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Reinoud Gosens - One of the best experts on this subject based on the ideXlab platform.
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Muscarinic m Receptors contribute to allergen induced airway remodeling in mice
American Journal of Respiratory Cell and Molecular Biology, 2014Co-Authors: Loes E M Kistemaker, Willemieke M Mudde, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Julius Wess, Reinoud GosensAbstract:Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the Muscarinic M3 Receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the Muscarinic M3 Receptor. The objective of this work was to study the contribution of the Muscarinic M3 Receptor to allergen-induced remodeling using Muscarinic M3 Receptor subtype–deficient (M3R−/−) mice. Wild-type (WT), M1R−/−, and M2R−/− mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R−/− mice (3...
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Muscarinic M₃ Receptors contribute to allergen-induced airway remodeling in mice
American journal of respiratory cell and molecular biology, 2014Co-Authors: Loes E M Kistemaker, Willemieke M Mudde, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Julius Wess, Sophie Bos, Reinoud GosensAbstract:Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the Muscarinic M3 Receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the Muscarinic M3 Receptor. The objective of this work was to study the contribution of the Muscarinic M3 Receptor to allergen-induced remodeling using Muscarinic M3 Receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the Muscarinic M3 Receptor, and not via M1 or M2 Receptors. No stimulatory role for Muscarinic M3 Receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.
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The Muscarinic M3 Receptor regulates allergen-induced airway remodeling in mice
European Respiratory Journal, 2013Co-Authors: Loes E M Kistemaker, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Jürgen Wess, Sophie Bos, Reinoud GosensAbstract:RATIONALE Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine (ACh) contributes to asthma symptoms by inducing bronchoconstriction via the Muscarinic M 3 Receptor. Recent evidence suggests that ACh also regulates airway inflammation and remodeling. Therefore, we studied the contribution of the M 3 Receptor to allergen-induced inflammation and remodeling using M 3 Receptor subtype deficient (M 3 R -/- ) mice. Wild type (WT), M 1 R -/- and M 2 R -/- mice were used as controls. METHODS Female C57Bl/6 mice (n=8-10) were sensitized and challenged with ovalbumin (twice weekly, for 4 weeks). Control animals were challenged with saline. Lungs were collected for analyses. RESULTS Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening, pulmonary vascular smooth muscle remodeling and increased deposition of collagen I and fibronectin in the airway wall of WT mice. These effects were partly to fully prevented in M 3 R -/- mice, whereas M 1 R -/- and M 2 R -/- mice responded similar to WT mice. In addition, airway smooth muscle mass and pulmonary vascular smooth muscle mass were lower in saline-challenged M 3 R -/- mice compared to WT mice. Interestingly, allergen-induced airway inflammation, measured as eosinophil infiltration and cytokine release (IL-4, IL-5, IL-13 and IL-17), was similar in all strains. CONCLUSION Our data indicate that ACh contributes to allergen-induced remodeling and smooth muscle mass via the M 3 Receptor, and not via M 1 or M 2 Receptors. Surprisingly, no role for individual Muscarinic Receptor subtypes in allergic inflammation was observed, suggesting that the role of ACh in remodeling is independent of the allergic inflammatory response.
