucous cells. When tracheal epithelial cells were allowed to attach to the substrate and grow, they became resistant to apoptosis induced by H 2O 2. These results suggest that H 2O 2 can induce apoptosis in airway epithelial cells.

Erythromycin inhibits ATP-induced intracellular calcium responses in bovine tracheal epithelial cells.

Erythromycin (EM) therapy is known to decrease airway secretion in chronic inflammatory airway diseases such as diffuse panbronchiolitis. Airway secretion is regulated by intracellular Ca2+ concentration ([Ca2+]i). To elucidate the intracellular site of action of EM in airway epithelium, we examined the effect of EM on Ca2+ dynamics in cultured bovine tracheal epithelial cells using fura-2. EM per se did not cause any change in [Ca2+]i. Adenosine triphosphate (ATP; 10(-4) M) induced a biphasic [Ca2+]i increase, consisting of a transient response followed by a sustained response. Pretreatment of cells with EM had little effect on the ATP-induced transient Ca2+ response but substantially reduced the sustained response in a dose-dependent manner. Clarithromycin, another 14-membered ring macrolide, likewise showed the inhibitory effect, but ampicillin and cephasolin did not. Uridine triphosphate (UTP; 10(-4) M) induced a biphasic [Ca2+]i increase similar to ATP, and the UTP-induced sustained Ca2+ response was also inhibited by EM. In Ca2+-deficient medium (1 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N, N'-tetraacetic acid [EGTA]) or in the presence of La3+, the sustained Ca2+ response disappeared, suggesting that EM may inhibit Ca2+ influx induced by P2u purinoceptor stimulation. In single-cell Ca2+ image analysis, low concentration of ATP (10(-6) M) induced Ca2+ oscillations, which were also inhibited by EM. The disappearance of [Ca2+]i oscillations after addition of EM was similar to that after addition of EGTA. These results suggest that EM may decrease Ca2+-dependent airway secretion by inhibiting agonist-stimulated Ca2+ influx.

Activation of bovine tracheal chloride channels by amino group‐specific reagents

1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.

Rapid Induction of Signal Transduction in Respiratory Epithelial Cells in Response to Pseudomonas aeruginosa Ligands ♦ 898

Studies of bronchoalveolar lavage from infants with cystic fibrosis (CF) have demonstrated increased amounts of proinflammatory cytokines and polymorphonuclear leukocytes even in the absence of detectable infection. Decreased clearance of secretions in the respiratory tree may lead to exposure of epithelial cells to environmental organisms that would be efficiently cleared in a normal host. We hypothesized that even transient exposure to Pseudomonas aeruginosa (PA) ligands, which recognize specific glycolipid receptors on respiratory epithelial cells, can lead to activation of host signal transduction pathways. Fluxes in intracellular calcium concentration ([Ca2+]i) are common components of proinflammatory signalling cascades and can be readily monitored by fluorescence imaging. We studied the kinetics of changes in [Ca2+]i of epithelial cells in response to wild-type PA as well as to several mutant strains, purified PA ligands, and antibody to the pilin receptor. Single cell imaging was performed on primary human nasal polyp, bovine tracheal epithelial cells, and SV-40 immortalized respiratory cell lines (1HAEo- and 16HBE). The cells were loaded with fura-2/AM, a calcium-sensitive, ratiometric fluorescent dye. Stimulation of the cells with wild-type PA (strain PAO1) triggered a 100-fold increase in [Ca2+]i that was maintained for several minutes. UV-killed organisms did not elicit a change in [Ca2+]i. Stimulation with a non-piliated strain (PAO-NP) or a non-piliated non-flagellated strain (PAO-NP fliA) led to a greatly attenuated calcium response by the epithelial cells. Purified PAO1 flagellin induced a more rapid increase in [Ca2+]i that returned to baseline in less than one minute. Treatment with purified pilin protein or antibody to asialoGM1 (the pilin receptor) led to a typical [Ca2+]i oscillatory response in the epithelial cells. All of the above responses were attenuated but not eliminated by chelation of extracellular calcium with EGTA, indicating that intracellular stores are likely to be responsible for these signals. These results demonstrate activation of signal transduction pathways in respiratory cells in response to only brief exposure to P. aeruginosa ligands.

Tannin inhibits the cAMP-beta-adrenergic receptor pathway in bovine tracheal epithelium.

Tannin, isolated from cotton bracts, inhibits chloride secretion in airway epithelium. In bovine tracheal epithelial cells, tannin (25 micrograms/ml) blunted isoproterenol (Iso)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Inhibition was time and dose dependent, with 52 +/- 5% (mean +/- SE, n = 6) inhibition at 60 min and 82 +/- 9% (n = 3) inhibition at 8 h. Inhibition was reversible starting at 4 h. Low-molecular-mass tannin (1,000-5,000 Da) had no effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP accumulation. Tannin exposure (25 micrograms/ml for 30 min) had no effect on the dissociation constant (Kd) for [3H]dihydroalprenolol (DHA) (0.41 +/- 0.03 nM, n = 3) but decreased maximal binding from 252 +/- 32 to 162 +/- 36 fmol/mg protein. Using single-point analysis and [3H]CGP-12177, we determined that tannin (25 micrograms/ml for 4 h) decreased surface beta-adrenergic receptor density from 26.4 +/- 4.3 (n = 12) to 11.9 +/- 3.0 fmol/mg protein and that the decrease was dose dependent. Agonist binding affinity by Iso displacement of DHA demonstrated a two-site model (Kd values = 27 +/- 9 and 2,700 +/- 600 nM) and a ratio of high- to low-affinity receptors of 1:1. Tannin (25 micrograms/ml) steepened the curve and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had no further effect on the shape or position of the displacement curve in the presence of tannin. In contrast, when polymer length was decreased by oxidation, tannin had no effect on the DHA displacement curve. These data demonstrate that tannin reversibly desensitizes bovine tracheal epithelial cells to Iso, decreases beta-adrenergic receptor density, and uncouples the receptor from its stimulatory G protein. These data also suggest that the polymer length of tannin and its interaction with cysteine residues are important for these effects. These studies provide additional evidence for the role of tannin in the occupational lung disease byssinosis.