Airway Smooth Muscle Contraction Research Articles

β-Adrenergic Receptor Antagonism of Cholinergic Stimulation of Airway Smooth Muscle Contraction: An Old Receptor Requires a Fresh Look.

β-Adrenergic Receptor Antagonism of Cholinergic Stimulation of Airway Smooth Muscle Contraction: An Old Receptor Requires a Fresh Look.

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

Exercise Reduces Airway Smooth Muscle Contraction in Asthmatic Rats via Inhibition of IL-4 Secretion and Store-Operated Ca2+ Entry Pathway.

Increased evidence has shown that aerobic exercise reduces airway hyperresponsiveness in asthmatic individuals. However, the underlying mechanisms of action remain elusive. This study aimed to investigate the effect of exercise on airway smooth muscle (ASM) contractile function in asthmatic rats, and uncover the possible involvement of interleukin 4 (IL-4) and the store-operated Ca2+ entry (SOCE) pathway. In this study, chicken ovalbumin was used to induce asthma in male Sprague-Dawley rats. The exercise group received moderate-intensity aerobic exercise training for 4 weeks. IL-4 concentrations in bronchoalveolar lavage fluid (BALF) samples were evaluated by enzyme linked immunosorbent assay. The contractile function of the ASM was investigated using tracheal ring tension experiments and intracellular Ca2+ imaging techniques. Western blot analysis was used to evaluate expression levels of calcium-release activated calcium (CRAC) channel protein (Orai) and stromal interaction molecule 1 (STIM1) in ASM. Our data showed that the carbachol-stimulated, SOCE-mediated contraction of rat ASM was significantly increased in asthmatic rats, which could be abolished by exercise. Pharmacological studies revealed that GSK5498A and BTP-2, selective blockers of CRAC channels significantly inhibited SOCE-induced ASM contraction. In addition, exercise inhibited the up-regulation of IL-4 in BALF as well as STIM1 and Orai expression in the ASM of asthmatic rats. In line with these observations, we demonstrated that pretreatment of the ASM with IL-4 up-regulated the expression level of STIM1, Orai1 and Orai2, thereby promoting SOCE-mediated ASM contraction. The data in this study reveal that aerobic exercise may improve the ASM contractile function in asthmatic rats by inhibiting IL-4 secretion and by down-regulating the expression of STIM1, Orai1 and Orai2, thus decreasing excessive SOCE-mediated ASM contraction in asthmatic rats.

In silico identification of a β2-adrenoceptor allosteric site that selectively augments canonical β2AR-Gs signaling and function

Activation of β2-adrenoceptors (β2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and β2AR agonists (β-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of β-agonists is limited by agonist-induced β2AR desensitization and noncanonical β2AR signaling involving β-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on β2AR that could modulate the activity of β-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated β2AR intermediate conformations and identified a putative allosteric binding site. Subsequent database screening using SILCS identified drug-like molecules with the potential to bind to the site. Experimental assays in HEK293 cells (expressing recombinant wild-type human β2AR) and human ASM cells (expressing endogenous β2AR) identified positive and negative allosteric modulators (PAMs and NAMs) of β2AR as assessed by regulation of β-agonist-stimulation of cyclic AMP generation. PAMs/NAMs had no effect on β-agonist-induced recruitment of β-arrestin to β2AR- or β-agonist-induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified site based on mutants of amino acids R131, Y219, and F282. Finally, functional studies revealed augmentation of β-agonist-induced relaxation of contracted human ASM cells and bronchodilation of contracted airways. These findings identify a allosteric binding site on the β2AR, whose activation selectively augments β-agonist-induced Gs signaling, and increases relaxation of ASM cells, the principal therapeutic effect of β-agonists.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of β-Adrenoceptors.

Beta-adrenoceptor (β-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting β-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of β-ARs. M2R-dependent contractions were apparent at low frequency (2Hz)electric field stimulation (EFS) and short (10s) stimulus intervals.The β1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 sand was more effective at inhibiting contractions evoked by EFS at 2than 20 Hz.Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the β3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of β1-ARs and that genetic ablation of M2Rs decreased the efficacy of β-AR agonists on cholinergic contractions.