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Muscarinic M3 Receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells
The European respiratory journal, 2009Co-Authors: Reinoud Gosens, Herman Meurs, Pieter S Hiemstra, D. Rieks, D. K. Ninaber, Klaus F. Rabe, J. Nanninga, Saeed Kolahian, Andrew J. Halayko, S. ZuyderduynAbstract:Acetylcholine is the primary parasympathetic neurotransmitter in the airways and is known to cause bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine also regulates aspects of remodelling and inflammation through its action on Muscarinic Receptors. In the present study, we aimed to determine the effects of Muscarinic Receptor stimulation on cytokine production by human airway smooth muscle cells (primary and immortalised cell lines). The Muscarinic Receptor agonists carbachol and methacholine both induced modest effects on basal interleukin (IL)-8 and -6 secretion, whereas the secretion of RANTES, eotaxin, vascular endothelial growth factor-A and monocyte chemoattractant protein-1 was not affected. Secretion of IL-8 and -6 was only observed in immortalised airway smooth muscle cells that express Muscarinic M3 Receptors. In these cells, methacholine also significantly augmented IL-8 secretion in combination with cigarette smoke extract in a synergistic manner, whereas synergistic effects on IL-6 secretion were not significant. Muscarinic M3 Receptors were the primary subtype involved in augmenting cigarette smoke extract-induced IL-8 secretion, as only tiotropium bromide and Muscarinic M3 Receptor subtype selective antagonists abrogated the effects of methacholine. Collectively, these results indicate that Muscarinic M3 Receptor stimulation augments cigarette smoke extract-induced cytokine production by airway smooth muscle. This interaction could be of importance in patients with chronic obstructive pulmonary disease.
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protective effects of tiotropium bromide in the progression of airway smooth muscle remodeling
American Journal of Respiratory and Critical Care Medicine, 2005Co-Authors: Reinoud Gosens, Johan Zaagsma, Herman MeursAbstract:Rationale: Recent findings have demonstrated that Muscarinic M3 Receptor stimulation enhances airway smooth muscle proliferation to peptide growth factors in vitro. Because both peptide growth factor expression and acetylcholine release are known to be augmented in allergic airway inflammation, it is possible that anticholinergics protect against allergen-induced airway smooth muscle remodeling in vivo. Objective: We investigated the effects of treatment with the long-acting Muscarinic Receptor antagonist tiotropium on airway smooth muscle changes in a guinea pig model of ongoing allergic asthma. Results: Twelve weekly repeated allergen challenges induced an increase in airway smooth muscle mass in the noncartilaginous airways. This increase was not accompanied by alterations in cell size, indicating that the allergen-induced changes were entirely from increased airway smooth muscle cell number. Morphometric analysis showed no allergen-induced changes in airway smooth muscle area in the cartilaginous airw...
Stefan R. Nahorski - One of the best experts on this subject based on the ideXlab platform.
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Effect of dimethylsphingosine on Muscarinic M3 Receptor signalling in SH-SY5Y cells
European Journal of Pharmacology, 2000Co-Authors: Kenneth W. Young, Deborah R. Channing, Stefan R. NahorskiAbstract:Abstract The sphingosine kinase inhibitor, dimethylsphingosine, is an important tool for investigating intracellular effects of the putative second messenger compound, sphingosine 1-phosphate. However, the specificity of action of dimethylsphingosine has not been fully investigated. In human SH-SY5Y neuroblastoma cells, dimethylsphingosine (30 μM), produced a 25-fold increase in the EC 50 for methacholine-induced Ca 2+ mobilisation, and reduced the maximum response by 57±5%, suggesting the involvement of sphingosine 1-phosphate production in the Ca 2+ signal. However, dimethylsphingosine also inhibited [ 3 H] N -methylscopolamine binding to whole SH-SY5Y cells and reduced methacholine-induced phosphoinositide turnover. Thus, this compound must be used with caution when investigating the role of sphingosine kinase in G-protein coupled Receptor-mediated Ca 2+ mobilisation responses.
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Differential regulation of Muscarinic M1 and M3 Receptors by a putative phosphorylation domain.
