Murine Tracheal Rings Research Articles

Secondary Metabolites in Allergic Plant Pollen Samples Modulate Afferent Neurons and Murine Tracheal Rings.

Plant pollens are strong airborne elicitors of asthma. Their proteinaceous allergens have been studied intensively, but little is known about a possible contribution of pollen secondary metabolites to the nonallergic exacerbation of asthma. Pollen samples originating from 30 plant species were analyzed by HPLC coupled to PDA, ESIMS, and ELSD detectors and off-line NMR spectroscopy. Polyamine conjugates, flavonoids, and sesquiterpene lactones were identified. Polyamine conjugates were characteristic of all Asteraceae species. The presence of sesquiterpene lactones in Asteraceae pollen varied between species and pollen lots. All plant pollen, including those from non-Asteraceae species, contained to some extent electrophiles as determined by their reaction with N-acetyl-l-cysteine. Selected pollen extracts and pure compounds were tested in murine afferent neurons and in murine tracheal preparations. Tetrahydrofuran extracts of Ambrosia artemisiifolia and Ambrosia psilostachya pollen and a mixture of sesquiterpene lactones coronopilin/parthenin increased the intracellular Ca2+ concentration in 15%, 32%, and 37% of cinnamaldehyde-responsive neurons, respectively. In organ bath experiments, only the sesquiterpene lactones tested induced a weak dilatation of naïve tracheas and strongly lowered the maximal methacholine-induced tracheal constriction. A tetrahydrofuran extract of A. psilostachya and coronopilin/parthenin led to a time-dependent relaxation of the methacholine-preconstricted trachea. These results provide the first evidence for a potential role of pollen secondary metabolites in the modulation of the tracheal tone.

Attenuation of airway smooth muscle contractility via flavonol-mediated inhibition of phospholipase-Cβ.

Enhanced contractility of airway smooth muscle (ASM) is a major pathophysiological characteristic of asthma. Expanding the therapeutic armamentarium beyond β-agonists that target ASM hypercontractility would substantially improve treatment options. Recent studies have identified naturally occurring phytochemicals as candidates for acute ASM relaxation. Several flavonoids were evaluated for their ability to acutely relax human and murine ASM ex vivo and murine airways in vivo and were evaluated for their ability to inhibit procontractile signaling pathways in human ASM (hASM) cells. Two members of the flavonol subfamily, galangin and fisetin, significantly relaxed acetylcholine-precontracted murine tracheal rings ex vivo (n = 4 and n = 5, respectively, P < 0.001). Galangin and fisetin also relaxed acetylcholine-precontracted hASM strips ex vivo (n = 6-8, P < 0.001). Functional respiratory in vivo murine studies demonstrated that inhaled galangin attenuated the increase in lung resistance induced by inhaled methacholine (n = 6, P < 0.01). Both flavonols, galangin and fisetin, significantly inhibited purified phosphodiesterase-4 (PDE4) (n = 7, P < 0.05; n = 7, P < 0.05, respectively), and PLCβ enzymes (n = 6, P < 0.001 and n = 6, P < 0.001, respectively) attenuated procontractile Gq agonists' increase in intracellular calcium (n = 11, P < 0.001), acetylcholine-induced increases in inositol phosphates, and CPI-17 phosphorylation (n = 9, P < 0.01) in hASM cells. The prorelaxant effect retained in these structurally similar flavonols provides a novel pharmacological method for dual inhibition of PLCβ and PDE4 and therefore may serve as a potential treatment option for acute ASM constriction.

Exploiting functional domains of GRK2/3 to alter the competitive balance of pro‐ and anticontractile signaling in airway smooth muscle

To clarify the potential utility of targeting GRK2/3-mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G-protein-coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA-mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist-induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (∼ 30-90%) increase in maximal β-agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C-terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase-dead holo-GRK2 diminished (∼ 30-70%) both PI hydrolysis and Ca(2+) mobilization by every Gq-coupled receptor examined. Under conditions of GRK2 C-terminal expression, β-agonist inhibition of methacholine-stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ∼ 30-50% greater β-agonist-mediated relaxation of methacholine-induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the β2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating β-agonist.

