Airway Smooth Muscle Relaxation Research Articles

Dehydroepiandrosterone Supplementation May Benefit Women with Asthma Who Have Low Androgen Levels: A Pilot Study.

IntroductionAmong individuals with severe asthma, FEV1 is low in individuals with low dehydroepiandrosterone (DHEA) sulfate (DHEAS) levels. In the Severe Asthma Research Program (SARP), no women with DHEAS > 200 μg/dL had an FEV1 < 60% predicted. DHEA has benefited patients with COPD and pulmonary hypertension in small trials. Therefore, we hypothesized that DHEA supplementation may improve FEV1 in asthmatic women with low DHEAS.MethodsPremenopausal, nonsmoking, otherwise healthy women, 18-50 years old, with mild or moderate asthma and baseline FEV1 > 60% predicted received 100 mg DHEA orally every 12 h for 2 weeks. Spirometry and DHEAS were measured at the initial visit and 2 weeks later, after completion of DHEA treatment. Based on our previous work, the primary outcome variable for this pilot study was post-albuterol spirometry in the low-DHEAS group. Subjects also continued their other routine asthma management.ResultsSerum DHEAS increased with DHEA treatment in women with starting DHEAS < 200 µg/dL: this increase was from 71 ± 23 to 725 ± 295 µg/dL (n = 10; p = 0.0001). The increase in the high-DHEAS group was smaller. Post-albuterol FEV1 increased by 51 mL, from 3.026 ± 0.5 to 3.077 ± 0.49 L (n = 10; p = 0.034 by paired t test, significant after Bonferroni), in women with low DHEAS. In the high-DHEAS group (baseline DHEAS ≥ 200 µg/dl), post-albuterol FEV1 did not change significantly (n = 3, p = NS). Three subjects were excluded: one had comorbid COPD, one could not perform spirometry, and one did not take the DHEA. There were no adverse effects of DHEA treatment in this trial.ConclusionsEndocrine treatments (corticosteroids) are a mainstay of anti-inflammatory management for moderate and severe asthma. Their use has improved asthma outcomes. Androgens also reduce airway inflammation and promote airway smooth muscle relaxation, but are rarely used clinically for asthma treatment. Our results suggest that the over-the-counter steroid DHEA may improve lung function in asthma outcomes among women with DHEAS < 200 ug/dL.

Estrogen receptors differentially regulate intracellular calcium handling in human nonasthmatic and asthmatic airway smooth muscle cells.

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERβ, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]i regulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]i response to histamine, whereas ERβ activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERβ activation was more effective than 17β-estradiol in reducing [Ca2+]i responses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERβ were mediated partially via L-type calcium channel inhibition and increased Ca2+ sequestration by sarcoplasmic reticulum. Overall, these data highlight the differential signaling of ERα and ERβ in ASM during inflammation. Specific ERβ activation reduces [Ca2+]i in the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.

NS8593 inhibits Ca2+ permeant channels reversing mouse airway smooth muscle contraction

AimsThis study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. Main methodsASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. Key findingsHigh K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). SignificanceOur results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.

Role of TRPP2 in mouse airway smooth muscle tension and respiration.

The transient receptor potential polycystin-2 (TRPP2) is encoded by the Pkd2 gene, and mutation of this gene can cause autosomal dominant polycystic kidney disease (ADPKD). Some patients with ADPKD experience extrarenal manifestations, including radiologic and clinical bronchiectasis. We hypothesized that TRPP2 may regulate airway smooth muscle (ASM) tension. Thus, we used smooth muscle-Pkd2 conditional knockout (Pkd2SM-CKO) mice to investigate whether TRPP2 regulated ASM tension and whether TRPP2 deficiency contributed to bronchiectasis associated with ADPKD. Compared with wild-type mice, Pkd2SM-CKO mice breathed more shallowly and faster, and their cross-sectional area ratio of bronchi to accompanying pulmonary arteries was higher, suggesting that TRPP2 may regulate ASM tension and contribute to the occurrence of bronchiectasis in ADPKD. In a bioassay examining isolated tracheal ring tension, no significant difference was found for high-potassium-induced depolarization of the ASM between the two groups, indicating that TRPP2 does not regulate depolarization-induced ASM contraction. By contrast, carbachol-induced contraction of the ASM derived from Pkd2SM-CKO mice was significantly reduced compared with that in wild-type mice. In addition, relaxation of the carbachol-precontracted ASM by isoprenaline, a β-adrenergic receptor agonist that acts through the cAMP/adenylyl cyclase pathway, was also significantly attenuated in Pkd2SM-CKO mice compared with that in wild-type mice. Thus, TRPP2 deficiency suppressed both contraction and relaxation of the ASM. These results provide a potential target for regulating ASM tension and for developing therapeutic alternatives for some ADPKD complications of the respiratory system or for independent respiratory disease, especially bronchiectasis.