European journal of pharmacology, 1999Co-Authors: Melinda S. Shockley, Stefan R. Nahorski, Andrew B. Tobin, Lara M. Tolbert, Wolfgang Sadee, Jelveh LamehAbstract:A motif consisting of several serine residues flanked N-terminally by acidic residues occurs in the third intracellular loop of both Muscarinic M1 and M3 Receptors (287SerLeuThrSerSer291 and 349SerAlaSerSer352, respectively). We examined the role of these domains in modulating agonist-induced desensitization and Receptor trafficking, and for the Muscarinic M3 Receptor, we assessed the contribution of phosphorylation to Receptor regulation. Mutation of the above residues did not affect desensitization of phosphoinositide hydrolysis signaling for either the Muscarinic M1 or M3 Receptor and did not alter the agonist-induced phosphorylation state of the Muscarinic M3 Receptor. Mutation of this domain (349SerAlaSerSer352/349AlaAlaAlaAla352) in the Muscarinic M3 Receptor completely abrogated Receptor internalization and subsequently, down-regulation. Mutation of the analogous domain (287SerLeuThrSerSer291/287AlaLeuAlaAlaAla291) in the Muscarinic M1 Receptor had no obvious effect on internalization, but led to a more rapid down-regulation. Thus, these serine-rich regions are not required for Receptor desensitization, but are differentially involved in Receptor trafficking for the Muscarinic M1 and M3 Receptors.
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Acute desensitization of phospholipase C-coupled Muscarinic M3 Receptors but not gonadotropin-releasing hormone Receptors co-expressed in alphaT3-1 cells: implications for mechanisms of rapid desensitization.
Biochemical Journal, 1998Co-Authors: Gary B. Willars, Craig A. Mcardle, Stefan R. NahorskiAbstract:In the present study we have expressed the Muscarinic M3 Receptor in an immortalized mouse pituitary cell line (alphaT3-1), which expresses an endogenous gonadotropin-releasing hormone (GnRH) Receptor, to examine potential differences in acute Receptor regulation. Both of these Receptors couple to the activation of phosphoinositide-specific phospholipase C (PLC) in these cells and we demonstrate that, despite expression in the same cell background, acute desensitization is a feature of Muscarinic M3 Receptors but not of GnRH Receptors. We show that, when the concentrations of GnRH and methacholine are matched to give approximately equivalent maximal elevations of Ins(1,4,5)P3, the GnRH Receptor is able to sustain PLC activity at the initial rate, whereas the Muscarinic M3 Receptor cannot. Thus PLC-activating G-protein-coupled Receptors are able to undergo rapid desensitization in this cell line, indicating that the desensitization profile is Receptor-specific rather than cell-specific. This argues strongly that post-Receptor regulatory features do not have a prominent role in mediating rapid desensitization in these cells. Furthermore GnRH Receptor-mediated PLC activity is sustained despite a marked and persistent depletion in the steady-state level of PtdIns(4,5)P2. In contrast, activation of Muscarinic Receptors is not sustained despite only a transient decrease in PtdIns(4,5)P2 concentration. Thus, whereas the contribution of PtdIns(4,5)P2 depletion to the temporal profile of Receptor-mediated PLC signalling has been difficult to assess, the present results demonstrate that this is unlikely to be of importance in these cells. We suggest that unique structural features of the GnRH Receptor result in a lack of appropriate regulatory phospho-acceptor sites and that the absence of agonist-dependent phosphorylation might underlie the lack of acute regulation.
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Muscarinic M3 Receptor coupling and regulation.
Life sciences, 1997Co-Authors: Stefan R. Nahorski, Andrew B. Tobin, Gary B. WillarsAbstract:Current concepts regarding the regulation and coupling of Muscarinic M3 Receptors to G-proteins and various effectors are discussed. The last few years have provided much evidence that although Muscarinic m1, M3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/11) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant Muscarinic Receptors even at relatively low levels. The significance of this diversity and the potential for agonist “trafficking” could open up opportunities for novel approaches to selective agonist action. Finally, mechanisms underlying the regulation of Muscarinic M3 coupling through Gq/11 to PIC are discussed. In particular, our recent studies on precursor lipid depletion, whether regulation is Receptor or cell specific and the identification and role of Receptor kinases are briefly reviewed.
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Rapid desensitization of Muscarinic M3 Receptor-stimulated polyphosphoinositide responses.
