β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.     

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma

β₂-adrenergic receptor (β₂AR) agonists (β₂-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β₂ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β₂ARs as a measure of resensitization. Despite significant accumulation of β₂ARs in the endosomes of asthmatic HASMCs, endosomal β₂ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β₂AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β₂AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.     

Characterising the Mechanism of Airway Smooth Muscle β2 Adrenoceptor Desensitization by Rhinovirus Infected Bronchial Epithelial Cells

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β₂ agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β₂ adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β₂ adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β₂ adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE₂, PGF_2α and PGI₂ had the ability to cause β₂ adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE₂ did not prevent ASMC β₂ adrenoceptor desensitization; however this medium induced PGE₂ from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β₂ adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE₂ and β₂ adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β₂ adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β₂ adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.     

Inhaled Long-Acting β2-Agonists Do Not Increase Fatal Cardiovascular Adverse Events in COPD: A Meta-Analysis

Background

The cardiovascular safety of inhaled long-acting β₂-agonists (LABAs) in patients with chronic obstructive pulmonary disease (COPD) is a controversial problem. Certain studies have suggested that inhaled LABAs lead to an increased risk of cardiovascular events in patients with COPD. This meta-analysis aimed to assess the cardiovascular safety of inhaled LABAs in COPD.

Methods

A meta-analysis of randomized, double-blind, parallel-group, placebo-controlled trials for LABA treatment of COPD with at least 3 months of follow-up was performed. The fixed-effects model was used to evaluate the effects of LABAs on fatal cardiovascular adverse events. Adverse events were collected for each trial, and the relative risk (RR) and 95% confidence intervals (CI) for LABA/placebo were estimated.

Results

There were 24 trials included in this meta-analysis. Compared with placebo, inhaled LABAs significantly decreased fatal cardiovascular adverse events in COPD patients (RR 0.65, 95% CI 0.50 to 0.86, P = 0.002). In sensitivity analysis, there was still no increased risk of fatal cardiovascular events (RR 0.68, 95%CI 0.46 to 1.01, P = 0.06) after excluding the trial with the largest weight. Among the different types of LABAs, only salmeterol had a significant effect (RR 0.64, 95% CI 0.46 to 0.90). In subgroup analyses, inhaled LABAs were able to significantly decrease fatal cardiovascular events in long-term trials (RR 0.64, 95% CI 0.47 to 0.87) and in trials with severe COPD patients (RR 0.69, 95% CI 0.50 to 0.96).

Conclusion

Inhaled LABAs do not increase the risk of fatal cardiovascular events in COPD patients.     

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48 h) PASMC were stimulated with IL-1β alone or with NO donor, l-arginine and/or NO synthase inhibitor l-NAME for 4 and 24 h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24 h. VEGF release was inhibited by l-NAME (531.8 ± 52 pg/ml), but restored and further elevated by l-arginine (1,529 ± 287 pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by l-NAME (1.1-fold) and augmented by l-arginine (3.8-fold) at 4 h. Restoration of a NO pathway by l-arginine in l-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [³H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.     

Differential Regulation of Extracellular Matrix and Soluble Fibulin-1 Levels by TGF-β1 in Airway Smooth Muscle Cells

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β₁) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×10⁴ cells/cm², and stimulated with or without TGF-β₁ (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β₁ decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β₁ did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β₁ induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β₁ induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β₁ is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.     

Real time analysis of β2-adrenoceptor-mediated signaling kinetics in Human Primary Airway Smooth Muscle Cells reveals both ligand and dose dependent differences

Background

β₂-adrenoceptor agonists elicit bronchodilator responses by binding to β₂-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β₂-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.

Methods

Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β₂-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.

Results

Temporal analysis revealed that in hASM cells the β₂-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.

Conclusions

We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β₂-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β₂-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.     

Constitutively Active Signaling by the G Protein βγ-Subunit Mediates Intrinsically Increased Phosphodiesterase-4 Activity in Human Asthmatic Airway Smooth Muscle Cells

Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4) activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM), as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM) cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.     

Interleukin-1β Stimulates Mitogen-Activated Protein Kinase in U373 Astrocytoma Cells without the Production of Lipid Second Messengers

IL-1 is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 activated only MAPK. In cultured rat astrocytes, IL-1 caused activation of MAPK without inducing proliferation.     

Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle

The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca²⁺-dependent mechanism and that intracellular Ca²⁺ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA.     

p38 Mitogen-Activated Protein Kinase Affects Transforming Growth Factor-β/Smad Signaling in Human Dental Pulp Cells

Transforming Growth Factor-β (TGF-β) plays an essential role in differentiation of dental pulp cells into odontoblasts during reparative dentine formation. However, the mechanism by which TGF-β stimulates dental repair remains rather obscure. Human dental pulp cells were used as an in vitro model in the present work. We showed that TGF-β signaled through mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38, along with Smad pathway. Distinct pathways exerted different time response. SB203580, a specific p38 MAPK inhibitor, reduced phosphorylation of Smad3, while it slightly enhanced phosphorylation of Smad2. Increased phosphorylation of ERK1/2 and p38 confirmed that SB203580 did not block activation of TGF-β receptors. In addition, the inhibition of ERK1/2 activity with MEK1/2 inhibitor U0126 increased TGF-β mediated phosphorylation of Smad3. Our results suggest that p38 affects the phosphorylation of Smad2 and Smad3 differentially during TGF-β signaling in human dental pulp cells and ERK1/2 might be involved in the process.     

Comparison of Functional β-Adrenoceptor Heterogeneity in Central and Peripheral Airway Smooth Muscle of Guinea Pig and Man

Abstract

The nature of the β-adrenoceptor population(s) mediating the relaxation of guinea pig and human airway smooth muscle was investigated. On the basis of a preferential blockade by β₁ and β₂ selective antagonists of the relaxation induced by β₁ and β₂- selective agonists, guinea pig tracheal strip relaxation was found to be mediated both by β₁ - and β₂ - adrenoceptors, the relative participation of which depending on the relative affinities of the agonist towards these two receptors. With highly selective antagonists the noradrenaline(NA)-induced relaxation could be split up biphasically into a β₁- and a β₂ - component. In contrast, no such differential blockade was observed with the guinea pig lung parenchyma strip relaxation which is mediated by a homogenous β₂ -adreno-ceptor population. On comparison of the tracheal, the spirally cut main bronchus- and intrapulmonary airway smooth muscle strips it could be shown that both the sensitivity of NA for neuronal uptake and the apparent affinity of the relaxation by NA decreased in the direction of the lung periphery. Using the same techniques it was ascertained that the relaxation of human tracheal smooth muscle (autopsy, obtained within 6 hours after death), main bronchus and intrapulmonary smooth muscle (operation) are mediated by homogenous β₂ -adrenoceptor populations. In addition, neuronal and extraneuron-al uptake sites were not operative in these preparations, whether obtained from operation or from autopsy.     

Salt Stress in Arabidopsis： Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant＇s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase （MAPK） cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein （LTP）-related hybrid proline-rich protein （HyPRP）, as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azil are hypersensitive to salt stress, while AZIl-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZIl-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.