Transforming Growth Factor-β1 Decreases β2-Agonist-induced Relaxation in Human Airway Smooth Muscle.

Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-β1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-β1 affects the ability of HASM cells to relax in response to β2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-β1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-β1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-β1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-β1 decreases HASM cell β2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying β2-agonist hyporesponsiveness in asthma, and suggest TGF-β1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.

Effects of intravenous phosphodiesterase inhibitors and corticosteroids on severe meconium aspiration syndrome.

Meconium aspiration syndrome (MAS) is a major cause of severe respiratory failure in near- and full-term neonates. Alleviating inflammation is key to successfully treating severe MAS. Phosphodiesterase (PDE) inhibitors are known to play a role in airway smooth muscle relaxation and alveolar inflammation inhibition. This study aimed to investigate the effects of various intravenous (IV) PDE inhibitors and corticosteroids on MAS. MAS was induced in newborn piglets by instilling human meconium in them. The piglets were randomly divided into five groups (n = 5 in each group): (1) control (sham treatment); (2) dexamethasone (Dex) (IV 0.6 mg/kg of dexamethasone); (3) aminophylline (Ami) (IV 6 mg/kg of aminophylline, followed by continuous infusion of 0.5 mg/kg/h of aminophylline; (4) milrinone (Mil) (IV 50 μg/kg of milrinone, followed by continuous infusion of 0.75 μg/kg/h of milrinone); and (5) rolipram (Rol) (IV 0.8 mg/kg of rolipram). The duration of the experimental period was 4 hours. Compared to the control group, all the four treatment groups revealed better oxygenation 3 hours and more after the start of treatment. The Rol group had a significantly elevated heart beat (p < 0.05) and relatively lower blood pressure compared to the other groups during the first 2 hours of the experiment. The Dex group had significantly lower interleukin (IL)-1β levels in the lung tissue compared to the other groups (p < 0.05) and significantly lower IL-6 levels compared to the Ami and Mil groups (p < 0.05). Lung histology showed slightly less inflammation and atelectasis in the Dex group compared to the other groups, but lung injury scores showed no significant between-group differences. Using IV corticosteroids or any type of PDE inhibitors has some beneficial effects in improving oxygenation in MAS. PDE inhibitors are not superior to IV corticosteroids; in fact, adverse cardiovascular effects occur with the phosphodiesterase type 4 (PDE4) inhibitor. Further investigations are required before using IV corticosteroids and PDE inhibitors in future clinical application.

Relaxant Action of Diclofenac Sodium on Mouse Airway Smooth Muscle.

Diclofenac sodium (DCF) is a nonsteroidal anti-inflammatory drug (NSAID) and is widely used as an analgesic and anti-inflammatory agent. Herein, we found that DCF could relax high K+ (80 mM K+)-/ACh-precontracted tracheal rings (TRs) in mice. This study aimed to elucidate the underlying mechanisms of DCF-induced relaxations. The effects of DCF on airway smooth muscle (ASM) cells were explored using multiple biophysiological techniques, such as isometric tension measurement and patch-clamping experiments. Both high K+- and ACh-evoked contraction of TRs in mice were relaxed by DCF in a dose-dependent manner. The results of isometric tension and patch-clamping experiments demonstrated that DCF-induced relaxation in ASM cells was mediated by cytosolic free Ca2+, which was decreased via inhibition of voltage-dependent L-type Ca2+ channels (VDLCCs), nonselective cation channels (NSCCs), and Na+/Ca2+ exchange. Meanwhile, DCF also enhanced large conductance Ca2+ activated K+ (BK) channels, which led to the relaxation of ASMs. Our data demonstrated that DCF relaxed ASMs by decreasing the intracellular Ca2+ concentration via inhibition of Ca2+ influx and Na+/Ca2+ exchange. Meanwhile, the enhanced BK channels also played a role in DCF-induced relaxation in ASMs. These results suggest that DCF is a potential candidate for antibronchospasmic drugs used in treating respiratory diseases such as asthma and chronic obstructive pulmonary disease.