Molecular pharmacology, 1992Co-Authors: Andrew B. Tobin, David G. Lambert, Stefan R. NahorskiAbstract:Chinese hamster ovary (CHO) cells transfected with human M3 Muscarinic Receptor cDNA (Bmax, 1343 +/- 46.8 fmol/mg of protein) were used to investigate agonist-mediated Muscarinic Receptor desensitization. Stimulation of CHO-M3 cells with a maximal dose of carbachol resulted in a biphasic production of mass inositol-1,4,5-trisphosphate [Ins(1,4,5)P3], measured by radioReceptor binding assay. The first phase comprises a rapid 8-10-fold increase in Ins(1,4,5)P3 that peaks after 10 sec and falls to levels 3-4-fold over basal within 1 min Ins(1,4,5)P3 rises again over the next 20 min to approximately 8-10-fold above basal, where levels are sustained for at least 2 hr. This later phase is, therefore, considered to be a desensitization-resistant component of M3 Receptor activation. A 5-min pre-exposure of CHO-M3 cells to carbachol resulted in attenuation of the initial peak Ins(1,4,5)P3 response to a subsequent application of agonist. The attenuation of the Ins(1,4,5)P3 response was reversible with a t1/2 of approximately 7.5 min. Desensitization and recovery of the peak Ins(1,4,5)P3 response correlated with a decrease and subsequent recovery of M3 Receptor-mediated mobilization of intracellular calcium stores, suggesting that the consequence of peak Ins(1,4,5)P3 desensitization is a reduced calcium mobilization response. N-[3H]Methylscopolamine binding to intact cells revealed that there was no change in cell surface M3 Receptors during the 5-min pre-exposure to agonist, indicating that the mechanism of Muscarinic Receptor desensitization described here is not sequestration or internalization of Receptors.
Herman Meurs - One of the best experts on this subject based on the ideXlab platform.
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Muscarinic m Receptors contribute to allergen induced airway remodeling in mice
American Journal of Respiratory Cell and Molecular Biology, 2014Co-Authors: Loes E M Kistemaker, Willemieke M Mudde, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Julius Wess, Reinoud GosensAbstract:Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the Muscarinic M3 Receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the Muscarinic M3 Receptor. The objective of this work was to study the contribution of the Muscarinic M3 Receptor to allergen-induced remodeling using Muscarinic M3 Receptor subtype–deficient (M3R−/−) mice. Wild-type (WT), M1R−/−, and M2R−/− mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R−/− mice (3...
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Muscarinic M₃ Receptors contribute to allergen-induced airway remodeling in mice
American journal of respiratory cell and molecular biology, 2014Co-Authors: Loes E M Kistemaker, Willemieke M Mudde, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Julius Wess, Sophie Bos, Reinoud GosensAbstract:Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the Muscarinic M3 Receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the Muscarinic M3 Receptor. The objective of this work was to study the contribution of the Muscarinic M3 Receptor to allergen-induced remodeling using Muscarinic M3 Receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the Muscarinic M3 Receptor, and not via M1 or M2 Receptors. No stimulatory role for Muscarinic M3 Receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.
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The Muscarinic M3 Receptor regulates allergen-induced airway remodeling in mice
European Respiratory Journal, 2013Co-Authors: Loes E M Kistemaker, Machteld N Hylkema, Herman Meurs, Pieter S Hiemstra, Huib A M Kerstjens, Jürgen Wess, Sophie Bos, Reinoud GosensAbstract:RATIONALE Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine (ACh) contributes to asthma symptoms by inducing bronchoconstriction via the Muscarinic M 3 Receptor. Recent evidence suggests that ACh also regulates airway inflammation and remodeling. Therefore, we studied the contribution of the M 3 Receptor to allergen-induced inflammation and remodeling using M 3 Receptor subtype deficient (M 3 R -/- ) mice. Wild type (WT), M 1 R -/- and M 2 R -/- mice were used as controls. METHODS Female C57Bl/6 mice (n=8-10) were sensitized and challenged with ovalbumin (twice weekly, for 4 weeks). Control animals were challenged with saline. Lungs were collected for analyses. RESULTS Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening, pulmonary vascular smooth muscle remodeling and increased deposition of collagen I and fibronectin in the airway wall of WT mice. These effects were partly to fully prevented in M 3 R -/- mice, whereas M 1 R -/- and M 2 R -/- mice responded similar to WT mice. In addition, airway smooth muscle mass and pulmonary vascular smooth muscle mass were lower in saline-challenged M 3 R -/- mice compared to WT mice. Interestingly, allergen-induced airway inflammation, measured as eosinophil infiltration and cytokine release (IL-4, IL-5, IL-13 and IL-17), was similar in all strains. CONCLUSION Our data indicate that ACh contributes to allergen-induced remodeling and smooth muscle mass via the M 3 Receptor, and not via M 1 or M 2 Receptors. Surprisingly, no role for individual Muscarinic Receptor subtypes in allergic inflammation was observed, suggesting that the role of ACh in remodeling is independent of the allergic inflammatory response.
