Airway Smooth Muscle Responsiveness Research Articles

Thymic Stromal Lymphopoietin (TSLP) and Airway Smooth Muscle

TSLP is a newly‐identified cytokine thought to be involved in airway inflammation. TSLP levels have been shown to significantly increase in airway smooth muscle (ASM) with chronic obstructive pulmonary disease and other inflammatory diseases. This study evaluated the expression of TSLP in human ASM cells and tissues exposed to cigarette smoke extract (CSE) or to pro‐inflammatory cytokines TNF‐α and IL‐13. In parallel studies, TSLP expression was evaluated in house dust mite sensitized mice with or without cigarette smoke exposure. The presence of TSLP and TSLP‐receptors were verified using Western blot analysis. Exposure to TNF‐α and IL‐13 (50 ng/mL each) for 24 hours resulted in increased TSLP and TSLP‐R expression. Simultaneous exposure to functional TSLP antibody blunted cytokine induced TSLP‐R expression. Interestingly, acute CSE (30 min) down‐regulated TSLP expression, while chornic exposure increased expression. Similarly, [Ca2+]i responses of human ASM to histamine were blunted with chronic CSE exposure, effects reversed by TSLP and TSLP‐R antibodies. Sensitized mice demonstrated an increase in TSLP expression in whole lung samples. This effect was even further enhanced in dust mite sensitized mice exposed to chronic cigarette smoke. These results indicate a complex role for TSLP in ASM inflammation with or without cigarette smoke exposure.Supported by NIH grants HL088029, HL090595, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

γ-Aminobutryic Acid

γ-Aminobutryic Acid

Extraction of membrane cholesterol disrupts caveolae and impairs serotonergic (5-HT2A) and histaminergic (H1) responses in bovine airway smooth muscle: role of Rho-kinase

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(2A)) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(2A) receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.

Modeling the Response of Airway Smooth Muscle to Cyclic Loading

Airway smooth muscle (ASM) exhibits complex contractile dynamics and has a highly disordered structure. This contrasts with skeletal muscle which contains ordered arrays of contractile filaments aligned with the long axes of the cells. Models of ASM, however, are often based on Huxley's cross-bridge model, which was developed for skeletal muscle and does not take into account the rheological properties of the non-contractile components of the tissue. Here we use a modeling approach to investigate the relative contributions of tissue viscoelasticity and crossbridge kinetics to the mechanical response of ASM to cyclic loading.Experiments were performed using rat trachealis muscle strips. Breathing was mimicked by applying sinusoidal length oscillations (frequency: 2Hz; amplitude: 1-4%). In unstimulated muscle, peak force during length oscillation followed a typical stress relaxation trajectory. In stimulated muscle, peak force decreased dramatically over the first 5-10 cycles to a level close to the isometric force at the mean length. Furthermore, steady-state peak force decreased as loading amplitude increased. ‘Sighs’ were mimicked by applying a large-amplitude loading cycle (5-25%). Sighs caused a transient but long-lasting reduction in peak force, with the degree of force reduction increasing with sigh amplitude.The response of unstimulated muscle to length oscillation could be reproduced well with a model consisting of a Hill-type contractile element and a parallel elastic element, both in series with a nonlinear Kelvin body (viscoelastic element). In order to reproduce the response of stimulated muscle to length oscillation, cross-bridge kinetics had to be included either using a Huxley-type model or by including first-order cross-bridge attachment and detachment kinetics in the Hill model. The decrement and slow recovery of force after a sigh, however, could not be reproduced by either model, indicating that additional mechanisms are required to explain this phenomenon.