In Vitro Effects of Beta-2 Agonists on Skeletal Muscle Differentiation, Hypertrophy, and Atrophy

Background Beta-2 agonists are widely used in the treatment of asthma and chronic obstructive pulmonary disease for their effect on airway smooth muscle relaxation. They also act on skeletal muscle, although their reported ergogenic effect is controversial.Aim To evaluate the in vitro effects of short-acting and long-acting beta-2 agonists on adrenergic receptor (ADR) expression, hypertrophy, and atrophy markers, in a skeletal muscle cell line.Methods The C2C12 cell line was used as a model of skeletal muscle differentiation. ADR messenger RNA expression was evaluated in proliferating myoblasts, committed cells, and differentiated myotubes, in basal conditions and after treatment with 10-6 M clenbuterol, salbutamol, salmeterol, and formoterol. Effect of beta-2 agonists on gene and protein expression of hypertrophy and atrophy markers was assessed in differentiated myotubes.Results Our study shows that beta-2 ADR messenger RNA was expressed and progressively increased during cell differentiation. Beta-2 agonist treatment did not affect its expression. Skeletal muscle hypertrophy markers (fast and slow myosin, myogenin) were not modulated by any of the beta-2 agonists evaluated. However, clenbuterol induced a significant, dose-dependent downregulation of skeletal muscle atrophy genes (atrogin-1, MuRF-1, and cathepsin L).Conclusions The reported ergogenic effect of beta-2 agonists, if any, should be considered as drug-specific and not class-specific and that of clenbuterol is mediated by the inhibition of the atrophic pathway.

Molecular Dynamics Simulations Reveal a Possible Membrane‐Involved Microkinetic Mechanism for Long‐Lasting Action of Salmeterol, a β2‐Adrenergic Receptor Agonist

ObjectiveThe exact mechanisms underlying the longer duration of action and capacity to “reassert” airway smooth muscle relaxation in vitro despite repeated washing, of several long‐acting β2‐adrenergic receptor (β2‐AR) agonists such as salmeterol remain unresolved. Recent experimental studies suggest that the “microkinetic model” in which membrane acts as a depot enriching local drug concentration around the receptor adequately explains the long‐lasting effects. Specifically, the 700‐fold higher potency of salmeterol to β2‐AR in comparison to the structurally related agonists was found to be majorly due to its interactions with the membrane. The ‘reduction of dimensionality’ effect whereby ligands move on or within the membrane by 2D lateral diffusion could likely produce further enhancement of drug association rates and corresponding affinity values. These studies also suggested that the “exosite” described before probably reflects the phospholipid membrane rather than specific residues on the receptor. Most recently, a crystal structure of salmeterol bound to the active β2‐AR revealed an exosite for its aryloxyalkyl tail at the extracellular vestibule of the receptor. However, the association and dissociation pathways through which salmeterol access and leave the binding site is unknown. In addition, the possibility of lipophilic salmeterol reaching the binding site through transmembrane helices from the surrounding membrane environment is compelling and warrants thorough investigation, especially with the availability of the bound crystal structure. Here, we present an extensive computational study investigating the plausible lipid‐mediated association and dissociation pathways of salmeterol.MethodsThe most favorable bilayer location, orientation, and conformation of salmeterol were investigated using a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) model membrane with 10% cholesterol by steered molecular dynamics (MD) and Umbrella Sampling simulations, using NAMD2.12 software. The dissociation and association pathways of salmeterol from the bound β2‐AR crystal structure (PDB ID 6CSY) were investigated by: 1) steered MD, 2) temperature accelerated MD, and 3) funnel metadynamics simulations. The protein, POPC lipids, and salmeterol were parameterized using the CHARMM36 and CGenFF force fields respectively.ResultsThe results thus far, reveal that the dissociation of salmeterol from its bound form proceeds through the extracellular side of the receptor, with a transient stay in the aqueous bulk before finally partitioning into the membrane (near the β2‐AR) in its energetically favorable bilayer depth (~15 Å from the bilayer center) determined by our partitioning studies. The specific interactions of the ligand with the receptor residues along the binding pathways and associated free energies were analyzed.ConclusionsComputational results suggest that the ‘microkinetic” mechanism attributed to the high membrane affinity of salmeterol may play a concerted role with its exosite binding towards the unique pharmacological properties.Support or Funding InformationNIH – R15GM131293This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A microphysiological model of the bronchial airways reveals the interplay of mechanical and biochemical signals in bronchospasm