Airway smooth muscle thickness and contraction are enhanced by intra-amniotic lipopolysaccharide in an ovine model of premature birth.

Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. We hypothesize that antenatal inflammation causes physiological abnormalities of the ASM that predisposes asthma. This study determined the short-term effects of antenatal inflammation on the developing ASM. Fourteen pregnant ewes were randomly assigned to one of three groups. Fetal lambs were exposed to intra-amniotic injections of lipopolysaccharide (LPS, n = 4) or saline (controls; n = 5) at 127 days' gestational age (GA). Preterm lambs were surgically delivered at 129 days' GA and received intensive care for 7 days before euthanasia. Naïve fetal controls (n = 5) were delivered and euthanized at 136 days' GA. ASM force to acetylcholine was measured in bronchial rings and normalized to ring length (tension) and ASM cross-sectional area (stress). Airway narrowing (% volume) to acetylcholine was assessed in bronchial segments. Fetal controls were structurally and functionally similar to saline-exposed lambs. Compared with saline, LPS-exposed lambs had increased macrophages in lung tissue (P = 0.0002) and interleukin-8 in alveolar wash (P = 0.003). LPS exposure increased ASM thickness (P = 0.005), airway narrowing (P = 0.003), ASM tension (P = 0.0002), and contractile stress (P < 0.0001). Notably, LPS-exposed lambs were more dependent on mechanical ventilation, and both LPS (P < 0.001) and ventilation (P = 0.012) were independent factors in increasing ASM stress. Only LPS independently increased ASM thickness (P = 0.045). Results indicate that antenatal exposure to LPS and subsequent mechanical ventilation promotes intrinsic changes to the ASM that enhances bronchoconstriction. If persistent into postnatal life, these developmental abnormalities may contribute to the known association between chorioamnionitis and asthma.NEW & NOTEWORTHY Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. Using an ovine model of antenatal inflammation, we demonstrate thickening and increased contraction of the premature ASM layer. If such physiological abnormalities persist throughout postnatal life, this represents a predisposition to an asthma diagnosis.

Aerobic Exercise Reduces Airway Smooth Muscle Contraction In Asthmatic Rats Via Inhibition Of Il-4 Secretion And Suppression Of Store-operated Ca2+ Entry

PURPOSE: Increasing evidence has shown that aerobic exercise training reduces airway hyperresponsiveness in asthmatic individuals. However, the underlying mechanisms remain elusive. This study aimed to investigate the effect of exercise on airway smooth muscle (ASM) contractile function in asthmatic rats and uncover the possible involvement of interleukin 4 (IL-4) and the store-operated Ca2+ entry (SOCE) pathway. METHODS: Chicken ovalbumin was used to stimulate asthma in male Sprague-Dawley rats. The exercise group received moderate-intensity aerobic exercise training for 4 weeks. IL-4 concentrations in bronchoalveolar lavage fluid (BALF) samples were evaluated by ELISA. The contractile function of the ASM was investigated using tracheal ring tension experiments and intracellular Ca2+ imaging techniques. Western-blot experiments were used to identify expression levels of the calcium-release activated calcium (CRAC) channel protein, Orai, in ASM. RESULTS: The carbachol-stimulated and SOCE-mediated contraction of rat ASM was significantly increased in asthmatic rats, but was abolished by exercise training. Pharmacological studies revealed that GSK5498A, a selective blocker of CRAC channels, significantly inhibited SOCE-induced ASM contraction. In addition, our data showed that aerobic exercise inhibits the up-regulation of IL-4 in BALF as well as Orai protein (subtypes 1 and 2) expression in the ASM of asthmatic rats. In line with these observations, we demonstrated that pretreatment of ASM with IL-4 both up-regulated the expression level of Orai (subtype 1, 2, 3) and promoted carbachol-induced and SOCE-mediated ASM contraction. CONCLUSIONS: The present study revealed that aerobic exercise improved ASM contractile function in asthmatic rats by inhibiting IL-4 secretion and down-regulating the expression levels of Orai, thus decreasing excessive SOCE-mediated ASM contraction in asthmatic rats. Supported by Natural Science Foundation of Guangdong Province (2019A1515012167).

