Gα12facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner (original) (raw)
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The Journal of Pharmacology and Experimental Therapeutics, 2010
Phosphoinositide 3-kinase ␥ (PI3K␥) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3K␥ can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3K␥ protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3K␥ inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 M. In contrast, inhibitors of PI3K␣, PI3K, or PI3K␦, at concentrations 40-fold higher than their reported IC 50 values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3K␥ inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3K␥-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca 2ϩ transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca 2ϩ oscillations that are critical for sustained airway contraction. This report is the first to show that PI3K␥ directly controls contractility of airways through regulation of Ca 2ϩ oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3K␥ inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.
KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase
American Journal of Physiology-Lung Cellular and Molecular Physiology, 2004
Airway smooth muscle (ASM) cells express voltage-dependent Ca2+channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+(fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+or using the selective L-type Ca2+channel blocker nifedipine; porcine bronchial preparat...
Proceedings of the National Academy of Sciences, 2016
G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to pr...
β-Agonist-associated Reduction in RGS5 Expression Promotes Airway Smooth Muscle Hyper-responsiveness
Journal of Biological Chemistry, 2011
Although short-acting and long-acting inhaled  2-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent  2-agonistinduced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to  2-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase C and myosin light chain). In contrast, -agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonistevoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5 ؊/؊ mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive  2-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.
Regulation of airway smooth muscle RhoA/ROCK activities by cholinergic and bronchodilator stimuli
European Respiratory Journal, 2006
The current study set out to compare the temporal relationships of Rho activity, Rho kinase (ROCK) activity and tone following cholinergic stimulation in the presence and absence of three different bronchodilators. Bovine trachea challenged with a half-maximally effective concentration of carbachol (CCh) was flash-frozen at different times, then assayed for Rho (rhotekin pull-down assay) and ROCK (Western blot; radiometric assay) activities. Rho was activated within 30 s, followed by ROCK (peak at 2 min); both returned to baseline by 20 min, although tone continued to rise over that period. Increasing the concentration of CCh greatly increased the magnitudes and rates of stimulation of Rho, ROCK and tone. These CChinduced changes were next compared in tissues pre-treated with isoproterenol, salmeterol or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Neither the time course nor the magnitude of Rho-activation were reduced by the b-agonists; SNAP slowed Rho activation but it did not alter the peak magnitude. These observations were mirrored in ROCK activation and contraction. When tissues were pre-constricted with CCh and then challenged with the bronchodilators, however, all three agonists reversed cholinergically stimulated Rho, ROCK and myosin light chain kinase activities as well as tone. In conclusion, bronchodilators can suppress RhoA and Rho kinase activities, although their major effect appears to be on myosin light chain kinase activity.
Rho-kinase and contractile apparatus proteins in murine airway hyperresponsiveness
Experimental and Toxicologic Pathology, 2008
Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Increased expression of smooth muscle contractile proteins or increased responsiveness of the contractile apparatus due to RhoA/Rho-kinase activation may contribute to AHR. BALB/c mice developed AHR following systemic sensitization by intraperitoneal injections of 20 mg ovalbumin (OVA) in presence of 2 mg Al(OH) 3 on days 1 and 14, and airway challenge by 1% OVA-inhalation for 20 min each on days 28, 29 and 30. As assessed by Western blot, protein expression of RhoA, MLC (myosin light chain) and smMLCK (smooth muscle myosin light chain kinase) was increased in lungs of OVA/OVA-animals with AHR, as well as in lungs of OVA-sensitized and sham-challenged animals (OVA/PBS) without AHR, compared with lungs of PBS/ PBS-animals. Pretreatment with the specific Rho-kinase inhibitor Y-27632 reduced MLC-phosphorylation and AHR. Contribution of Rho-kinase to bronchoconstriction was increased in lungs of OVA/OVA-animals compared with OVA/PBS-and PBS/PBS-animals, respectively. Furthermore, bronchoconstriction following MCh stimulation was significantly reduced after Y-27632 application. In conclusion, systemic allergen-sensitization increased pulmonary expression of proteins involved in smooth muscle contraction, which may contribute to development of AHR. However, this observation was independent from local allergen challenge, suggesting that additional cofactors may be required for the activation of Rho-kinase and thereby the induction of AHR. Rho-kinase may play an important role in murine AHR, and the bronchodilating action of Rho-kinase inhibition may offer a new therapeutic perspective in obstructive airway disease.
