Airway Smooth Muscle Contraction Research Articles

Epithelium‐dependent modulation of responsiveness of airways from caveolin‐1 knockout mice is mediated through cyclooxygenase‐2 and 5‐lipoxygenase

Acute silencing of caveolin-1 (Cav-1) modulates receptor-mediated contraction of airway smooth muscle. Moreover, COX-2- and 5-lipoxygenase (5-LO)-derived prostaglandin and leukotriene biosynthesis can influence smooth muscle reactivity. COX-2 half-life can be prolonged through association with Cav-1. We suggested that lack of Cav-1 modulated levels of COX-2 which in turn modulated tracheal contraction, when arachidonic acid signalling was disturbed by inhibition of COX-2. Using tracheal rings from Cav-1 knockout (KO) and wild-type mice (B6129SF2/J), we measured isometric contractions to methacholine and used PCR, immunoblotting and immunohistology to monitor expression of relevant proteins. Tracheal rings from Cav-1 KO and wild-type mice exhibited similar responses, but the COX-2 inhibitor, indomethacin, increased responses of tracheal rings from Cav-1 KO mice to methacholine. The phospholipase A₂ inhibitor, eicosatetraynoic acid, which inhibits formation of both COX-2 and 5-LO metabolites, had no effect on wild-type or Cav-1 KO tissues. Indomethacin-mediated hyperreactivity was ablated by the LTD₄ receptor antagonist (montelukast) and 5-LO inhibitor (zileuton). The potentiating effect of indomethacin on Cav-1 KO responses to methacholine was blocked by epithelial denudation. Immunoprecipitation showed that COX-2 binds Cav-1 in wild-type lungs. Immunoblotting and qPCR revealed elevated levels of COX-2 and 5-LO protein, but not COX-1, in Cav-1 KO tracheas, a feature that was prevented by removal of the epithelium. The indomethacin-induced hypercontractility observed in Cav-1 KO tracheas was linked to increased expression of COX-2 and 5-LO, which probably enhanced arachidonic acid shunting and generation of pro-contractile leukotrienes when COX-2 was inhibited.

Counterpoint: Alterations in airway smooth muscle phenotype do not cause airway hyperresponsiveness in asthma

We have been assigned the task of showing that “alterations in airway smooth muscle phenotype DO NOT cause the airways hyperresponsiveness of asthma.” In the following paragraphs we will briefly review the overwhelming lack of evidence that any fundamental change in the intrinsic properties of

Models to study airway smooth muscle contraction in vivo, ex vivo and in vitro: Implications in understanding asthma

Asthma is a chronic obstructive airway disease characterised by airway hyperresponsiveness (AHR) and airway wall remodelling. The effector of airway narrowing is the contraction of airway smooth muscle (ASM), yet the question of whether an inherent or acquired dysfunction in ASM contractile function plays a significant role in the disease pathophysiology remains contentious.The difficulty in determining the role of ASM lies in limitations with the models used to assess contraction. In vivo models provide a fully integrated physiological response but ASM contraction cannot be directly measured. Ex vivo and in vitro models can provide more direct assessment of ASM contraction but the loss of factors that may modulate ASM responsiveness and AHR, including interaction between multiple cell types and disruption of the mechanical environment, precludes a complete understanding of the disease process.In this review we detail key advantages of common in vivo, ex vivo and in vitro models of ASM contraction, as well as emerging tissue engineered models of ASM and whole airways. We also highlight important findings from each model with respect to the pathophysiology of asthma.

Substance P Regulates Environmental Tobacco Smoke-Enhanced Tracheal Smooth Muscle Responsiveness in Mice

Environmental tobacco smoke (ETS) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness (AHR) in susceptible individuals and animals, but the underlying mechanism is not fully understood. Substance P (SP) release from sensory nerve fibers has been linked to AHR. The present experiments characterize the role of SP in tracheal smooth muscle on ETS-increased airway responses. The mice were exposed to either sidestream tobacco smoke (SS), a surrogate to ETS, or filtered air (FA) for 1 day or 5 consecutive days. Contractions of tracheal smooth muscle to SP and electrical field stimulation (EFS) were not significantly altered in 1 of day SS-exposed mice. However, 5 of days SS exposure significantly increased airway smooth muscle contractions to SP and EFS. Administration of CP-99994, an antagonist of the neurokinin (NK)1 receptor, attenuates the SS exposure-enhanced tracheal smooth muscle responses to EFS. Furthermore, the immunohistochemistry showed that SP nerve fibers were increased in tracheal smooth muscle after 5 of days SS exposure. These results suggest that the increased SP production may contribute to SS-enhanced smooth muscle responsiveness in mice trachea.