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Muscarinic M3 Receptor stimulation increases cigarette smoke-induced IL-8 secretion by human airway smooth muscle cells
The European respiratory journal, 2009Co-Authors: Reinoud Gosens, Herman Meurs, Pieter S Hiemstra, D. Rieks, D. K. Ninaber, Klaus F. Rabe, J. Nanninga, Saeed Kolahian, Andrew J. Halayko, S. ZuyderduynAbstract:Acetylcholine is the primary parasympathetic neurotransmitter in the airways and is known to cause bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine also regulates aspects of remodelling and inflammation through its action on Muscarinic Receptors. In the present study, we aimed to determine the effects of Muscarinic Receptor stimulation on cytokine production by human airway smooth muscle cells (primary and immortalised cell lines). The Muscarinic Receptor agonists carbachol and methacholine both induced modest effects on basal interleukin (IL)-8 and -6 secretion, whereas the secretion of RANTES, eotaxin, vascular endothelial growth factor-A and monocyte chemoattractant protein-1 was not affected. Secretion of IL-8 and -6 was only observed in immortalised airway smooth muscle cells that express Muscarinic M3 Receptors. In these cells, methacholine also significantly augmented IL-8 secretion in combination with cigarette smoke extract in a synergistic manner, whereas synergistic effects on IL-6 secretion were not significant. Muscarinic M3 Receptors were the primary subtype involved in augmenting cigarette smoke extract-induced IL-8 secretion, as only tiotropium bromide and Muscarinic M3 Receptor subtype selective antagonists abrogated the effects of methacholine. Collectively, these results indicate that Muscarinic M3 Receptor stimulation augments cigarette smoke extract-induced cytokine production by airway smooth muscle. This interaction could be of importance in patients with chronic obstructive pulmonary disease.
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protective effects of tiotropium bromide in the progression of airway smooth muscle remodeling
American Journal of Respiratory and Critical Care Medicine, 2005Co-Authors: Reinoud Gosens, Johan Zaagsma, Herman MeursAbstract:Rationale: Recent findings have demonstrated that Muscarinic M3 Receptor stimulation enhances airway smooth muscle proliferation to peptide growth factors in vitro. Because both peptide growth factor expression and acetylcholine release are known to be augmented in allergic airway inflammation, it is possible that anticholinergics protect against allergen-induced airway smooth muscle remodeling in vivo. Objective: We investigated the effects of treatment with the long-acting Muscarinic Receptor antagonist tiotropium on airway smooth muscle changes in a guinea pig model of ongoing allergic asthma. Results: Twelve weekly repeated allergen challenges induced an increase in airway smooth muscle mass in the noncartilaginous airways. This increase was not accompanied by alterations in cell size, indicating that the allergen-induced changes were entirely from increased airway smooth muscle cell number. Morphometric analysis showed no allergen-induced changes in airway smooth muscle area in the cartilaginous airw...
Vladlen Z. Slepak - One of the best experts on this subject based on the ideXlab platform.
