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Bitter taste receptors (TAS2Rs) of the tongue likely evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants, leading to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased ASM [Ca2+]i in a Gβγ, PLCβ and IP3-receptor dependent manner which would be expected (like acetylcholine) to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM, and dilation of airways that was 3-fold greater than β-agonists. Relaxation by TAS2Rs is from a localized [Ca2+]i response at the cell membrane which opens BKCa channels leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in an asthma mouse model. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation.

Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca2+-activated K+ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca2+ events nor alter spontaneous local Ca2+ transients. Interestingly, they increase global intracellular [Ca2+]i, although to a much lower level than bronchoconstrictors. We show that these Ca2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca2+]i induced by bronchoconstrictors, and this lowering of the [Ca2+]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca2+ channels (VDCCs), resulting in reversal in [Ca2+]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca2+ signaling pathways via Gβγ to increase [Ca2+]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca2+]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

Author Summary

Bitter taste receptors (TAS2Rs), a G-protein-coupled receptor family long thought to be solely expressed in taste buds on the tongue, have recently been detected in airways. Bitter substances can activate TAS2Rs in airway smooth muscle to cause greater bronchodilation than β2 adrenergic receptor agonists, the most commonly used bronchodilators. However, the mechanisms underlying this bronchodilation remain elusive. Here we show that, in resting primary airway smooth muscle cells, bitter tastants activate a TAS2R-dependent signaling pathway that results in an increase in intracellular calcium levels, albeit to a level much lower than that produced by bronchoconstrictors. In bronchoconstricted cells, however, bitter tastants reverse the bronchoconstrictor-induced increase in calcium levels, which leads to the relaxation of smooth muscle cells. We find that this reversal is due to inhibition of L-type calcium channels. Our results suggest that under normal conditions, bitter tastants can activate TAS2Rs to modestly increase calcium levels, but that when smooth muscle cells are constricted, they can block L-type calcium channels to induce bronchodilation. We postulate that this novel mechanism could operate in other extraoral cells expressing TAS2Rs.

Background

Chronic use of long-acting β2-adrenergic receptor (β2AR) agonists (LABAs), resulting in β2AR desensitization, has been associated with increased asthma morbidity. When LABAs are used in combination with inhaled glucocorticoids (GCs), however, asthma control is improved, raising the question: Do GCs inhibit the proasthmatic mechanism that mediates altered contractility in LABA-exposed airway smooth muscle (ASM)?

Objective

This study aimed to identify the potential protective role and mechanism of action of GCs in mitigating the effects of prolonged LABA exposure on ASM constrictor and relaxation responsiveness.

Methods

Cultured human ASM (HASM) cells and isolated rabbit ASM tissues were examined for induced changes in agonist-mediated cAMP accumulation, constrictor and relaxation responsiveness, and expression of specific GC-regulated molecules following 24h exposure to the LABA, salmeterol, in the absence and presence of dexamethasone (DEX).

Results

Salmeterol-exposed ASM exhibited impaired cAMP and relaxation responses to isoproterenol and increased acetylcholine-induced contractility. These pro-asthmatic effects of prolonged LABA exposure were attributed to upregulated phosphodiesterase 4 (PDE4) activity, and ablated by pretreatment with DEX. Further studies demonstrated that: 1) DEX suppressed activation of the mitogen-activated protein kinase (MAPK), ERK1/2, which upregulates PDE4 expression in salmeterol-exposed ASM; and 2) the inhibitory actions of DEX on salmeterol-induced ERK1/2 activation and resultant PDE4-mediated changes in ASM responsiveness were prevented by gene silencing or pharmacological inhibition of DEX-induced expression of MAPK phosphatase-1 (MKP-1), an endogenous deactivator of ERK1/2 signaling.

Conclusion

GCs prevent the adverse proasthmatic effects of prolonged LABA exposure on airway responsiveness due to GC-induced upregulation of MKP-1, which inhibits proasthmatic ERK1/2 signaling in the LABA-exposed ASM.