Inhibition of angiotensin II-induced contraction of human airway smooth muscle cells by angiotensin-(1-7) via downregulation of the RhoA/ROCK2 signaling pathway

Sustained renin-angiotensin system (RAS) activation in asthmatic patients plays a crucial role in airway hyperresponsiveness and airflow limitation. Angiotensin II (Ang II), as a key peptide of RAS, contributes to the contraction of human airway smooth muscle by activating the RhoA/Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling pathway. Angiotensin-(1-7) [Ang-(1-7)], is a component of the angiotensin I converting enzyme 2 (ACE2)-Ang-(1-7)-Mas axis which counteracts the detrimental effects of the ACE- Ang II-angiotensin type 1 receptor (AT1R) axis in vivo; however, whether Ang-(1-7) can inhibit the effect of Ang II in the contraction of human airway smooth muscle cells (HASMCs) is unknown. In our study, collagen gel lattices and immunofluorescence were used to evaluate the contraction of HASMCs induced by Ang II. Real-time PCR and western blot analysis were performed to confirm the regulatory mechanism and the participating signaling pathway. Ang II caused the contraction of HASMCs; this effect was reversed by Ang‑(1‑7). In addition, irbesartan and A779, which are inhibitors of AT1R and Mas, respectively, attenuated the effect of Ang II and Ang-(1-7). Furthermore, Y-27632, an inhibitor of ROCK2, attenuated the Ang II-induced contraction of HASMCs by blocking the RhoA/ROCK2 signaling pathway which is involved in this contraction, and thus may be a major regulator involved in the basal maintenance of contractility in HASMCs. These data demonstrate that Ang II induces the contraction of HASMCs and that this effect can be reversed by Ang-(1-7), partially through the downregulation of of the RhoA/ROCK2 signaling pathway.

Muscarinic receptors on airway mesenchymal cells: Novel findings for an ancient target

Since ancient times, anticholinergics have been used as a bronchodilator therapy for obstructive lung diseases. Targets of these drugs are G-protein-coupled muscarinic M1, M2 and M3 receptors in the airways, which have long been recognized to regulate vagally-induced airway smooth muscle contraction and mucus secretion. However, recent studies have revealed that acetylcholine also exerts pro-inflammatory, pro-proliferative and pro-fibrotic actions in the airways, which may involve muscarinic receptor stimulation on mesenchymal, epithelial and inflammatory cells. Moreover, acetylcholine in the airways may not only be derived from vagal nerves, but also from non-neuronal cells, including epithelial and inflammatory cells. Airway smooth muscle cells seem to play a major role in the effects of acetylcholine on airway function. It has become apparent that these cells are multipotent cells that may reversibly adopt (hyper)contractile, proliferative and synthetic phenotypes, which are all under control of muscarinic receptors and differentially involved in bronchoconstriction, airway remodeling and inflammation. Cholinergic contractile tone is increased by airway inflammation associated with asthma and COPD, resulting from exaggerated acetylcholine release as well as increased expression of contraction related proteins in airway smooth muscle. Moreover, muscarinic receptor stimulation promotes proliferation of airway smooth muscle cells as well as fibroblasts, and regulates cytokine, chemokine and extracellular matrix production by these cells, which may contribute to airway smooth muscle growth, airway fibrosis and inflammation. In line, animal models of chronic allergic asthma and COPD have recently demonstrated that tiotropium may potently inhibit airway inflammation and remodeling. These observations indicate that muscarinic receptors have a much larger role in the pathophysiology of obstructive airway diseases than previously thought, which may have important therapeutic implications.

Integrin α9β1 in airway smooth muscle suppresses exaggerated airway narrowing

Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9β1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9β1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9β1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9β1 or treated with integrin α9β1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9β1. Therefore, integrin α9β1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.

