A new perspective on muscarinic receptor antagonism in obstructive airways diseases

<p>Acetylcholine has traditionally only been regarded as a neurotransmitter of the parasympathetic nervous system, causing bronchoconstriction and mucus secretion in asthma and COPD by muscarinic receptor activation on airway smooth muscle and mucus-producing cells. Recent studies in experimental models indicate that muscarinic receptor stimulation in the airways also induces pro-inflammatory, pro-proliferative and pro-fibrotic effects, which may involve activation of airway structural and inflammatory cells by neuronal as well as non-neuronal acetylcholine. In addition, mechanical changes caused by muscarinic agonist-induced bronchoconstriction may be involved in airway remodeling. Crosstalk between muscarinic receptors and beta(2)-adrenoceptors on airway smooth muscle causes a reduced bronchodilator response to beta(2)-agonists, and a similar mechanism could possibly apply to the poor inhibition of inflammatory and remodeling processes by these drugs. Collectively, these findings provide novel perspectives for muscarinic receptor antagonists in asthma and COPD, since these drugs may not only acutely affect cholinergic airways obstruction, but also have important beneficial effects on beta(2)-agonist responsiveness, airway inflammation and remodeling. The clinical relevance of these findings is presently under investigation and starting to emerge.</p>

Regulation of airway inflammation and remodeling by muscarinic receptors: Perspectives on anticholinergic therapy in asthma and COPD

Acetylcholine is the primary parasympathetic neurotransmitter in the airways and an autocrine/paracrine secreted hormone from non-neuronal origins including inflammatory cells and airway structural cells. In addition to the well-known functions of acetylcholine in regulating bronchoconstriction and mucus secretion, it is increasingly evident that acetylcholine regulates inflammatory cell chemotaxis and activation, and also participates in signaling events leading to chronic airway wall remodeling that is associated with chronic obstructive airway diseases including asthma and COPD. As muscarinic receptors appear responsible for most of the pro-inflammatory and remodeling effects of acetylcholine, these findings have significant implications for anticholinergic therapy in asthma and COPD, which is selective for muscarinic receptors. Here, the regulatory role of acetylcholine in inflammation and remodeling in asthma and COPD will be discussed including the perspectives that these findings offer for anticholinergic therapy in these diseases. (C) 2012 Elsevier Inc. All rights reserved

De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth

beta-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of beta-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in increased beta-catenin protein stability. Here, we report on a novel pathway that regulates the expression and nuclear presence of beta-catenin. In proliferating human airway smooth muscle cells, we observed increased expression of beta-catenin, which was required for proliferation. Interestingly, increased beta-catenin expression was accompanied by an increase in beta-catenin mRNA and was independent of beta-catenin liberation from the plasma membrane, suggesting a role for de novo synthesis. This was confirmed using actinomycin D and cycloheximide, which abrogated the induction and nuclear localization of beta-catenin protein. GSK-3 inhibition using SB216763 failed to regulate beta-catenin mRNA. However, expression of dominant negative H-Ras or pharmacological inhibition of MEK reduced serum and TGF-beta-induced beta-catenin mRNA and protein. Collectively, these data indicate that beta-catenin is an important signaling intermediate in airway smooth muscle growth and that its cellular accumulation and nuclear localization require de novo protein synthesis effected, in part, via H-Ras and MEK.-Gosens, R., Baarsma, H. A., Heijink, I. H., Oenema, T. A., Halayko, A. J., Meurs, H., Schmidt, M. De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth. FASEB J. 24, 757-768 (2010). www.fasebj.or

Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells

Oenema TA, Smit M, Smedinga L, Racke K, Halayko AJ, Meurs H, Gosens R. Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L589-L597, 2012. First published August 3, 2012; doi:10.1152/ajplung.00400.2011.-Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-beta(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M-2 and M-3 receptors were stimulated with methacholine (MCh), TGF-beta(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-alpha-actin and calponin by TGF-beta(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-alpha-actin stress fibers by TGF-beta(1). MCh did not augment TGF-beta(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-beta(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3 beta and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-beta(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD

Delivery system for budesonide based on lipid-DNA.