Malat1 deficiency prevents hypoxia-induced lung dysfunction by protecting the access to alveoli.

Hypoxia is common in lung diseases and a potent stimulator of the long non-coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1). Herein, we investigated the impact of Malat1 on hypoxia-induced lung dysfunction in mice. Malat1-deficient mice and their wild-type littermates were tested after 8 days of normoxia or hypoxia (10% oxygen). Hypoxia decreased elastance of the lung by increasing lung volume and caused in vivo hyperresponsiveness to methacholine without altering the contraction of airway smooth muscle. Malat1 deficiency also modestly decreased lung elastance but only when tested at low lung volumes and without altering lung volume and airway smooth muscle contraction. The in vivo responsiveness to methacholine was also attenuated by Malat1 deficiency, at least when elastance, a readout sensitive to small airway closure, was used to assess the response. More impressively, in vivo hyperresponsiveness to methacholine caused by hypoxia was virtually absent in Malat1-deficient mice, especially when hysteresivity, a readout sensitive to small airway narrowing heterogeneity, was used to assess the response. Malat1 deficiency also increased the coefficient of oxygen extraction and decreased ventilation in conscious mice, suggesting improvements in gas exchange and in clinical signs of respiratory distress during natural breathing. Combined with a lower elastance at low lung volumes at baseline, as well as a decreased propensity for small airway closure and narrowing heterogeneity during a methacholine challenge, these findings represent compelling evidence suggesting that the lack of Malat1 protects the access to alveoli for air entering the lung.

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction invivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

The impact of long-acting muscarinic antagonists on mucus hypersecretion and cough in chronic obstructive pulmonary disease: a systematic review.

Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway mucus hypersecretion as sputum expectoration and cough. Evidence accumulated in the past decade has shown that the cholinergic system not only regulates airway smooth muscle contraction but also the activity of inflammatory and airway epithelial cells, including goblet cells, and submucosal gland activity. Long-acting muscarinic antagonists (LAMAs) with the most favourable M3/M2 muscarinic acetylcholine (ACh) receptors residency properties are not only excellent bronchodilators but potentially also mucus-modifying agents, able to positively impact on mucus hypersecretion and cough. The aim of this systematic review was to investigate the impact of LAMAs on mucus hypersecretion and cough in COPD patients. The evidence confirmed that LAMAs, mainly tiotropium and aclidinium, improved sputum production and cough in moderate to severe COPD. Thus, LAMAs not only antagonise the ACh-induced bronchoconstriction of the airways but also appear to limit the production of mucus secreted in response to ACh by airway goblet cells and/or submucosal glands. Further clinical studies are necessary to evaluate the impact of LAMAs exclusively on sputum symptoms and cough as primary end-points and to investigate whether LAMAs have a modulatory action on the rheological properties of mucus.

SPLUNC1 is a negative regulator of the Orai1 Ca2+ channel.

Orai1 is a ubiquitously‐expressed plasma membrane Ca2+ channel that is involved in store‐operated Ca2+ entry (SOCE): a fundamental biological process that regulates gene expression, the onset of inflammation, secretion, and the contraction of airway smooth muscle (ASM). During SOCE, Ca2+ leaves the endoplasmic reticulum, which then stimulates a second, amplifying wave of Ca2+ influx through Orai1 into the cytoplasm. Short Palate LUng and Nasal epithelial Clone 1 (SPLUNC1; gene name BPIFA1) is a multi‐functional, innate defense protein that is highly abundant in the lung. We have previously reported that SPLUNC1 was secreted from epithelia, where it bound to and inhibited Orai1, leading to reduced SOCE and ASM relaxation. However, the underlying mechanism of action is unknown. Here, we probed the SPLUNC1‐Orai1 interactions in ASM and HEK293T cells using biochemical and imaging techniques. We observed that SPLUNC1 caused a conformational change in Orai1, as measured using Forster resonance energy transfer (FRET). SPLUNC1 binding also led to Nedd4‐2 dependent ubiquitination of Orai1. Moreover, SPLUNC1 internalized Orai1 to lysosomes, leading to Orai1 degradation. Thus, we conclude that SPLUNC1 is an allosteric regulator of Orai1. Our data indicate that SPLUNC1‐mediated Orai1 inhibition could be utilized as a therapeutic strategy to reduce SOCE.

