Induced Contraction Of Airway Smooth Muscle Research Articles

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Inhibiting Airway Smooth Muscle Contraction Using Pitavastatin: A Role for the Mevalonate Pathway in Regulating Cytoskeletal Proteins.

Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred via reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the β2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma—contraction, failure to adequately relax in response to a deep breath, and inflammation—pitavastatin may represent a unique asthma therapeutic.

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.

Epithelium-generated neuropeptide Y induces smooth muscle contraction to promote airway hyperresponsiveness.

Asthma is one of the most common chronic diseases globally and can be divided into presenting with or without an immune response. Current therapies have little effect on nonimmune disease, and the mechanisms that drive this type of asthma are poorly understood. Here, we have shown that loss of the transcription factors forkhead box P1 (Foxp1) and Foxp4, which are critical for lung epithelial development, in the adult airway epithelium evokes a non-Th2 asthma phenotype that is characterized by airway hyperresponsiveness (AHR) without eosinophilic inflammation. Transcriptome analysis revealed that loss of Foxp1 and Foxp4 expression induces ectopic expression of neuropeptide Y (Npy), which has been reported to be present in the airways of asthma patients, but whose importance in disease pathogenesis remains unclear. Treatment of human lung airway explants with recombinant NPY increased airway contractility. Conversely, loss of Npy in Foxp1- and Foxp4-mutant airway epithelium rescued the AHR phenotype. We determined that NPY promotes AHR through the induction of Rho kinase activity and phosphorylation of myosin light chain, which induces airway smooth muscle contraction. Together, these studies highlight the importance of paracrine signals from the airway epithelium to the underlying smooth muscle to induce AHR and suggest that therapies targeting epithelial induction of this phenotype may prove useful in treatment of noneosinophilic asthma.

Sarcoplasmic reticulum Ca2+ refilling is determined by L-type Ca2+ and store operated Ca2+ channels in guinea pig airway smooth muscle

Sarcoplasmic reticulum Ca2+ refilling (SRREF) is crucial to sustain the agonists induced airway smooth muscle contraction. Nevertheless, its mechanisms have not been clearly described yet, although L-type voltage dependent, store operated, receptor operated channels and the Na+/Ca2+ exchanger in its reverse mode (NCXREV) have been proposed as Ca2+ handling proteins participating in this process. We found that in guinea pig and bovine tracheal smooth muscle, SRREF induced by caffeine was completely abolished by thapsigargin, even in the presence of Bay K8644, an activator of the L-type Ca2+ channel. Activation of NCXREV in guinea pig tracheal myocytes increased SRREF in ~70%, while opening of the L-type Ca2+ channels with Bay K8644 and favoring the capacitative Ca2+ entry with 2-APB (32μM) also augmented the SRREF by ~170% and ~71%, respectively. Methoxyverapamil (D-600, an L-type Ca2+ channel blocker), 2-APB (100µM, antagonist of the capacitative Ca2+ entry) and PPADS (NCXREV blocker) diminished the SRREF by ~63%, ~72% and ~31%, respectively. The simultaneous addition of D-600 and 2-APB annulled SRREF. These last results were also seen when carbachol was used instead of caffeine. In tracheal rings, 2-APB and nifedipine abolished the carbachol-induced contraction. We concluded that the sarcoplasmic reticulum Ca2+ pump is the only mechanism involved in the SRREF and that L-type Ca2+ voltage dependent and store operated Ca2+ channels are the principal membranal Ca2+ handling proteins that provide extracellular Ca2+ for SRREF and carbachol-induced contraction in the guinea pig airway smooth muscle; NCXREV seems to play a minor role.

The Sophora flavescens flavonoid compound trifolirhizin inhibits acetylcholine induced airway smooth muscle contraction.

Asthma is a serious health problem worldwide, particularly in industrialized countries. Despite a better understanding of the pathophysiology of asthma, there are still considerable gaps in knowledge as well as a need for classes of drugs. ASHMI™ (Anti-asthma Herbal Medicine Intervention) is an aqueous extract of Ganoderma lucidum (Fr.) P. Karst (Ling Zhi), Sophora flavescens Aiton (Ku Shen) and Glycyrrhiza uralensis Fisch. ex DC (Gan Cao). It prevents allergic asthma airway hyper-reactivity in mice and inhibits acetylcholine (ACh) induced airway smooth muscle (ASM) contraction in tracheal rings from allergic asthmatic mice. The purpose of this research was to identify individual herb(s) and their active compound(s) that inhibit ASM contraction. It was found that S. flavescens, but not G. lucidum or G. uralensis aqueous extracts, inhibited ASM contraction in tracheal rings from asthmatic mice. Bioassay-guided isolation and identification of flavonoid fractions/compound(s) via methylene chloride extraction, preparative HPLC fractionation, and LC-MS and NMR spectroscopic analyses showed that trifolirhizin is an active constituent that inhibits acetylcholine mediated ASM contraction or directly relaxes pre-contracted ASM independent of β2-adrenoceptors.