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Regulator of G-protein signaling Gβ5-R7 is a crucial activator of Muscarinic M3 Receptor-stimulated insulin secretion.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2017Co-Authors: Qiang Wang, Alexey Pronin, Konstantin Levay, Joana Almaça, Alessia Fornoni, Alejandro Caicedo, Vladlen Z. SlepakAbstract:In pancreatic β cells, Muscarinic cholinergic Receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in β cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gβ5, such that the knockout of the Gnb5 gene results in degradation of all R7 subunits. We found that Gnb5 knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gβ5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in Gnb5-/- MIN6 cells showed that cAMP, diacylglycerol, or Ca2+ levels were not significantly affected. There was no reduction in the amplitude of free Ca2+ responses in islets from the Gnb5-/- mice, but the frequency of Ca2+ oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of Gnb5 impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in Gnb5-/- cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gβ5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic β-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gβ5-R7 is a crucial activator of Muscarinic M3 Receptor-stimulated insulin secretion.
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Helix 8 and the i3 loop of the Muscarinic M3 Receptor are crucial sites for its regulation by the Gβ5-RGS7 complex.
Biochemistry, 2015Co-Authors: Darla Karpinsky-semper, Claude-henry Volmar, Konstantin Levay, Tayou, Brett J. Schuchardt, Vikas Bhat, Amjad Farooq, Vladlen Z. SlepakAbstract:The Muscarinic M3 Receptor (M3R) is a Gq-coupled Receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the Receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.
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differential effects of the gβ5 rgs7 complex on Muscarinic M3 Receptor induced ca2 influx and release
Molecular Pharmacology, 2014Co-Authors: Darla Karpinskysemper, Claude-henry Volmar, Vladlen Z. SlepakAbstract:The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca2+ signaling mediated by the Muscarinic M3 Receptor (M3R), the route of Ca2+ entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca2+ release, Gβ5-RGS7 enhanced Ca2+ influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain–deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca2+ channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca2+ influx and release elicited by different Muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca2+ response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca2+ release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.
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Effects of the Gβ5-RGS7 complex on Muscarinic M3 Receptor-induced Ca2+ signaling and insulin secretion (843.12)
The FASEB Journal, 2014Co-Authors: Vladlen Z. Slepak, Darla Karpinsky-semper, Qiang WangAbstract:The G protein subunit Gβ5 forms obligatory heterodimers with regulators of G protein signaling (RGS) proteins of R7 family (RGS6, 7, 9 and 11). These complexes are expressed in neurons and glands. Previously, we showed that Gβ5-RGS7 attenuated signaling downstream of Muscarinic M3 Receptor, M3R. A predicted consequence of Gβ5 ablation on pancreatic beta cells would be an increase in insulin secretion. However, we found that Gβ5-R7 knockout strongly attenuated, while its overexperession in Min6 insulinoma cells enhanced M3R-induced insulin secretion. We studied isolated mouse islets, transfected Min6 and CHO-K1 cells, measuring insulin secretion and changes in intracellular Ca2+ in the presence of various pharmacological agents, Muscarinic agonists and functional mutants of Gβ5-RGS7 and M3R. The results showed that Gβ5-RGS7 has a dual effect on M3R-mediated Ca2+ transients. While it attenuated Ca2+ release from internal stores, it stimulated Ca2+ influx across the plasma membranes. This pathway involves Gq...
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Differential Effects of the Gβ5-RGS7 Complex on Muscarinic M3 Receptor–Induced Ca2+ Influx and Release
Molecular pharmacology, 2014Co-Authors: Darla Karpinsky-semper, Claude-henry Volmar, Vladlen Z. SlepakAbstract:The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca2+ signaling mediated by the Muscarinic M3 Receptor (M3R), the route of Ca2+ entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca2+ release, Gβ5-RGS7 enhanced Ca2+ influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain–deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca2+ channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca2+ influx and release elicited by different Muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca2+ response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca2+ release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.
Roberto Maggio - One of the best experts on this subject based on the ideXlab platform.