Mast Cell-Airway Smooth Muscle Crosstalk: The Role of Thymic Stromal Lymphopoietin

The mast cell localization to airway smooth muscle (ASM) bundle in asthma is important in the development of disordered airway physiology. Thymic stromal lymphopoietin (TSLP) is expressed by airway structural cells. Whether it has a role in the crosstalk between these cells is uncertain. We sought to define TSLP expression in bronchial tissue across the spectrum of asthma severity and to investigate the TSLP and TSLP receptor (TSLPR) expression and function by primary ASM and mast cells alone and in coculture. TSLP expression was assessed in bronchial tissue from 18 subjects with mild to moderate asthma, 12 with severe disease, and nine healthy control subjects. TSLP and TSLPR expression in primary mast cells and ASM was assessed by immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assay, and its function was assessed by calcium imaging. The role of TSLP in mast cell and ASM proliferation, survival, differentiation, synthetic function, and contraction was examined. TSLP expression was increased in the ASM bundle in mild-moderate disease. TSLP and TSLPR were expressed by mast cells and ASM and were functional. Mast cell activation by TSLP increased the production of a broad range of chemokines and cytokines, but did not affect mast cell or ASM proliferation, survival, or contraction. TSLP expression by the bronchial epithelium and ASM was upregulated in asthma. TSLP promoted mast cell synthetic function, but did not contribute to other functional consequences of mast cell-ASM crosstalk.

Regulation of GPCR-mediated smooth muscle contraction: Implications for asthma and pulmonary hypertension

Contractile G-protein-coupled receptors (GPCRs) have emerged as key regulators of smooth muscle contraction, both under healthy and diseased conditions. This brief review will discuss some key topics and novel insights regarding GPCR-mediated airway and vascular smooth muscle contraction as discussed at the 7th International Young Investigators' Symposium on Smooth Muscle (2011, Winnipeg, Manitoba, Canada) and will in particular focus on processes driving Ca2+-mobilization and -sensitization.

<em>In vitro</em> 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.

Effect of length oscillations on airway smooth muscle reactivity and cross-bridge cycling

Excessive airway narrowing due to airway smooth muscle (ASM) hyperconstriction is a major symptom in many respiratory diseases. In vitro imposition of length oscillations similar to those produced by tidal breathing on contracted ASM have shown to reduce muscle active forces, which is usually attributed to unconfirmed disruption of actomyosin cross-bridges. This research focuses on an in vitro investigation of the effect of mechanical oscillations on ASM reactivity and actomyosin cross-bridges. A computerized organ bath system was used to test maximally precontracted bovine ASM subjected to length oscillations at frequencies in the range of 10-100 Hz superimposed on tidal breathing oscillation. Using an immunofluorescence technique, two specific antibodies against the phospho-serine19 myosin light chain and the α-smooth muscle actin were used to analyze the colocalization between these two filaments. Data were processed using the plug-in "colocalization threshold" of ImageJ 1.43m software. The results demonstrate that both tidal and superimposed length oscillations reduce the active force in contracted ASM for a relatively long term and that the latter enhances the force reduction of the former. This reduction was also found to be frequency and time dependent. Additionally colocalization analysis indicates that length oscillations cause the detachment of the actomyosin connections and that this condition is sustained even after the cessation of the length oscillations.

Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness

Drugs targeting individual G protein-coupled receptors are used as asthma therapies, but this strategy is limited because of G protein-coupled receptor signal redundancy. Regulator of G protein signaling 2 (RGS2), an intracellular selective inhibitor of multiple bronchoconstrictor receptors, may play a central role in the pathophysiology and treatment of asthma. We defined functions and mechanisms of RGS2 in regulating airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Real-time PCR and Western blot were used to determine changes in RGS2 expression in ovalbumin-sensitized/-challenged mice. We also used immunohistochemistry and real-time PCR to compare RGS2 expression between human asthmatic and control subjects. The AHR of RGS2 knockout mice was assessed by using invasive tracheostomy and unrestrained plethysmography. Effects of loss of RGS2 on mouse airway smooth muscle (ASM) remodeling, contraction, intracellular Ca(2+), and mitogenic signaling were determined in vivo and in vitro. RGS2 was highly expressed in human and murine bronchial epithelium and ASM and was markedly downregulated in lungs of ovalbumin-sensitized/-challenged mice. Lung tissues and blood monocytes from asthma patients expressed significantly lower RGS2 protein (lung) and mRNA (monocytes) than from nonasthma subjects. The extent of reduction of RGS2 on human monocytes correlated with increased AHR. RGS2 knockout caused spontaneous AHR in mice. Loss of RGS2 augmented Ca(2+) mobilization and contraction of ASM cells. Loss of RGS2 also increased ASM mass and stimulated ASM cell growth via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. We identified RGS2 as a potent modulator of AHR and a potential novel therapeutic target for asthma.