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane–spanning receptors activated during asthma, or by treatment with bronchodilators such as β2–adrenergic receptor (β2AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP1 receptor, since its endogenous agonist prostaglandin E2 is abundant in the airway, but its functional implications are poorly defined. Activation of EP1 failed to elicit ASM contraction in mouse trachea via this Gαq-coupled receptor. However, EP1 activation markedly reduced the bronchodilatory function of β2AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP1 reduced β2AR-stimulated cAMP in ASM but did not promote or augment β2AR phosphorylation or alter β2AR trafficking. Bioluminescence resonant energy transfer showed EP1 and β2AR formed heterodimers, which were further modified by EP1 agonist. In cell membrane [35S]GTPγS binding studies, the presence of the EP1 component of the dimer uncoupled β2AR from Gαs, an effect accentuated by EP1 agonist activation. Thus alone, EP1 does not appear to have a significant direct effect on airway tone but acts as a modulator of the β2AR, altering Gαs coupling via steric interactions imposed by the EP1:β2AR heterodimeric signaling complex and ultimately affecting β2AR-mediated bronchial relaxation. This mechanism may contribute to β-agonist resistance found in asthma.

Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.

Background

β2-adrenoceptor agonists elicit bronchodilator responses by binding to β2-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β2-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.

Methods

Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β2-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.

Results

Temporal analysis revealed that in hASM cells the β2-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.

Conclusions

We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β2-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.

In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.

In asthma, the increase in airway smooth muscle (ASM) can contribute to inflammation, airway wall remodeling and airway hyperresponsiveness (AHR). Targetting peroxisome proliferator-activated receptor γ (PPARγ), a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPARγ ligands, such as rosiglitazone (RGZ) and pioglitazone (PGZ), inhibit proliferation and inflammatory cytokine production from ASM in vitro. In addition, inhaled administration of these ligands reduces inflammatory cell infiltration and airway remodelling in mouse models of allergen-induced airways disease. PPARγ ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPARγ-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to β2-adrenoceptor agonists and inhibit the development of AHR associated with exposure to nicotine in utero or following allergen challenge. Of particular interest, a small clinical trial has shown that oral RGZ treatment improves lung function in smokers with asthma, a group that is generally unresponsive to conventional steroid treatment. These combined findings support further investigation of the potential for PPARγ agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.

Background and Objective

Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.

Objective

To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.

Methods

We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.

Results

In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.

Conclusion

Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

Bronchodilators are the first line therapy during acute asthmatic exacerbations to reverse airway obstruction primarily by relaxing airway smooth muscle. Only three categories of bronchodilators exist in clinical practice: β-adrenergic agonists, anticholinergics, and methylxanthines. Each of these categories have specific drugs dating back to the early 20th century, raising the question of whether or not we can find better bronchodilators. While caffeine, theophylline, atropine, and epinephrine were the first generations of therapeutics in each of these drug classes, there is no question that improvements have been made in the bronchodilators in each of these classes. In the following editorial, we will briefly describe new classes of potential bronchodilators including: novel PDE inhibitors, natural phytotherapeutics, bitter taste receptor ligands, and chloride channel modulators, which have the potential to be used alone or in combination with existing bronchodilators to reverse acute airway obstruction in the future.

Airway smooth muscle (ASM) plays a pivotal role in modulating bronchomotor tone but also orchestrates and perpetuates airway inflammation and remodeling. Despite substantial research, there remain important unanswered questions. In 2006, the National Heart, Lung, and Blood Institute sponsored a workshop to define new directions in ASM biology. Important questions concerning the key functions of ASM include the following: Does developmental dysregulation of ASM function promote airway disease, what key signaling pathways in ASM evoke airway hyperresponsiveness in vivo, do alterations in ASM mass affect excitation–contraction coupling, and can ASM modulate airway inflammation and remodeling in a physiologically relevant manner? This workshop identified critical issues in ASM biology to delineate areas for scientific investigation in the identification of new therapeutic and diagnostic approaches in asthma, chronic obstructive pulmonary disease, and cystic fibrosis.