Budesonide is a hydrophobic glucocorticoid with high anti-inflammatory activity for the treatment of asthma, inflammatory bowel disease and rheumatoid arthritis. A micellar drug-delivery system based on lipid-DNA may provide a strategy to maximize its drug efficacy and reduce adverse effects. In this work, we report the use of lipid-DNAA (UU11mer), featuring two hydrophobic alkyl chains and forming micelles at a comparatively low critical micelle concentration, to render budesonide water-soluble with a high loading capacity (LC). The inhibition of interleukin-8 (IL-8) release shows that the new delivery system retains the inhibitory activity in cell-based assays. In conclusion, this research provides a novel approach to formulate and administer budesonide in a non-invasive manner, which dramatically improves its water-solubility while retaining its bioavailability

Cross-Talk between Transforming Growth Factor-beta(1) and Muscarinic M-2 Receptors Augments Airway Smooth Muscle Proliferation

<p>Transforming growth factor-beta(1) (TGF-beta(1)) is a central mediator in tissue remodeling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia, as observed in asthma. The mechanisms underlying this response, however, remain unclear because TGF-beta(1) exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-beta(1) on ASM is indirect and requires prolonged exposure to allow for extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-beta(1), alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-beta(1) exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-beta(1) increased ASM cell proliferation and potentiated the platelet-derived growth factor-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-beta(1). Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin alpha 5 beta(1) function-blocking antibodies, inhibited the effects of TGF-beta(1) and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-beta(1) increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-beta(1)-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M-2 receptor subtype-selective antagonist gallamine, but not the M-3-selective antagonist DAU5884. In line with these findings, the irreversible G(i) protein inhibitor pertussis toxin also prevented the potentiation of TGF-beta(1)-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-beta(1) enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M-2 receptor stimulation.</p>

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle

Abstract Background Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown. Methods The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β. Results Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc. Conclusions We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.</p

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 μM) or TGF-β1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-β release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-β1-induced remodeling, but rather, it inhibited methacholine-induced TGF-β release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-β release and bronchoconstriction

Bronchoconstriction induces the release of biologically active TGF-β leading to contractile protein expression.

<p>Human MRC-5 fibroblasts were stimulated for 1 hour with TGF-β₁ (2 ng/mL), methacholine (MCh; 10 µM) or medium (basal), or with conditioned media obtained from lung slice cultures treated for 2 days with and without 10 µM methacholine. MRC-5 cell lysates were analysed for phosphorylated (ser 423/425) and total Smad-3. Representative blots and quantified data of Smad-3 phosphorylation in response to conditioned media are shown in (A). Data shown are the means ± SE of 4 independent experiments. ^*: p<0.05, compared to basal (paired Student's <i>t</i>-test with two-tailed distribution). Lung slices were pre-treated with latrunculin A (0.3 µM), SB431542 (0.3 µM), or medium (basal) for 30 min, followed by 2 days of treatment with methacholine (MCh; 10 µM), TGF-β₁ (2 ng/mL), or medium (basal) (B). Lung slice lysates were analysed for the presence of sm-myosin, using ß-actin as a loading control. Blots shown are representative of 3 experiments. Human MRC-5 fibroblasts were stimulated for 1 hour with conditioned media obtained from lung slice cultures after treatment with methacholine (MCh; 10 µM), TGF-β (2 ng/mL) or medium (basal), in the absence and presence of latrunculin A (0.3 µM) or SB431542 (0.3 µM) (C). MRC-5 cell lysates were analysed for phosphorylated (ser 423/425) and total Smad-3. Blots shown are representative of 3 experiments. Lung slices were pre-incubated with latrunculin A (0.3 µM), SB431542 (0.3 µM), or medium (basal) for 30 min, followed by 2 days stimulation with methacholine (MCh; 10 µM), TGF-β₁ (2 ng/mL), histamine (His, 1 µM), KCl (K⁺, 60 mM) or medium (basal) (D). Lung slice lysates were analysed for sm-myosin, using β-actin as a loading control. Blots shown are representative of 3 experiments.</p

Localization of sm-myosin expression after TGF-β₁ and methacholine treatment.

<p>Lung slices were treated with TGF-β₁ (2 ng/mL), methacholine (MCh; 10 µM) or medium (basal) for 2 days. Lung slices were then fixed, stained for sm-myosin and analysed by confocal immunofluorescence microscopy. The images shown in (A) are taken from the peripheral airways (diameter smaller than 100 µm). Staining intensity within the muscle bundle was quantified and data shown from the peripheral airways (B) and central airways (diameter larger than 400 µm; C) are the means ± SE of 3 independent experiments. ^*: p<0.05 compared to basal (one-way ANOVA, posthoc Newman-Keuls).</p