Desflurane and neural control of airway tone

The study by Myers et al. in this edition of the Journal adds to our understanding of the role of desflurane in reactive airway disease. The authors compare the ability of sevoflurane with that of desflurane to alter respiratory mechanics following a cholinergic contraction in normal or sensitized rabbits whose lungs were ventilated through a tracheostomy. This study is an important addition to a growing body of literature which addresses an important clinical question: Does the choice of volatile anesthetic matter in patients with reactive airway disease, and more specifically, is desflurane a bad choice in patients with reactive airway disease? The airway is exposed to two primary irritants during general anesthesia with a volatile anesthetic, i.e., a foreign body (endotracheal tube or laryngeal mask airway device [LMAD]) acting as a mechanical irritant and a volatile anesthetic which can elicit a chemical irritation. C-fibres, including excitatory non-adrenergic non-cholinergic (eNANC) nerves, innervate the upper airway and are activated by transient receptor potential (TRP) channels to release tachykinins which can elicit smooth muscle contraction. Desflurane has been shown to activate TRP A1 receptors on C-fibres, inducing a tachykinin-mediated airway response. A secondary important neural reflex pathway which responds to airway irritation is the cholinergic pathway whereby parasympathetic nerve fibres travelling within the vagus nerve release acetylcholine which induces airway smooth muscle contraction. The current study was designed to evaluate the effect of desflurane on the neurotransmitter of this latter neural pathway, namely, acetylcholine. The authors demonstrate that desflurane is comparable with sevoflurane in its ability to attenuate bronchoconstriction induced by activation of muscarinic receptors on airway smooth muscle by intravenous methacholine. These results are consistent with previous studies which implicated TRP channels on C-fibres as the culprit of desflurane-induced bronchoconstriction observed in clinical studies. Although not a topic of the current study, desflurane activation of C-fibres would also account for the frequently observed clinical complication of cough. In isolated human bronchi, desflurane was equally potent as halothane and isoflurane at relaxing proximal but not distal bronchi. This finding is also consistent with the present study since neural innervation is thought to be primarily cholinergic in large central airways and eNANC in peripheral airways. In previous studies of isolated airways or tracheostomized and ventilated lungs of animals from multiple species, desflurane was shown to be at least as effective as other volatile anesthetics in directly relaxing airway smooth muscle. Taken together, these studies, which are performed in the absence of intact innervation, suggest that clinical difficulties with desflurane and the airway are not likely related to direct airway smooth muscle effects but are mediated by differential effects on irritant neural reflexes. The current study is an important addition to our mechanistic understanding of differential effects of volatile anesthetics on bronchoconstriction. This study demonstrates that sevoflurane and desflurane are similar in their abilities to prevent bronchoconstriction of cholinergic origin occurring in sensitized airways. It should be emphasized that desflurane was delivered to an intubated trachea (or isolated airway tissue) in this and previous C. W. Emala, MD (&) Department of Anesthesiology, Columbia University, 622 West 168th St., P&S Box 46, New York, NY 10032, USA e-mail: cwe5@columbia.edu

Differentially Expressed MicroRNAs in Allergic Asthma Target Genes underlying Airway Hyper-Responsiveness and Goblet Cell Hyperplasia (136.22)