Chronic use of inhaled beta-agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta-agonist-promoted desensitization of airway smooth muscle beta-2-adrenergic receptors, mediated by G protein-coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta-arrestin-2 gene ablation augments beta-agonist-mediated airway smooth muscle relaxation, while augmenting beta-agonist-stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA-mediated knockdown of arrestins also augments beta-agonist-stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E2 receptors were not affected by either beta-arrestin-2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta-2-adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.—Deshpande, D. A., Theriot, B. S., Penn, R. B., Walker, J. K. L. β-Arrestins specifically constrain β2-adrenergic receptor signaling and function in airway smooth muscle.

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed Gs-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gβγ sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via β-agonist stimulation of the beta-2-adrenergic receptor (β2AR). Conversely, neither 5′-(N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the β-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to Gs-coupled receptors in ASM, GRK2/3 selectively attenuates β2AR signaling, yet relief of GRK2/3-dependent β2AR desensitization does not influence at least one important physiological function of the receptor.

T-helper type 2 (Th2) cytokines have been implicated in the pathogenesis of the pulmonary inflammatory response and altered bronchial responsiveness in allergic asthma. To elucidate the mechanism of Th2-dependent mediation of altered airway responsiveness in the atopic asthmatic state, the expression and actions of specific cytokines were examined in isolated rabbit and human airway smooth muscle (ASM) tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal isometric contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with either an IL-5 receptor blocking antibody (IL-5ra) or the human recombinant IL-1 receptor antagonist (IL-1ra), whereas an IL-4 neutralizing antibody had no effect. Moreover, relative to controls, atopic asthmatic sensitized ASM cells demonstrated an initial, early (after 3 hours of incubation) increased mRNA expression and protein release of IL-5. This was followed (after 6 hours of incubation) by an enhanced mRNA expression and release of IL-1β protein, an effect that was inhibited in sensitized cells pretreated with IL-5ra. Extended studies demonstrated that naive ASM exposed to exogenously administered IL-5 exhibited an induced upregulated mRNA expression and protein release of IL-1β associated with proasthmatic-like changes in ASM constrictor and relaxant responsiveness, and that these effects were ablated in tissues pretreated with IL-1ra. Taken together, these observations provide new evidence that (a) the Th2 cytokine IL-5 and the pleiotropic proinflammatory cytokine IL-1β are endogenously released by atopic asthmatic sensitized ASM and mechanistically interact to mediate the proasthmatic perturbations in ASM responsiveness; and (b) the nature of this interaction is given by an initial endogenous release of IL-5, which then acts to induce the autologous release of IL-1β by the sensitized ASM itself, resulting in its autocrine manifestation of the proasthmatic phenotype.

Asthma is characterized by the association of airway hyperresponsiveness (AHR), inflammation, and remodelling. The aim of the present article is to review the pivotal role of airway smooth muscle (ASM) in the pathophysiology of asthma. ASM is the main effector of AHR. The mechanisms of AHR in asthma may involve a larger release of contractile mediators and/or a lower release of relaxant mediators, an improved ASM cell excitation/contraction coupling, and/or an alteration in the contraction/load coupling. Beyond its contractile function, ASM is also involved in bronchial inflammation and remodelling. Whereas ASM is a target of the inflammatory process, it can also display proinflammatory and immunomodulatory functions, through its synthetic properties and the expression of a wide range of cell surface molecules. ASM remodelling represents a key feature of asthmatic bronchial remodelling. ASM also plays a role in promoting complementary airway structural alterations, in particular by its synthetic function.

Airway hyperresponsiveness (AHR) is one of the cardinal features of asthma. Contraction of airway smooth muscle (ASM) cells that line the airway wall is thought to influence aspects of AHR, resulting in excessive narrowing or occlusion of the airway. ASM contraction is primarily controlled by agonists that bind G protein-coupled receptor (GPCR), which are expressed on ASM. Integrins also play a role in regulating ASM contraction signaling. As therapies for asthma are based on symptom relief, better understanding of the crosstalk between GPCRs and integrins holds good promise for the design of more effective therapies that target the underlying cellular and molecular mechanism that governs AHR. In this paper, we will review current knowledge about integrins and GPCRs in their regulation of ASM contraction signaling and discuss the emerging concept of crosstalk between the two and the implication of this crosstalk on the development of agents that target AHR.