In asthma, airway smooth muscle (ASM) contraction and the subsequent decrease in airflow involve a poorly understood set of mechanical and biochemical events. Organ-level and molecular-scale models of the airway are frequently based on purely mechanical or biochemical considerations and do not account for physiological mechanochemical couplings. Here, we present a microphysiological model of the airway that allows for the quantitative analysis of the interactions between mechanical and biochemical signals triggered by compressive stress on epithelial cells. We show that a mechanical stimulus mimicking a bronchospastic challenge triggers the marked contraction and delayed relaxation of ASM, and that this is mediated by the discordant expression of cyclooxygenase genes in epithelial cells and regulated by the mechanosensor and transcriptional co-activator YAP (Yes-associated protein). A mathematical model of the intercellular feedback interactions recapitulates aspects of obstructive disease of the airways, including pathognomonic features of severe, difficult-to-treat asthma. The microphysiological model could be used to investigate the mechanisms of asthma pathogenesis and to develop therapeutic strategies that disrupt the positive feedback loop that leads to persistent airway constriction.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

Mechanopharmacology and Synergistic Relaxation of Airway Smooth Muscle.

Asthmatic airways are stiffer than normal. We have shown that the cytoskeletal passive stiffness of airway smooth muscle (ASM) can be regulated by intracellular signaling pathways, especially those associated with Rho kinase (ROCK). We have also shown that an oscillatory strain reduces the passive stiffness of ASM and its ability to generate force. Here, we investigated the combined effect of inhibiting the ASM contraction with β2 agonist and decreasing the ASM cytoskeletal stiffness with ROCK inhibitor and/or force oscillation (FO) on the relaxation of contracted ASM. We hypothesize that the ASM relaxation can be synergistically enhanced by the combination of these interventions, because drug-induced softening of the cytoskeleton enhances the FO-induced relaxation and vice versa. Sheep tracheal strips were isotonically contracted to acetylcholine (3 × 10−5 M). At the plateau of shortening, β2 agonist salbutamol (10−7 M), ROCK inhibitor H1152 (10−7 M), and FO (square wave, 1 Hz, amplitude 6% maximal active force) were applied either alone or in combination. After adjusting for nonspecific time-dependent variation, relengthening by individual interventions with low-dose salbutamol or H1152, or small amplitude FO was not significantly different from zero. However, significant relengthening was observed in all combination treatments. The relengthening was greater than the mathematical sum of relengthening caused by individual treatments thereby demonstrating synergistic relaxation. The ASM stiffness did not change with salbutamol or H1152 treatments, but was lower with FO in combination with H1152. The results suggest that the mechanopharmacological treatment can be an effective therapy for asthma.

Saponins of Dioscorea Nipponicae Inhibits IL-17A-Induced Changes in Biomechanical Behaviors of In Vitro Cultured Human Airway Smooth Muscle Cells.

Airway hyperresponsiveness (AHR) is one of the main pathologic features of bronchial asthma, which is largely attributable to enhanced contractile response of asthmatic airway smooth muscle. Although β2 adrenergic receptor agonists are commonly used to relax airway smooth muscle for treating AHR, there are side effects such as desensitization of long-term use. Therefore, it is desirable to develop alternative relaxant for airway smooth muscle, preferably based on natural products. One potential candidate is the inexpensive and widely available natural herb saponins of Dioscorea nipponicae (SDN), which has recently been reported to suppress the level of inflammatory factor IL-17A in ovalbumin-induced mice, thereby alleviating the inflammation symptoms of asthma. Here, we evaluated the biomechanical effect of SDN on IL-17A-mediated changes of cultured human airway smooth muscle cells (HASMCs) in vitro. The stiffness and traction force of the cells were measured by optical magnetic twisting cytometry (OMTC), and Fourier transform traction microscopy (FTTM), respectively. The cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry, the cell migration was measured by cell scratch test, and the changes of cell cytoskeleton were assessed by laser confocal microscopy. We found that the stiffness and traction force of HASMCs were enhanced along with the increases of IL-17A concentration and exposure time, and SDN treatment dose-dependently reduced these IL-17A-induced changes in cell mechanical properties. Furthermore, SDN alleviated IL-17A-mediated effects on HASMCs proliferation, migration, and cytoskeleton remodeling. These results demonstrate that SDN could potentially be a novel drug candidate as bronchodilator for treating asthma-associated AHR.