CAMP regulation of airway smooth muscle function

Agonists activating β2-adrenoceptors (β2ARs) on airway smooth muscle (ASM) are the drug of choice for rescue from acute bronchoconstriction in patients with both asthma and chronic obstructive pulmonary disease (COPD). Moreover, the use of long-acting β-agonists combined with inhaled corticosteroids constitutes an important maintenance therapy for these diseases. β-Agonists are effective bronchodilators due primarily to their ability to antagonize ASM contraction. The presumed cellular mechanism of action involves the generation of intracellular cAMP, which in turn can activate the effector molecules cAMP-dependent protein kinase (PKA) and Epac. Other agents such as prostaglandin E2 and phosphodiesterase inhibitors that also increase intracellular cAMP levels in ASM, can also antagonize ASM contraction, and inhibit other ASM functions including proliferation and migration. Therefore, β2ARs and cAMP are key players in combating the pathophysiology of airway narrowing and remodeling. However, limitations of β-agonist therapy due to drug tachyphylaxis related to β2AR desensitization, and recent findings regarding the manner in which β2ARs and cAMP signal, have raised new and interesting questions about these well-studied molecules. In this review we discuss current concepts regarding β2ARs and cAMP in the regulation of ASM cell functions and their therapeutic roles in asthma and COPD.

The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH

Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar 'dose'-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.

Targeting Phosphoinositide 3-Kinase γ in Airway Smooth Muscle Cells to Suppress Interleukin-13-Induced Mouse Airway Hyperresponsiveness

We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca(2+) oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca(2+) transient and increased Ca(2+) oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca(2+) transient by 20 to 30% but markedly attenuated Ca(2+) oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma.

Allergen and Ozone Exacerbate Serotonin-Induced Increases in Airway Smooth Muscle Contraction in a Model of Childhood Asthma

Background: Serotonin (5-HT) modulates cholinergic neurotransmission and exacerbates airway smooth muscle (ASM) contraction in normal animal and nonasthmatic human tissue. Exposure to house dust mite allergen (HDMA) and ozone (O₃) leads to airway hyperreactivity and 5-HT-positive cells in the airway epithelium of infant rhesus monkeys. Research shows that concomitant exposure in allergic animals has an additive effect on airway hyperreactivity. Objectives: In this study, the hypothesis is that the exposure of allergic infant rhesus monkeys to HDMA, O₃ and in combination, acting through 5-HT receptors, enhances 5-HT modulation of postganglionic cholinergic ASM contraction. Methods: Twenty-four HDMA-sensitized infant monkeys were split into 4 groups at the age of 1 month, and were exposed to filtered air (FA), HDMA, O₃ or in combination (HDMA+O₃). At the age of 6 months, airway rings were harvested and postganglionic, and parasympathetic-mediated ASM contraction was evaluated using electrical-field stimulation (EFS). Results: 5-HT exacerbated the EFS response within all exposure groups, but had no effect in the FA group. 5-HT₂, 5-HT₃ and 5-HT₄ receptor agonists exacerbated the response. 5-HT concentration-response curves performed after incubation with specific receptor antagonists confirmed the involvement of 5-HT₂, 5-HT₃ and 5-HT₄ receptors. Conversely, a 5-HT₁ receptor agonist attenuated the tension across all groups during EFS, and in ASM contracted via exogenous acetylcholine. Conclusions: HDMA, O₃ and HDMA+O₃ exposure in a model of childhood allergic asthma enhances 5-HT exacerbation of EFS-induced ASM contraction through 5-HT₂, 5-HT₃ and 5-HT₄ receptors. A nonneurogenic inhibitory pathway exists, unaffected by exposure, mediated by 5-HT₁ receptors located on ASM.

Supplementation of L‐arginine and/or inhibition of arginase enhance the effect of inhaled NO on airway function of hyperoxia‐exposed rat pups