Background

Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.

Methodology/Principal Findings

In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.

Conclusion/Significance

Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.

Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human δ opioid receptors (hDOR) by measuring guanosine 5′-O-(3-[35S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of β-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of β-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced β-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.

Background

Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines.

Methods

Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)‐1β, IL‐4, and IL‐13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF‐β, and SCF in the supernatants were measured and the effect of non‐asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined.

Results

Human lung mast cells and HMC‐1 cells migrated towards Th2 stimulated ASM from asthmatics but not non‐asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non‐asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant.

Conclusion

Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non‐asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma.

CD4+ T helper (TH)1- and TH2-type cytokines reportedly play an important role in the pathobiology of asthma. Recent evidence suggests that proasthmatic changes in airway smooth muscle (ASM) responsiveness may be induced by the autocrine release of certain proinflammatory cytokines by the ASM itself. We examined whether TH1- and TH2-type cytokines are expressed by atopic asthmatic sensitized ASM and serve to autologously regulate the proasthmatic phenotype in the sensitized ASM. Expression of these cytokines and their receptors was examined in isolated rabbit and human ASM tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or control subjects. Relative to controls, atopic sensitized ASM cells exhibited an early increased mRNA expression of the TH2-type cytokines, interleukin-5 (IL-5) and granulocyte–macrophage colony-stimulating factor (GM-CSF), and their receptors. This was later followed by enhanced mRNA expression of the TH1-type cytokines, IL-2, IL-12, and interferon-γ (IFN-γ), as well as their respective receptors. In experiments on isolated ASM tissue segments (a) exogenous administration of IL-2 and IFN-γ to atopic asthmatic serum–sensitized ASM ablated both their enhanced constrictor responsiveness to acetylcholine (ACh) and their attenuated relaxation responsiveness to β-adrenoceptor stimulation with isoproterenol, and (b) administration of IL-5 and GM-CSF to naive ASM induced significant increases in their contractility to ACh and impaired their relaxant responsiveness to isoproterenol. Collectively, these observations provide new evidence demonstrating that human ASM endogenously expresses both TH1- and TH2-type cytokines and their receptors, that these molecules are sequentially upregulated in the atopic asthmatic sensitized state, and that they act to downregulate and upregulate proasthmatic perturbations in ASM responsiveness, respectively.

Background

Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Methods

Primary Human ASM cells were isolated from explants of trachealis muscle from individuals with no history of respiratory disease. The effect of cytokine or asthma medication on histamine or bradykinin induced IPx signalling was assessed by [3H] inositol incorporation. Quantitative Real Time PCR was used to measure mRNA levels of receptors and downstream signalling components. Transcriptional mechanisms were explored using a combination of 5'Rapid Amplification of cDNA Ends (5'RACE) and promoter-reporter techniques.

Results

Treatment of Human ASM cells with IL-13, IFNγ or salmeterol for 24 hours lead to a modest augmentation of histamine induced IPx responses (144.3 +/- 9.3, 126.4 +/- 7.5 and 117.7 +/- 5.2%, p < 0.05). Similarly, TNFα, IFNγ or salmeterol treatment augmented bradykinin induced IPx responses (127.4 +/- 8.3, 128.0 +/- 8.4 and 111.7 +/- 5.0%, P < 0.05). No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus. Analyses of mRNA expression of components of the IPx pathway i.e. H1 Histamine Receptor (HRH1), B2 Bradykinin Receptor (BDKRB2), Gαq/11 and PLC-β1 identified that a significant induction of receptor mRNA (>2 fold) was a feature of these responses explaining the cytokine and spasmogen specificity. The HRH1 and BDKRB2 promoter regions were mapped in ASM and promoter-reporter analyses identified that salmeterol can induce HRH1 (>2 fold) and BDKRB2 (2–5 fold) transcription. The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.