Artesunate attenuates airway resistance in vivo and relaxes airway smooth muscle cells in vitro via bitter taste receptor-dependent calcium signalling.

What is the central question of this study? The aim of this study was to evaluate artesunate for its use as a bronchodilator in asthma treatment. What is the main finding and its importance? We found that artesunate reduces airway resistance in both normal and ovalbumin-treated Balb/c mice in vivo. Artesunate reduces traction force while inducing Ca2+ influx into cultured airway smooth muscle cells in vitro, most probably via the bitter taste receptor. These findings provide important evidence at both animal and cellular levels that artesunate might potentially be used as a bronchodilator for treating obstructive airway diseases, such as asthma. Following the surprising discovery that bitter taste receptors (TAS2Rs) expressed in the lung and can be stimulated to relax airway smooth muscle cells (ASMCs), there is great interest in searching for a bitter taste receptor agonist as a new bronchodilator for asthma therapy. Among the great many other natural bitter substances, artesunate is of special interest to be evaluated for this purpose because of its pharmacological value as a derivative from the well-known anti-malarial, artemisinin. Therefore, in this study we treated either normal or ovalbumin (OVA)-induced asthmatic Balb/c mice in vivo with artesunate (30, 60 or 120μg) via aerosol inhalation. Subsequently, we measured the airway resistance of the mice in the presence or absence of artesunate. In addition, we treated either mouse or human ASMCs cultured in vitro with artesunate (0.25-2.0mM) and then measured the traction force and [Ca2+ ]i flux of the cells in the presence or absence of artesunate. The results demonstrate that artesunate attenuated airway resistance in a dose-dependent manner in both the normal and the OVA-treated mice, but more potently in the latter. The in vivo efficacy of artesunate at 120μg was comparable to that of the conventional bronchodilator, salbutamol, at 3μg in terms of the reduction in airway resistance. Artesunate also reduced traction force and induced an increase in [Ca2+ ]i in the cultured ASMCs, which was mediated, at least in part, by TAS2R signalling in the human ASMCs. These results together suggest that artesunate might potentially be a cheap and safe bronchodilator to complement the current therapy of asthma.

A novel GABAA receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.

Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.

Attenuation of murine and human airway contraction by a peptide fragment of the cytoskeleton regulatory protein gelsolin.

We have previously reported that mice genetically deficient in the actin binding protein gelsolin exhibit impaired airway smooth muscle (ASM) relaxation. Primary cultured ASM cells from these mice demonstrate enhanced inositol triphosphate (IP3) synthesis and increased intracellular calcium in response to Gq-coupled agonists. We hypothesized that this was due to increased intracellular availability of unbound phosphatidylinositol 4,5-bisphosphate (PIP2), based on the fact that gelsolin contains a short peptide region that binds PIP2, presumably making it a less available substrate. We now questioned whether a peptide that corresponds to the PIP2 binding region of gelsolin could modulate ASM signaling and contraction. The 10 amino acid sequence of the gelsolin peptide within the PIP2-binding region was incubated with primary cultures of human ASM cells, and IP3 synthesis was measured in response to a Gq-coupled agonist. Gelsolin peptide-treated cells generated less IP3 under basal and bradykinin or acetylcholine (Gq-coupled) conditions. Acetylcholine-induced contractile force measured in isolated tracheal rings from mice and human tracheal muscle strips in organ baths was attenuated in the presence of the gelsolin peptide. The gelsolin peptide also attenuated methacholine-induced airway constriction in murine precision-cut lung slices. Furthermore, this peptide fragment delivered to the respiratory system of mice via nebulization attenuated subsequent methacholine-induced increases in airway resistance in vivo. The current study demonstrates that introduction of this small gelsolin peptide into the airway may be a novel therapeutic option in bronchoconstrictive diseases.

Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one derivatives as potent β2-adrenoceptor agonists.