Thermally-induced Activation Of TRPV2 Channels Causes Cell Death In Airway Smooth Muscle

“Bronchial thermoplasty” — the direct application of thermal energy to the airway wall in the clinical setting — leads to reduction of the smooth muscle mass within the airway wall, reduced potential for bronchoconstriction and improvement in asthma symptoms. However the mechanism underlying this response has not yet been elucidated. We found a steep thermal sensitivity of isometric contractions in bovine airway smooth muscle: ∼0%, ∼50% and ∼100% reduction at 55°C, respectively. These changes in contractility developed within minutes after thermal treatment. This thermal sensitivity was shifted to lower temperatures by the TRPV2 agonist 2-APB (2-aminoethoxydiphenyl borate, 10-4 M). Likewise, the TRPV2 agonists 2-APB and tetrahydrocannabinol (10-4 M) evoked a large membrane conductance with linear current-voltage relationship and reversal potential of ∼0 mV. Immunohistochemistry showed TRPV2 to be distributed around the smooth muscle. These observations are all consistent with the involvement of TRPV2 in the thermal response of airway smooth muscle. Oddly, however, temperature-induced ablation of contractions was not prevented by agents which block conductance through TRPV2 channels (ruthenium red; La3+, Gd3+, capsaicin, removal of external Ca2+). We conclude that bronchial thermoplasty activates TRPV2 channels in the muscle, and that this in some way transduces into a disappearance of the smooth muscle cell (and thus loss of contractility). Furthermore, the data suggest these changes are not dependent upon the ionic currents per se through those channels; instead, they may involve a direct interaction between the channels and some intracellular entity(s).

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis.

BackgroundMany cystic fibrosis (CF) patients display airway hyperresponsiveness and have symptoms of asthma such as cough, wheezing and reversible airway obstruction. Chronic airway bacterial colonization, associated with neutrophilic inflammation and high levels of interleukin-8 (IL-8) is also a common occurrence in these patients. The aim of this work was to determine the responsiveness of airway smooth muscle to IL-8 in CF patients compared to non-CF individuals.MethodsExperiments were conducted on cultured ASM cells harvested from subjects with and without CF (control subjects). Cells from the 2nd to 5th passage were studied. Expression of the IL-8 receptors CXCR1 and CXCR2 was assessed by flow cytometry. The cell response to IL-8 was determined by measuring intracellular calcium concentration ([Ca2+]i), cell contraction, migration and proliferation.ResultsThe IL-8 receptors CXCR1 and CXCR2 were expressed in both non-CF and CF ASM cells to a comparable extent. IL-8 (100 nM) induced a peak Ca2+ release that was higher in control than in CF cells: 228 ± 7 versus 198 ± 10 nM (p < 0.05). IL-8 induced contraction was greater in CF cells compared to control. Furthermore, IL-8 exposure resulted in greater phosphorylation of myosin light chain (MLC20) in CF than in control cells. In addition, MLC20 expression was also increased in CF cells. Exposure to IL-8 induced migration and proliferation of both groups of ASM cells but was not different between CF and non-CF cells.ConclusionASM cells of CF patients are more contractile to IL-8 than non-CF ASM cells. This enhanced contractility may be due to an increase in the amount of contractile protein MLC20. Higher expression of MLC20 by CF cells could contribute to airway hyperresponsiveness to IL-8 in CF patients.

GSK-3/β-catenin signaling axis in airway smooth muscle: role in mitogenic signaling

Beta-catenin plays a dual role in cellular signaling by stabilizing cadherin-mediated cell-cell contact and by regulating gene transcription associated with cell cycle progression. Nonetheless, its presence and function in airway smooth muscle have not been determined. We hypothesized a central role for beta-catenin in mitogenic signaling in airway smooth muscle in response to growth factor stimulation. Immunocytochemical and biochemical analysis revealed that human airway smooth muscle cells indeed express abundant beta-catenin, which was localized primarily to the plasma membrane in quiescent cells. Treatment of airway smooth muscle cells with PDGF or FBS induced sustained phosphorylation of glycogen synthase kinase-3 (GSK-3), a negative regulator in its unphosphorylated form that promotes beta-catenin degradation. GSK-3 phosphorylation was also increased in airway smooth muscle cells with a proliferative phenotype compared with quiescent airway smooth muscle cells with a mature phenotype. Parallel with the increase in GSK-3 phosphorylation, growth factor treatment induced an increased expression and nuclear presence of beta-catenin and activated promitogenic signaling in airway smooth muscle, including the phosphorylation of retinoblastoma protein, DNA synthesis ([(3)H]thymidine incorporation), and cell proliferation. Importantly, small interfering RNA knockdown of beta-catenin strongly reduced retinoblastoma protein phosphorylation, [(3)H]thymidine incorporation, and cell proliferation induced by PDGF and FBS. Collectively, these data reveal the existence of a GSK-3/beta-catenin signaling axis in airway smooth muscle that is regulated by growth factors and of central importance to mitogenic signaling.