Phosphatidylinositol 3-Kinase Isoform-Specific Effects in Airway Mesenchymal Cell Function
Journal of Pharmacology and Experimental Therapeutics, 2011
The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in the airway remodeling associated with asthma. The class IA PI3K isoforms are known to be activated by growth factors and cytokines. Because this pathway is a possible site of pharmacological intervention for treating the disease, it is important to know which isoforms contribute to this process. Therefore, we used a pharmacological approach to investigate the roles of the three class IA PI3K isoforms (p110␣, p110, and p110␦) in airway remodeling using airway smooth muscle (ASM) cells derived from asthmatic subjects and ASM cells and lung fibroblasts from nonasthmatic subjects. These studies used the inhibitors NЈ-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylidene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (PIK75) (which selectively inhibits p110␣), 7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (TGX221) (which selectively inhibits p110), and 2-[(6-amino-9H-purin-9yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (IC87114) (which selectively inhibits p110␦). Cells were stimulated with transforming growth factor- (TGF) and/or 10% fetal bovine serum in the presence or absence of inhibitor or vehicle control (dimethyl sulfoxide). PIK75, but not TGX221 or IC87114, attenuated TGF-induced fibronectin deposition in all cell types tested. PIK75 and TGX221 each decreased secretion of vascular endothelial growth factor and interleukin-6 in nonasthmatic ASM cells and lung fibroblasts, whereas TGX221 was not as effective in asthmatic ASM cells. In addition, PIK75 decreased cell survival in TGF-stimulated asthmatic, but not nonasthmatic, ASM cells. In conclusion, specific PI3K isoforms may play a role in pathophysiological events relevant to airway wall remodeling.
Journal of Clinical Investigation, 2007
Receptor-mediated airway smooth muscle (ASM) contraction via G αq , and relaxation via G αs , underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G αi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M 3 -muscarinic receptors and diminished relaxation mediated by β 2 -adrenergic receptors (β 2 ARs). A causal effect between G i expression and phenotype has not been established, nor have mechanisms whereby G i modulates G q /G s signaling. To delineate isolated effects of altered G i , transgenic mice were generated overexpressing G αi2 or a G αi2 peptide inhibitor in ASM. Unexpectedly, G αi2 overexpression decreased contractility to methacholine, while G αi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G q signaling network: decreased phospholipase C and increased PKCα, respectively. G αi2 overexpression decreased β 2 AR-mediated airway relaxation, while G αi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G s and G i . IL-13 transgenic mice (a model of asthma), which developed increased ASM G αi , displayed marked increases in airway hyperresponsiveness when G αi function was inhibited. Increased G αi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G s /G q activities could provide new asthma therapies.
Rho kinase inhibitors: A novel therapeutical intervention in asthma?
European Journal of Pharmacology, 2008
In asthma, inflammatory mediators that are released in the airways by recruited inflammatory cells and by resident structural cells result in airway hyperresponsiveness caused by increased bronchoconstriction. In addition, chronic inflammation appears to drive remodelling of the airways that contributes to the development of fixed airway obstruction and airway hyperresponsiveness in chronic asthma. Airway remodelling includes several key features such as excessive deposition of extracellular matrix proteins in the airway wall (fibrosis) and increased abundance of contractile airway smooth muscle encircling the airways. Current asthma therapy fails to inhibit these features satisfactorily. This review focuses on Rho kinase as a potential drug target in asthma, as compelling evidence from animal models and ex vivo studies suggests a central role for this enzyme and its associated signalling in acute and chronic airway hyperresponsiveness.