A series of novel β2-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent β2-adrenoceptor agonistic effects and high β2/β1-selectivity with EC50 values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea pig trachea model of bronchodilation. These results support further development of the two compounds into drug candidates.

Airway smooth muscle relaxant activity of Cordia dodecandra A. DC. mainly by cAMP increase and calcium channel blockade

Ethnopharmacological relevanceThe fight against chronic respiratory diseases needs the exploration of new active compounds with properties that contribute to diminish the symptoms or resolve the disease alongside current therapy. Materials and methodsEight extracts obtained from the bark and leaves of a Mayan medicinal plant used to treat asthma, Cordia dodecandra A. DC., were investigated for their relaxant effect on rat isolated tracheal rings pre-contracted with carbachol [1 µM]. The underlying functional mode of action of the most effective extract was assessed and the chromatographic fingerprints of more active extracts were analyzed. ResultsThe dichloromethane bark extract (DECd-b) was the most effective and potent (Emax= 87.57 ± 1.32 %; EC50 = 392.7 ± 5.18 µg/mL). DECd-b relaxant effect was maximized in presence of isoproterenol (β-adrenergic agonist, [10 µM]) and theophylline (phosphodiesterase unspecific inhibitor, [10 µM]). DECd-b also showed efficient relaxation of KCl [80 mM]-induced contraction and inhibition of CaCl2-induced contraction. Pre-incubation with propranolol (non-selective β-adrenergic antagonist, [10 µM]), SQ22536 (adenylyl cyclase inhibitor; [100 µM]), ODQ (guanylyl cyclase inhibitor; [10 µM]), l-NAME (nitric oxide synthase inhibitor; [10 µM]), indomethacin (a cyclooxygenase unspecific inhibitor; [10 µM]), glibenclamide (ATP-sensitive potassium channel blocker; [10 µM]) and 2-aminopyridine (voltage-gated potassium channel blocker [100 µM]) did not modify the DECd-b relaxant-effect curve. The chromatographic analysis of DECd-b suggests the cordiaquinones presence with double conjugated bounds such as menaquinone. ConclusionsResults suggest that DECd-b induces relaxation mainly by cAMP increase and Ca2+ channel blockade. The chromatographic profiles and UV spectrum of DECd-b and HECd-l suggest the presence of molecules with structure of meroterpenoid naphthoquinones. This work report scientific evidence of C. dodecandra medicinal specie, which contributes to the pharmacological and phytochemical background of C. dodecandra providing an added value to the traditional use of this specie.

Pleiotropic effects and pharmacological properties of penehyclidine hydrochloride.

BackgroundPenehyclidine hydrochloride (PHC) is an anticholinergic drug manufactured in China. It is used widely in clinics as a reversal agent in cases of organic phosphorus poisoning and as a preanesthetic medication. Compared with other anticholinergic agents, PHC confers substantial advantages. Here, in this review, we focus on its important clinical effects for organic phosphorus poisoning, preanesthetic medication, and the protective effects on certain visceral organs.Materials and methodsOur bibliographic sources include the PubMed and China National Knowledge Infrastructure (CNKI) databases, updated in March 2018. To assess the data in detail, we used the search terms “penehyclidine hydrochloride,” “preanesthetic medication,” and “organic phosphorus.” Papers were restricted to those published in the English and Chinese languages, and to “paper” and “review” as the document type.ResultsPHC can effectively antagonize the symptoms of central and peripheral poisoning caused by organophosphorus poisoning. As a preanesthetic medication, it can not only effectively reduce mucus secretion and vascular infiltration but can also relax airway smooth muscles, dilate bronchioles in pulmonary conditions such as bronchiectasis, and increase pulmonary dynamic compliance. It can also prevent reflexive actions of the vagus nerve caused by excessive acetylcholine release such as abnormal airway contraction. Furthermore, it can strengthen sedation, bidirectionally regulate heart rate, and effectively inhibit respiratory secretions. In recent studies, PHC was shown to also have protective effects on various organs, such as the heart, lungs, brain, kidneys, intestines, and liver.ConclusionPHC has beneficial pharmacological properties used in the treatment of organophosphorus poisoning and as a preanesthetic medication for its few side effects. It also has protective effects on multiple organs, suggesting that PHC has extensive clinical application value which is worth further research. This review should be of help to those intending to research these topics further.