A Contraction Assay System Using Established Human Bronchial Smooth Muscle Cells

Background: To further understand the mechanisms of airway obstruction in asthma, it is crucial to investigate contractile responses of human airway smooth muscle. An in vitro assay system employing collagen gels embedded with well-established bronchial smooth muscle cells of human origin was explored in the present study. Methods: Commercially available cultured human bronchial smooth muscle cells were embedded into a collagen gel. Well-known constrictors, histamine and methacholine, were added to the gel. The gel images were captured by an image analyzer, and contractile responses were evaluated. Results: Histamine and methacholine induced contraction of the gels in a dose-dependent manner. Pyrilamine, an H₁ receptor antagonist, inhibited gel contraction in an agonist-specific manner. Conclusion: Our contraction assay system, employing widely distributed cultured cells, was highly reproducible and precise. It may go a long way toward understanding mechanisms of asthmatic responses and evaluation of antiasthma drugs.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca(2+) release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca(2+) response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca(2+) responses to histamine. The augmentation of Ca(2+) signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca(2+) responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

The Effects of Leptin on Airway Smooth Muscle Responses

Obesity is associated with asthma and airway hyperresponsiveness. Leptin modulates some of the proinflammatory effects observed in obesity. The objective of this study was to determine the effects of leptin on airway smooth muscle responses. The effect of leptin (0.1-100 ng/ml) on migration (toward platelet-derived growth factor [PDGF], 10 ng/ml, across collagen-coated membrane in Transwell culture plates), proliferation (by BrDU incorporation), and cytokine production (by Bioplex bead assay) of cultured human airway smooth muscle cells from nine nonasthmatic donors was assessed. Effects of leptin on the contractile responses were studied in bovine tracheal smooth muscle rings. Leptin receptor expression and activation of STAT-3, Src kinase, Suppressor of Cytokine Signaling-3 (SOCS-3), and COX were evaluated by Western blotting and PCR. PGE(2) levels in supernatant were assessed by enzyme immunoassay. Human airway smooth muscle cells express leptin receptor, which, when engaged, phosphorylated STAT-3. Leptin inhibited PDGF-induced human airway smooth muscle migration and proliferation and IL-13-induced eotaxin production. Leptin did not stimulate cytokine synthesis and did not evoke contractile responses or inhibit isoproterenol-induced relaxation of carbachol-induced contraction of bovine tracheal rings. The inhibitory effects on migration and eotaxin production are not due to activation of SOCS-3 but are partly due to increased production of PGE(2) because they were attenuated by indomethacin. In conclusion, leptin inhibited human airway smooth muscle proliferation, migration toward PDGF, and IL-13-induced eotaxin production. This is partly mediated by PGE(2) secretion from smooth muscle cells induced by leptin. The association between obesity and asthma is unlikely to be due to a direct effect of leptin on airway smooth muscle.

Alternative splicing of the G protein-coupled receptor superfamily in human airway smooth muscle diversifies the complement of receptors

G protein-coupled receptors (GPCRs) are the largest signaling family in the genome, serve an expansive array of functions, and are targets for approximately 50% of current therapeutics. In many tissues, such as airway smooth muscle (ASM), complex, unexpected, or paradoxical responses to agonists/antagonists occur without known mechanisms. We hypothesized that ASM express many more GPCRs than predicted, and that these undergo substantial alternative splicing, creating a highly diversified receptor milieu. Transcript arrays were designed detecting 434 GPCRs and their predicted splice variants. In this cell type, 353 GPCRs were detected (including 111 orphans), with expression levels varying by approximately 900-fold. Receptors used for treating airway disease were expressed lower than others with similar signaling properties, indicating potentially more effective targets. A disproportionate number of Class-A peptide-group receptors, and those coupling to G(q)/(11) or G(s) (vs. G(i)), was found. Importantly, 192 GPCRs had, on average, five different expressed receptor isoforms because of splicing events, including alternative splice donors and acceptors, novel introns, intron retentions, exon(s) skips, and novel exons, with the latter two events being most prevalent. The consequences of splicing were further investigated with the leukotriene B4 receptor, known for its aberrant responsiveness in lung. We found transcript expression of three variants because of alternative donor and acceptor splice sites, representing in-frame deletions of 38 and 100 aa, with protein expression of all three isoforms. Thus, alternative splicing, subject to conditional, temporal, and cell-type regulation, is a major mechanism that diversifies the GPCR superfamily, creating local recepteromes with specialized environments.