Hyperoxia‐induced lung injury in neonates is characterized by both enhanced contraction and impaired relaxation of airway smooth muscle. We have demonstrated that hyperoxia‐induced lung reactivity is due to impaired NO‐mediated signaling. We hypothesize that supplementation of L‐arginine and/or blockade of arginase will enhance the effect of inhaled NO (iNO). The rat pups (P0) were exposed for 7 days to room air (RA), 50% O2, or 50% O2 plus NO (2 or 20 PPM) in presence or absence of L‐arginine and nor‐NOHA administration. 50% O2 significantly (p&lt;0.001) increased the EFS‐induced contraction of tracheal rings as compared to RA exposure. The iNO reversed the hyperoxia‐induced increased contraction of tracheal rings in a dose dependent manner. The reduction effect of iNO was enhanced significantly by arginase inhibition but not by L‐arginine supplementation. The nor‐NOHA in combination with 2 PPM iNO decreased contraction of tracheal rings equal to 20 PPM iNO alone. Moderate hyperoxic exposure significant enhanced arginase I expression in tracheal epithelium. The iNO (20 PPM) has inhibited arginase I expression below RA level in hyperoxic animals. The arginase I expression was also downregulated by nor‐NOHA in hyperoxic group receiving 20 PPM iNO. In conclusion, arginase inhibition may provide a useful therapeutic tool for the increased airway reactivity that occurs in hyperoxia exposed neonates. (IKARIA‐ZAIDS1)

Effects of latrunculin‐B in smooth muscle contraction, Ca2+ oscillations and Ca2+ sensitization in small intrapulmonary airways

The role of actin filament dynamics in the regulation of airway smooth muscle contraction is not fully understood. We have investigated the effects of impairing actin filament polymerization with latrunculin‐B (Lat‐B) –a G‐actin binding toxin, on agonist‐induced Ca2+ signaling, Ca2+ sensitivity and contraction in small intrapulmonary airways in mouse lung slices. Incubation of lung slices with Lat‐B (0.1 μM) for 60 min caused a reduction of airway contraction induced by 0.1 μM serotonin (5‐HT) and 0.1 μM acetylcholine (ACh) by 54 ± 8% and 42 ± 6%, respectively. In addition, Lat‐B induced relaxation of airways pre‐contracted with agonists. Maximal airway relaxation was 55 ± 5% and 53 ± 8% in airways pre‐contracted with 5‐HT and ACh, respectively. Furthermore, Lat‐B affected the Ca2+ signaling induced by ACh and 5‐HT by reducing the frequency of agonists‐induced Ca2+ oscillations. Finally, in Ca2+ permeabilized lung slices, Lat‐B induced relaxation of airways pre‐contracted with either 5‐HT or ACh indicating Ca2+ desensitization. Together these results suggest that actin filament dynamics are important for the regulation of airway smooth muscle contraction and signaling.

Targeting phosphoinositide 3‐kinase γ in airway smooth muscle cells to suppress interleukin‐13‐induced mouse airway hyperresponsiveness

Excessive activation of different bronchoconstrictor G‐protein coupled receptors (GPCRs) in airway smooth muscle (ASM) contributes to airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. We previously reported that phosphoinositide 3‐kinase γ (PI3Kγ), a down‐stream effector of multiple types of GPCRs, directly regulates ASM contraction by modulating Ca2+ oscillations. The aim of this study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR induced by interleukin‐13 (IL‐13) in a murine model of asthma. We found that intranasal administration of IL‐13 into mice induced AHR after 48 h, as assessed by invasive tracheostomy. Acute treatment with a PI3Kγ‐specific inhibitor attenuated IL‐13 effects in vivo and IL‐13‐augmented airway contractility of precision‐cut mouse lung slices in vitro. In addition, silencing PI3Kγ expression in isolated mouse ASM cells markedly attenuated IL‐13‐augmented Ca2+ oscillations and contractility of ASM cells induced by different bronchoconstrictor GPCRs. Our studies demonstrate that PI3Kγ in ASM cells is important for AHR and acute treatment with a PI3Kγ inhibitor can ameliorate AHR by a broader‐based suppression of unwanted GPCR signaling in a murine model of asthma. This work was supported by American Asthma Foundation Early Excellence Award and State of Nebraska Research Funds.

IL-17A produced by αβ T cells drives airway hyper-responsiveness in mice and enhances mouse and human airway smooth muscle contraction

Emerging evidence suggests that the TH17 subset of αβ T cells contributes to the development of allergic asthma. In this study we found that mice lacking αvβ8 on dendritic cells failed to generate TH17 cells in the lung and were protected from AHR in response to house dust mite and ovalbumin sensitization and challenge. Because loss of TH17 cells inhibited airway narrowing without obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of TH17 cytokines on mouse and human airway smooth muscle function. IL-17A enhanced contractile force generation through a NF-κB/RhoA/ROCK2 signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after OVA sensitization and challenge, and the diminished contraction of tracheal rings from these mice was reversed by IL-17A. These data indicate that IL-17A produced by TH17 cells contributes to allergen-induced AHR through direct effects on airway smooth muscle.