&lt;em&gt;In vitro&lt;/em&gt; Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways(1,2). In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations. Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are G(i/o ;)and Gq coupled receptors, respectively(3,4,5). M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum(3,6,7). M2/G(i/o ;)signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels(5,8,9,10). These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle(11). In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")(4,7). More detailed reviews on signaling pathways controlling airway constriction can be found(4,12). The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine(13,14), both well-known ASM modulators in humans and other species(5,15). Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K(+), Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

The αvβ6 integrin modulates airway hyperresponsiveness in mice by regulating intraepithelial mast cells

Allergic asthma is the most common form of asthma, affecting more than 10 million Americans. Although it is clear that mast cells have a key role in the pathogenesis of allergic asthma, the mechanisms by which they regulate airway narrowing in vivo remain to be elucidated. Here we report that mice lacking αvβ6 integrin are protected from exaggerated airway narrowing in a model of allergic asthma. Expression microarrays of the airway epithelium revealed mast cell proteases among the most prominent differentially expressed genes, with expression of mouse mast cell protease 1 (mMCP-1) induced by allergen challenge in WT mice and expression of mMCP-4, -5, and -6 increased at baseline in β6-deficient mice. These findings were most likely explained by loss of TGF-β activation, since the epithelial integrin αvβ6 is a critical activator of latent TGF-β, and in vitro-differentiated mast cells showed TGF-β-dependent expression of mMCP-1 and suppression of mMCP-4 and -6. In vitro, mMCP-1 increased contractility of murine tracheal rings, an effect that depended on intact airway epithelium, whereas mMCP-4 inhibited IL-13-induced epithelial-independent enhancement of contractility. These results suggest that intraepithelial activation of TGF-β by the αvβ6 integrin regulates airway responsiveness by modulating mast cell protease expression and that these proteases and their proteolytic substrates could be novel targets for improved treatment of allergic asthma.

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFα and IL-13

BackgroundDuring pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.MethodExpression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.ResultsPCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.ConclusionTaken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.

Pharmacology and immunological actions of a herbal medicine ASHMITM on allergic asthma

AbstractAllergic asthma is a chronic and progressive inflammatory disease for which there is no satisfactory treatment. Studies reported tolerability and efficacy of an anti‐asthma herbal medicine intervention (ASHMI) for asthma patients, developed from traditional Chinese medicine. To investigate the pharmacological actions of ASHMI on early‐ and late‐phase airway responses (EAR and LAR), Ovalbumin (OVA)‐sensitized mice received 6 weeks of ASHMI treatment beginning 24 h following the first intratracheal OVA challenge. EAR were determined 30 min following the fourth challenge and LAR 48 h following the last challenge. ASHMI effects on cytokine secretion, murine tracheal ring contraction and human bronchial smooth muscle cell prostaglandin (PG) production were also determined.ASHMI abolished EAR, which was associated with significantly reduced histamine, leukotriene C4, and OVA‐specific IgE levels, as well as LAR, which was associated with significantly reduced bronchoalveolar lavage fluid (BALF) eosinophils, decreased airway remodeling, and lower Th2 cytokine levels in BALF and splenocyte cultures. Furthermore, ASHMI inhibited contraction of murine tracheal rings and increased production of the potent smooth muscle relaxer PGI2. ASHMI abrogation of allergic airway responses is associated with broad effects on asthma pathological mechanisms. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Essential role of IFN β and CD38 in TNF α-induced airway smooth muscle hyper-responsiveness

We recently identified autocrine interferon (IFN) β as a novel mechanism mediating tumor necrosis factor (TNF) α-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFN β in regulating TNF α-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNF α-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFN β antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNF α-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFN β prevented TNF α enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus ( Af)), we found heightened expression of both IFN β and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFN β and CD38 may play a significant role in the development of TNF α-associated AHR.