Prolonged heterologous β2-adrenoceptor desensitization promotes proasthmatic airway smooth muscle function via PKA/ERK1/2-mediated phosphodiesterase-4 induction

Beta2-adrenergic receptor (beta2AR) agonists acutely relieve bronchoconstriction via cAMP-mediated relaxation of airway smooth muscle (ASM). Airway constrictor responsiveness may be significantly heightened, however, following protracted exposure to these agents, presumably reflecting the effects of beta2AR desensitization in ASM accompanying prolonged cAMP signaling. Because cAMP phosphodiesterase (PDE) activity can significantly modulate ASM contractility, we investigated the mechanism regulating PDE expression and its potential role in mediating changes in agonist-induced constrictor and relaxation responsiveness in ASM following its heterologous beta2AR desensitization by prolonged exposure to cAMP-elevating agents. Isolated rabbit ASM tissues and cultured human ASM cells treated for 24 h with the receptor- or nonreceptor-coupled cAMP-stimulating agent, prostaglandin E(2) (PGE(2)) or forskolin, respectively, exhibited constrictor hyperresponsiveness to acetylcholine and impaired beta2AR-mediated relaxation and cAMP accumulation. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity, reflective of increased transcription of the PDE4D5 isoform, and were prevented by pretreatment of the ASM with a PDE4 inhibitor. Extended studies using gene silencing and pharmacological approaches to inhibit specific intracellular signaling molecules demonstrated that the mechanism underlying PGE(2)-induced transcriptional upregulation of PDE4D5 involves PKA-dependent activation of G(i) protein signaling via the betagamma-subunits, the latter eliciting downstream activation of ERK1/2 and its consequent induction of PDE4D5 transcription. Collectively, these findings identify that beta2AR desensitization in ASM following prolonged exposure to cAMP-elevating agents is associated with proasthmatic-like changes in ASM responsiveness that are mediated by upregulated PDE4 expression induced by activated cross talk between the PKA and ERK1/2 signaling pathways.

The Expression of Adiponectin Receptors and the Effects of Adiponectin and Leptin on Airway Smooth Muscle Cells

PurposeObesity is a major risk factor for asthma and it influences airway smooth muscle function and responsiveness. Adiponectin is inversely associated with obesity and its action is mediated through at least 2 cell membrane receptors (AdipoR1 and AdipoR2). Leptin is positively associated with obesity. We investigated whether human airway smooth muscle (ASM) cells express adiponectin receptors and whether adiponectin and leptin regulate human ASM cell proliferation and vascular endothelial growth factor (VEGF) release.Materials and MethodsHuman ASM cells were growth-arrested in serum-deprived medium for 48 hours and then stimulated with PDGF, adiponectin and leptin. After 48 hours of stimulation, proliferation was determined using a cell proliferation ELISA kit. Human AdipoR1 and -R2 mRNA expressions were determined by RT-PCR using human-specific AdipoR1 and -R2 primers. Concentrations of VEGF, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α in cell culture supernatant were determined by ELISA.ResultsBoth AdipoR1 and AdipoR2 mRNA were expressed in the cultured human ASM cells. However, adiponectin did not suppress PDGF-enhanced ASM cell proliferation, nor did leptin promote ASM cell proliferation. Leptin promoted VEGF release by human ASM cells, while adiponectin did not influence VEGF release. Neither leptin nor adiponectin influenced MCP-1 secretion from human ASM cells. Adiponectin and MIP-1α were not secreted by human ASM cells.ConclusionHuman ASM cells expressed adiponectin receptors. However, adiponectin did not regulate human ASM cell proliferation or VEGF release, while leptin stimulated VEGF release by human ASM cells.