Abstract Asthma is an allergic disease that results in the obstruction of airways as a result of goblet cell hyperplasia and airway hyper-reactivity (AHR). Broad-spectrum P2-receptor antagonists and glucocorticoids are commonly used therapeutic agents for this disease. Niflumic acid (NA), an inhibitor of calcium-activated chloride channels is used to overcome corticosteroid-resistant asthma. Using Next Generation Sequencing, we dissected the asthma-related small RNAome in the lung before and after (FC) allergen challenge. We find a significant induction of microRNAs in FC from 40% to 74% suggesting an important role for miRNAs in asthma. Also, we uncovered 460 miRNA-up:mRNA-down and 17 miRNA-down:mRNA-up pairs that are significantly correlated in naïve and FC lung. The latter category contains a number of key genes induced during asthma and include miR-106b:P2X, miR-205:P2Y, miR-145:CLCA3 and miR-133a:GM-CSF. We also uncovered two novel miRNAs Asth-miR-1 and Asth-miR-2. Asth-miR-1 is predicted to target Toll-like receptor (TLR) adaptor TIRAP and IRAK which associates to activate NF-Kb, AP-1 and IRFs, and under some conditions, induce allergic lung disease. Asth-miR-2 is predicted to target ryanodine receptor 2 (RyR) that mediates Ca2+ release that induces airway smooth muscle contraction and bronchoconstriction. The integration of miR-106b, 205, 145 a, 133a and Asth-miR-1 and Asth-miR-1 2 with current therapies can potentially significantly enhance the efficacy and specificity of drugs used to combat asthma.

Insulin induces airway smooth muscle contraction

Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations. It was found that insulin (1 nM-1 microM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 microM), Rho-kinase (Y-27632, 1 microM) and p42/44 MAP kinase (PD-98059, 30 microM and U-0126, 3 microM), but not of PI-3-kinase (LY-294002,10 microM). In addition, insulin significantly increased PGF(2alpha)-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 microM) and the EP(1)-receptor antagonist AH-6809 (10 microM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins. Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.

Diacylglycerol-Containing Docosahexaenoic Acid in Acyl Chain Modulates Airway Smooth Muscle Tone

We synthesized and assessed the role of a diacylglycerol (DAG)-containing docosahexaenoic acid (DHA), that is, 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDHG), in the contraction of guinea pig airway smooth muscle (ASM). We compared its action with 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and 1,2-dioctanoyl-sn-glycerol (1,2-DiC8), a stable DAG analog. The three DAGs (SAG, SDHG, and 1,2-DiC8) induced reversible concentration-dependent contraction of ASM. SDHG induced higher guinea pig ASM contraction than did SAG and 1,2-DiC8. The effects of SDHG were blocked, to different extents, by nifedipine (L-type Ca2+ channel blocker). By employing GF-109203X (protein kinase C [PKC] inhibitor) and lanthanum (La3+), a nonselective cation channel blocker, we observed that SDHG evoked ASM contractile response via PKC-dependent and PKC-independent (but Ca2+-dependent) pathways. Interestingly, SAG exerted its action only by increasing [Ca2+]i and did not require PKC activation. To probe the implication of calcium mobilization, we employed thapsigargin (TG), which also induced ASM contraction in a calcium-dependent manner. SDHG and 1,2-DiC8, in a PKC-dependent manner, induced the phosphorylation of CPI-17 (myosin light chain phosphatase inhibitor of 17 kD). Furthermore, SAG and TG failed to phosphorylate CPI-17 in ASM cells. Our results suggest that different DAG species, produced during a dietary supplementation with fatty acids, could modulate the reactivity of airway smooth muscles in a PKC-dependent and -independent manner, and hence, may play a critical role in health and disease.

NO does not mediate inhibitory neural responses in sheep airway and bronchial vascular smooth muscle.

Endogenous nitric oxide (NO) influences acetylcholine-induced bronchovascular dilation in sheep and is a mediator of the airway smooth muscle inhibitory nonadrenergic, noncholinergic neural response in several species. This study was designed to determine the importance of NO as a neurally derived modulator of ovine airway and bronchial vascular smooth muscle. We measured the response of pulmonary resistance (RL) and bronchial blood flow (Qbr) to vagal stimulation in 14 anesthetized, ventilated, open-chest sheep during the following conditions: 1) control; 2) infusion of the alpha-agonist phenylephrine to reduce baseline Qbr by the same amount as would be produced by infusion of Nomega-nitro-L-arginine (L-NNA), a NO synthase inhibitor; 3) infusion of L-NNA (10(-2) M); and 4) after administration of atropine (1.5 mg/kg). The results showed that vagal stimulation produced an increase in RL and Qbr in periods 1, 2, and 3 (P < 0.01) that was not affected by L-NNA. After atropine was administered, there was no increase in Qbr or RL. In vitro experiments on trachealis smooth muscle contracted with carbachol showed no effect of L-NNA on neural relaxation but showed a complete blockade with propranolol (P < 0.01). In conclusion, the vagally induced airway smooth muscle contraction and bronchial vascular dilation are not influenced by NO, and the sheep's trachealis muscle, unlike that in several other species, does not have inhibitory nonadrenergic, noncholinergic innervation.