In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function

Objective &amp; HypothesisActivation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.MethodsWe employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.ResultsDatabase screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.ConclusionThese findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.

Inhibitory Effect of S100A11 on Airway Smooth Muscle Contraction and Airway Hyperresponsiveness.

S100A11 is a member of the S100 calcium-binding protein family and has intracellular and extracellular regulatory activities. We previously reported that S100A11 was differentially expressed in the respiratory tracts of asthmatic rats as compared with normal controls. Here, we aimed to analyze the potential of S100A11 to regulate both allergen-induced airway hyperresponsiveness (AHR) as well as acetylcholine (ACh)-induced hypercontractility of airway smooth muscle (ASM) and contraction of ASM cells (ASMCs). Purified recombinant rat S100A11 protein (rS100A11) was administered to OVA-sensitized and challenged rats and then the AHR of animals was measured. The relaxation effects of rS100A11 on ASM were detected using isolated tracheal rings and primary ASMCs. The expression levels of un-phosphorylated myosin light chain (MLC) and phosphorylated MLC in ASMCs were analyzed using Western blotting. Treatment with rS100A11 attenuated AHR in the rats. ASM contraction assays showed that rS100A11 reduced the contractile responses of isolated tracheal rings and primary ASMCs treated with ACh. In addition, rS100A11 markedly decreased the ACh-induced phosphorylation of the myosin light chain in ASMCs. Moreover, rS100A11 also suppressed the contractile response of tracheal rings in calcium-free buffer medium. These results indicate that S100A11 protein can relieve AHR by relaxing ASM independently of extracellular calcium. Our data support the idea that S100A11 is a potential therapeutic target for reducing airway resistance in asthma patients.

Requirements and limitations of imaging airway smooth muscle throughout the lung in vivo

Clinical visualization and quantification of the amount and distribution of airway smooth muscle (ASM) in the lungs of individuals with asthma has major implications for our understanding of airway wall remodeling as well as treatments targeted at the ASM. This paper theoretically investigates the feasibility of quantifying airway wall thickness (focusing on the ASM) throughout the lung in vivo by means of bronchoscopic polarization-sensitive optical coherence tomography (PS-OCT). Using extensive human biobank data from subjects with and without asthma in conjunction with a mathematical model of airway compliance, we define constraints that airways of various sizes pose to any endoscopic imaging technique and how this is impacted by physiologically relevant processes such as constriction, inflation and deflation. We identify critical PS-OCT system parameters and pinpoint parts of the airway tree that are conducive to successful quantification of ASM. We further quantify the impact of breathing and ASM contraction on the measurement error and recommend strategies for standardization and normalization

An in silico and in vitro integrated analysis method to reveal the curative mechanisms and pharmacodynamic substances of Bufei granule on chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with high disability and mortality. Clinical studies have shown that the Traditional Chinese Medicine Bufei Granule (BFG) has conspicuous effects on relieving cough and improving lung function in patients with COPD and has a reliable effect on the treatment of COPD, whereas the therapeutic mechanism is vague. In the present study, the latent bronchodilators and mechanism of BFG in the treatment of COPD were discussed through the method of network pharmacology. Then, the molecular docking and molecular dynamics simulation were performed to calculate the binding efficacy of corresponding compounds in BFG to muscarinic receptor. Finally, the effects of BFG on bronchial smooth muscle were validated by in vitro experiments. The network pharmacology results manifested the anti-COPD effect of BFG was mainly realized via restraining airway smooth muscle contraction, activating cAMP pathways and relieving oxidative stress. The results of molecular docking and molecular dynamics simulation showed alpinetin could bind to cholinergic receptor muscarinic 3. The in vitro experiment verified both BFG and alpinetin could inhibit the levels of CHRM3 and acetylcholine and could be potential bronchodilators for treating COPD. This study provides an integrating network pharmacology method for understanding the therapeutic mechanisms of traditional Chinese medicine, as well as a new strategy for developing natural medicines for treating COPD.