Conclusion

Our results indicate that the spasmogen specific receptor locus may be a key site of regulation determining the magnitude of spasmogen mediated ASM IPx responses during airway inflammation or following asthma medication. These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

Airway hyperresponsiveness is a cardinal feature of asthma but remains largely unexplained. In asthma, the key end-effector of acute airway narrowing is the airway smooth muscle (ASM) cell. Here we report novel biophysical properties of the ASM cell isolated from the relatively hyporesponsive Lewis rat versus the relatively hyperresponsive Fisher rat. We focused upon the ability of the cytoskeleton (CSK) of the ASM cell to stiffen, to generate contractile forces, and to remodel. We used optical magnetic twisting cytometry to measure cell stiffness and traction microscopy to measure contractile forces. To measure remodeling dynamics, we quantified spontaneous nanoscale motions of a microbead tightly anchored to the CSK. In response to a panel of contractile and relaxing agonists, Fisher ASM cells showed greater stiffening, bigger contractile forces, and faster CSK remodeling; they also exhibited higher effective temperature of the CSK matrix. These physical differences measured at the level of the single cell in vitro were consistent with strain-related differences in airway responsiveness in vivo. As such, comprehensive biophysical characterizations of CSK dynamics at the level of the cell in culture may provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma.

Airway hyperresponsiveness is a cardinal feature of asthma but remains largely unexplained. In asthma, the key end-effector of acute airway narrowing is the airway smooth muscle (ASM) cell. Here we report novel biophysical properties of the ASM cell isolated from the relatively hyporesponsive Lewis rat versus the relatively hyperresponsive Fisher rat. We focused upon the ability of the cytoskeleton (CSK) of the ASM cell to stiffen, to generate contractile forces, and to remodel. We used optical magnetic twisting cytometry to measure cell stiffness and traction microscopy to measure contractile forces. To measure remodeling dynamics, we quantified spontaneous nanoscale motions of a microbead tightly anchored to the CSK. In response to a panel of contractile and relaxing agonists, Fisher ASM cells showed greater stiffening, bigger contractile forces, and faster CSK remodeling; they also exhibited higher effective temperature of the CSK matrix. These physical differences measured at the level of the single cell in vitro were consistent with strain-related differences in airway responsiveness in vivo. As such, comprehensive biophysical characterizations of CSK dynamics at the level of the cell in culture may provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma.

Background

The response to β2-adrenoceptor agonists is reduced in asthmatic airways. This desensitization may be in part due to inflammatory mediators and may involve cysteinyl-leukotrienes (cysteinyl-LTs). Cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. We tested the hypothesis that leukotriene D4 (LTD4) and allergen challenge cause β2-adrenoceptor desensitization through the activation of protein kinase C (PKC).

Methods

The isoproterenol-induced cAMP accumulation was evaluated in human airway smooth muscle cell cultures challenged with exogenous LTD4 or the PKC activator phorbol-12-myristate-13-acetate with or without pretreatments with the PKC inhibitor GF109203X or the CysLT1R antagonist montelukast. The relaxant response to salbutamol was studied in passively sensitized human bronchial rings challenged with allergen in physiological salt solution (PSS) alone, or in the presence of either montelukast or GF109203X.

Results

In cell cultures, both LTD4 and phorbol-12-myristate-13-acetate caused significant reductions of maximal isoproterenol-induced cAMP accumulation, which were fully prevented by montelukast and GF109203X, respectively. More importantly, GF109203X also prevented the attenuating effect of LTD4 on isoproterenol-induced cAMP accumulation. In bronchial rings, both montelukast and GF109203X prevented the rightward displacement of the concentration-response curves to salbutamol induced by allergen challenge.

Conclusion

LTD4 induces β2-adrenoceptor desensitization in human airway smooth muscle cells, which is mediated through the activation of PKC. Allergen exposure of sensitized human bronchi may also cause a β2-adrenoceptor desensitization through the involvement of the CysLT1R-PKC pathway.