Effects of estrogen on bronchial hyperresponsiveness of ovariectomized murine tracheal rings sensitized with human asthmatic serum.

Exogenous estrogen or increased estrogen levels in pregnancy decrease asthma severity, suggesting that sex hormones may modulate the frequency and severity of asthma exacerbations and raising the possibility that estrogen may affect airway smooth muscle function and hyper-responsiveness in asthma. Therefore, we studied the effects of estrogen replacement therapy on carbachol-induced constriction of mouse tracheal rings. Female mice were ovariectomized (OVX) and implanted subcutaneously with an estrogen pellet releasing either 5μg or 15μg estradiol/day. After the 3 weeks estrogen treatment, animals were sacrificed and tracheal rings were sensitized overnight in medium containing serum from asthmatic (IgE> 1,000U) or non asthmatic (IgE <70U) humans and used for tension studies. The maximal contractile force (Fmax) to carbachol in rings from OVX mice sensitized with serum from asthmatic patients (ATR) was much higher than the Fmax in ATR from OVX mice receiving estrogen replacement and similar to the Fmax of ATR from male mice. In male mice, Fmax to carbachol in tracheal rings treated with serum from control patients (CTR) was 3.8±0.6g, compared to 6.1 ± 1.0g in ATR from male mice and 5.6±0.9g in ATR from female OVX mice. Conversely, Fmax to carbachol was 2.4 ± 0.3g in ATR from female OVX mice treated with 5μg estradiol/day and 2.7 ± 0.4 g in ATR from female OVX mice treated with 15μg estradiol/day. These results indicate that ovariectomized female mice are more susceptible to the asthmatic phenotype than ovariectomized female mice receiving estrogen replacement therapy, and suggest a protective role of estrogen replacement therapy in asthma.

TGF-β potentiates airway smooth muscle responsiveness to bradykinin

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-beta has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-beta can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-beta directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-beta alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-beta-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B(2)) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-beta on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-beta, in a dose- (0.5-10 ng/ml) and time- (2-24 h) dependent manner, increased mRNA and protein expression of the B(2) receptor in cultured human ASM cells. Maximal B(2) receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-beta treatment and was further confirmed using fluorescence microscopy. TGF-beta (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-beta-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-beta may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B(2) receptor expression and signaling.

IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle.

Growing evidence suggests that interleukin (IL)-13, a Th2-type cytokine, plays a critical role in the development of bronchial hyper-responsiveness (BHR), an essential feature of asthma, although the underlying mechanisms remain unknown. In the present study, we investigated whether IL-13 directly affects airway smooth muscle (ASM) function. In murine tracheal rings, IL-13 (100 ng ml-1, 24 h) significantly increased both the carbachol- and KCl-induced maximal force generation without affecting ASM sensitivity. In cultured human ASM cells, IL-13 (50 ng ml-1, 24 h) also augmented cytosolic calcium levels to bradykinin, histamine and carbachol by 60, 35 and 26%, respectively. The present study demonstrates that IL-13 may promote BHR by directly modulating ASM contractility, an effect that may be due to enhanced G protein-coupled receptor (GPCR)-associated calcium signaling.