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Modulation of carbachol-induced Ca2+ oscillations in airway smooth muscle cells by PGE2

PGE2 is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE2.Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE2 (300 nM) and the EP2R agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE2 were reversed by application of the EP2R antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE2. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE2. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE2.These data suggest that PGE2 inhibits CCh-induced Ca2+ oscillations in murine ASMC via stimulation of EP2Rs and a mechanism involving activation of PKA and EPAC.

Role of airway smooth muscle cell phenotypes in airway tone and obstruction in guinea pig asthma model

BackgroundAirway obstruction (AO) in asthma is driven by airway smooth muscle (ASM) contraction. AO can be induced extrinsically by direct stimulation of ASM with contractile agonists as histamine, or by indirect provocation with antigens as ovalbumin, while the airway tone is dependent on intrinsic mechanisms. The association of the ASM phenotypes involved in different types of AO and airway tone in guinea pigs was evaluated.MethodsGuinea pigs were sensitized to ovalbumin and challenged with antigen. In each challenge, the maximum OA response to ovalbumin was determined, and before the challenges, the tone of the airways. At third challenge, airway responsiveness (AR) to histamine was evaluated and ASM cells from trachea were disaggregated to determinate: (a) by flow cytometry, the percentage of cells that express transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b), (b) by RT-PCR, the SERCA2B gene expression, (c) by ELISA, reduced glutathione (GSH) and, (d) Ca2+ sarcoplasmic reticulum refilling rate by microfluorometry. Control guinea pig group received saline instead ovalbumin.ResultsAntigenic challenges in sensitized guinea pigs induced indirect AO, AR to histamine and increment in airway tone at third challenge. No relationship was observed between AO induced by antigen and AR to histamine with changes in airway tone. The extent of antigen-induced AO was associated with both, TGF-β1 expression in ASM and AR degree. The magnitude of AR and antigen-induced AO showed an inverse correlation with GSH levels in ASM. The airway tone showed an inverse association with SERCA2b expression.ConclusionsOur data suggest that each type of AO and airway tone depends on different ASM phenotypes: direct and indirect AO seems to be sensitive to the level of oxidative stress; indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 and the SERCA2b expression level plays a role in the airway tone.

Postjunctional M2 Muscarinic Receptors Augment Neurally Mediated Cholinergic Contractions of Murine Airway Smooth Muscle.

Postjunctional M2 Muscarinic Receptors Augment Neurally Mediated Cholinergic Contractions of Murine Airway Smooth Muscle.

Effects of airway smooth muscle contraction and inflammation on lung tissue compliance.

There are renewed interests in using the parameter K of Salazar-Knowles' equation to assess lung tissue compliance. K either decreases or increases when the lung's parenchyma stiffens or loosens, respectively. However, whether K is affected by other common features of respiratory diseases, such as inflammation and airway smooth muscle (ASM) contraction, is unknown. Herein, male C57BL/6 mice were treated intranasally with either saline or lipopolysaccharide (LPS) at 1 mg/kg to induce pulmonary inflammation. They were then subjected to either a multiple or a single-dose challenge with methacholine to activate ASM to different degrees. A quasi-static pressure-driven partial pressure-volume (P-V) maneuver was performed before and after methacholine. The Salazar-Knowles' equation was then fitted to the deflation limb of the P-V loop to obtain K, as well as the parameter A, an estimate of lung volume (inspiratory capacity). The fitted curve was also used to derive the quasi-static elastance (Est) at 5 cmH2O. The results demonstrate that LPS and both methacholine challenges increased Est. LPS also decreased A, but did not affect K. In contradistinction, methacholine decreased both A and K in the multiple-dose challenge, whereas it decreased K but not A in the single-dose challenge. These results suggest that LPS increases Est by reducing the open lung volume (A) and without affecting tissue compliance (K), whereas methacholine increases Est by decreasing tissue compliance with or without affecting lung volume. We conclude that lung tissue compliance, assessed using the parameter K of Salazar-Knowles' equation, is insensitive to inflammation but sensitive to ASM contraction.