Exploration of the β2-adrenergic receptor regulatory regions: the next step in the holy grail of asthma pharmacogenetics research

exploration of the β2-adrenergic receptor (ADRB2) has been the holy grail of asthma pharmacogenetics research since the gene was cloned two decades ago. The evolution in our understanding of genetic variants in the ADRB2 reflects both the progress and the limitations in exploring the contributions

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+mobilization and contraction in airway smooth muscle

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

E-Ring Isoprostanes Stimulate a Cl− Conductance in Airway Epithelium via Prostaglandin E2-Selective Prostanoid Receptors

Isoprostanes comprise a class of membrane lipid metabolites produced during oxidative stress, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. They are widely recognized to evoke a variety of biological responses in airway and pulmonary vascular smooth muscle, lymphatics, and innervation. However, their effects on airway epithelium are largely unstudied. We examined the electrophysiological responses evoked by several different isoprostane species in bovine airway epithelium using the Ussing chamber technique. The E-ring isoprostanes 15-E(1t)-IsoP and 15-E(2t)-IsoP evoked a substantial increase in short-circuit current (I(SC)), whereas four different F-ring isomers were ineffective. 15-E(2t)-IsoP-evoked I(SC) was mimicked by the prostaglandin E(2)-selective prostanoid receptor (EP)-agonist prostaglandin E(2) but not by agonists of EP(1)/EP(3)-, FP-, or TP receptors (sulprostone, fluprostenol, and U46619, respectively). This response was significantly reduced by the EP(4)-receptor blocker GW627386 but not by blockers of other prostanoid receptors (ICI 192,605 [TP-selective], SC19220 [EP(1)-selective], AH6809 [DP/EP(1)/EP(2)-selective], and AL8810 [FP-selective]). 15-E(2t)-IsoP-evoked I(SC) was reduced by blockers of Cl(-) channels (niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid), of Na(+)/K(+)/2Cl(-) co-transport (furosemide and bumetanide), of adenylate cyclase (MDL 12,330A), or of guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by blockers of Na(+) conductances (amiloride). We conclude that 15-E(2t)-IsoP activates a transepithelial Cl(-) conductance in bovine airway epithelium through an EP(4) receptor coupled to adenylate cyclase and soluble guanylate cyclase.

In vivo adenosine A2B receptor desensitization in guinea-pig airway smooth muscle: Implications for asthma

This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

A new mechanism for respiratory syncytial virus-induced β2-adrenergic receptor insensitivity

respiratory syncytial virus (RSV) is the most common infectious cause of wheezing in infants and children and is a frequent initiator of acute asthma exacerbations ([13][1]). The mechanism by which RSV results in airway obstruction is not completely understood but includes an increase in airway

Superantigen Presentation by Airway Smooth Muscle to CD4+ T Lymphocytes Elicits Reciprocal Proasthmatic Changes in Airway Function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.

Inhibition of myosin light‐chain phosphorylation inverts the birefringence response of porcine airway smooth muscle

Muscle birefringence, caused mainly by parallel thick filaments, increases in smooth muscle during stimulation, signalling thick filament formation upon activation. The reverse occurs in skeletal muscle, where a decrease in birefringence has been correlated with crossbridge movement away from the thick filaments. When force generation by trachealis muscle was inhibited with wortmannin, which inhibits myosin light-chain phosphorylation and thick-filament formation, but not the calcium increase caused by stimulation, the birefringence response inverted, suggesting crossbridge movement similar to that of skeletal muscle. Resistance to quick stretches was much greater in stimulated muscle than in unstimulated muscle before wortmannin treatment and no different in stimulated and unstimulated muscle after force inhibition by wortmannin. Before wortmannin treatment, stimulation reduced thick-filament cross-sectional areas in electron micrographs by 44%. After force inhibition by wortmannin, filament areas were not significantly different in stimulated and unstimulated muscle and not significantly different from those of relaxed muscle without wortmannin treatment. These results suggest that myofibrillar-space calcium causes crossbridges to move away from the thick filaments without firmly attaching to thin filaments.