Commentary on Selected Contributions

HIGHLIGHTED TOPICSAirway Hyperresponsiveness: From Molecules to BedsideCommentary on Selected ContributionsGary C. Sieck, Gary C. SieckJournal of Applied Physiology, EditorPublished Online:01 Aug 2003https://doi.org/10.1152/japplphysiol.00576.2003MoreSectionsPDF (22 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Growing evidence suggests that changes in airway smooth muscle function play a role in the pathogenesis of asthma. Recently, great attention has been paid to the links between airway inflammation and airway smooth muscle function in order to better understand the development of airway hyperresponsiveness, a defining feature of asthma. Studies, using cultured airway smooth muscle cells or isolated human and animal airway preparations, presently suggest that modulation of G-protein-coupled receptor signaling in airway smooth muscle may represent one potential mechanism by which cytokines regulate the development of bronchial hyperresponsiveness. In this context, it has been suggested that tumor necrosis factor-α (TNF-α) potentially causes airway hyperresponsiveness via engagement of TNFR1, TNFR2, and CD38/cADP ribose pathways.In the first Highlighted Topics article featured in this issue of the Journal of Applied Physiology, “TNF-α modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCL,” Chen et al. examined the involvement of structural or functional alterations of airway smooth muscle induced by TNF-α in isolated murine tracheal rings. These investigators found that TNF-α acting via TNFR1 and a PTX-sensitive G-protein-coupled receptor pathway enhanced force generation in response to both cholinergic stimulation and KCl stimulation while impairing the relaxation induced by a β-adrenoceptor agonist. These results support the concept that modulation of airway smooth muscle contractility may play an important role in TNF-α (TNFR1)-induced airway hyperresponsiveness in chronic lung diseases by altering G-protein-coupled receptor responsiveness. Understanding the critical pathways associated with TNF-α and TNFR1 activation in murine tracheal preparations may represent a useful model to investigate the molecular mechanisms leading to airway hyperresponsiveness and may offer new therapeutic approaches to treatment of chronic lung diseases characterized by high levels of TNF-α in the airways.In the second article featured in this issue, “How does airway inflammation modulate asthmatic airway constriction? An antigen challenge study,” Henderson et al. examined how the extent of airway inflammation may, or may not, dictate the phenotypic expression of asthma in a given patient. Motivating this study were the recent findings of these investigators, which revealed that asthmatic lungs constrict heterogeneously and that asthmatic airways cannot dilate to the same degree as those from normal lungs during a deep inspiration. Indeed, the latter finding implies that airway smooth muscle resides in a distinctly stiff and reactive state in the asthmatic but not the normal lung. The question posed was “How does this apparent smooth muscle defect interact with inflammation to produce the clinical features of asthma?” In nine asthmatic patients, these investigators performed an allergen study to measure mechanical phenotype before and after enhanced airway inflammation. The earlyphase response to inhaled allergen reflects the impact of acute release of bronchoconstrictor mediators (e.g., histamine), whereas, during the late phase, mediators from neutrophils and eosinophils are released within the airways, resembling what occurs during an asthma attack. These results show that, after allergen exposure, heterogeneity and reduced capacity to maximally dilate airways are key features of the physiological response, regardless of whether provoked acutely (early phase) or with conditions akin to chronic inflammation (late phase). The implication of these results is that the endogenous constrictor milieu of chronic inflammation may serve to sustain smooth muscle tone and conditions that permit hyperreactivity and the resulting heterogeneity. In particular, heterogeneity motivates concern. Local marked constriction affecting only a small percentage of airways not only can greatly amplify dynamic lung resistance and elastance at typical breathing rates but can cause hypoxemia by altering ventilation-perfusion matching. It can also diminish dilator response to a deep inspiration and can produce refractoriness to inhaled medications by preventing their distribution to the most severely affected regions of the lung. Defining the biological basis for this pronounced and consistent heterogeneity and the diminished airway dilatory response may provide insight into key features of the asthmatic phenotype.This article has no references to display. Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 95Issue 2August 2003Pages 863-863 Copyright & PermissionsCopyright © 2003 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00576.2003History Published online 1 August 2003 Published in print 1 August 2003 PDF download Metrics Downloaded 45 times

TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl.

Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-alpha, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF-alpha also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-alpha, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF-alpha also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF-alpha on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF-alpha receptor (TNFR)1-deficient mice and in control rings treated with a TNF-alpha mutant that solely activates TNFR2. TNF-alpha also attenuated relaxation responsiveness to isoproterenol but not to PGE2 or forskolin. TNF-alpha modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of Gialpha proteins. Taken together, these data suggest that TNF-alpha may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and beta-adrenoceptor GPCR agonists.