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Paired activation of two components within Muscarinic M3 Receptor dimers is required for recruitment of β-arrestin-1 to the plasma membrane
The Journal of biological chemistry, 2005Co-Authors: Francesca Novi, Laura Stanasila, Franco Giorgi, Giovanni Corsini, Susanna Cotecchia, Roberto MaggioAbstract:Abstract β-Arrestins regulate the functioning of G protein-coupled Receptors in a variety of cellular processes including Receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled Receptor-mediated recruitment of β-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via β-arrestin recruitment entails paired activation of Receptor dimers. To address this question, we investigated the ability of different Muscarinic Receptor dimers to recruit β-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated Muscarinic M3 Receptor, which is deleted in most of the third intracellular loop (M3-short). Although still capable of activating phospholipase C, this Receptor loses almost completely the ability to recruit β-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M3-short was co-expressed with the M3 Receptor. Under these conditions, the M3/M3-short heterodimer could not recruit β-arrestin-1 to the plasma membrane, even though the control M3/M3 homodimer could. We next tested the ability of chimeric adrenergic Muscarinic α2/M3 and M3/α2 heterodimeric Receptors to co-immunoprecipitate with β-arrestin-1 following stimulation with adrenergic and Muscarinic agonists. β-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each Receptor may exert on the M2/M3 heterodimer to recruit β-arrestin-1. Remarkably, we observed that M2/M3 heterodimers recruit significantly greater amounts of β-arrestin-1 than their respective M3/M3 or M2/M2 homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of β-arrestin-1 to Muscarinic M3 Receptors requires paired stimulation of two Receptor components within the same Receptor dimer.
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the paired activation of the two components of the Muscarinic M3 Receptor dimer is required for induction of erk1 2 phosphorylation
Journal of Biological Chemistry, 2004Co-Authors: Francesca Novi, Marco Scarselli, Giovanni Umberto Corsini, Roberto MaggioAbstract:Abstract Muscarinic M3 Receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the Muscarinic M3 Receptor in which most of the third intracellular (i3) loop had been deleted (M3-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the Receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M3-short greatly reduced the ability of M3 to activate ERK1/2. In another set of experiments we tested the ability of a mutant M3/M2(16aa) Receptor, in which the first 16 amino acids of the i3 loop of the M3 Receptor were replaced with the corresponding segment of the Muscarinic M2 Receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Gαq, but it is weakly coupled to Gαi. Despite its coupling modification this Receptor was able to stimulate ERK1/2 phosphorylation. Again, M3-short greatly reduced the ability of M3/M2(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M3 cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 μm and a second increase starting from 10 μm. In CHO M3-short and in double-transfected CHO M3/M3-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M3-short prevents the effect of the higher doses of carbachol on the M3 Receptor. In a final experiment we tested the ability of co-transfected chimeric α2/M3 and M3/α2 Receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric Receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a Receptor dimer.
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The paired activation of the two components of the Muscarinic M3 Receptor dimer is required for induction of ERK1/2 phosphorylation.
The Journal of biological chemistry, 2003Co-Authors: Francesca Novi, Marco Scarselli, Giovanni Umberto Corsini, Roberto MaggioAbstract:Abstract Muscarinic M3 Receptors stimulate ERK1/2, the mitogen-activated protein kinase pathway. A mutant of the Muscarinic M3 Receptor in which most of the third intracellular (i3) loop had been deleted (M3-short) completely lost the ability to stimulate the ERK1/2 phosphorylation in COS-7 cells. This loss was evident despite the fact that the Receptor was able to couple efficiently to the phospholipase C second messenger pathway. In co-transfected cells, M3-short greatly reduced the ability of M3 to activate ERK1/2. In another set of experiments we tested the ability of a mutant M3/M2(16aa) Receptor, in which the first 16 amino acids of the i3 loop of the M3 Receptor were replaced with the corresponding segment of the Muscarinic M2 Receptor to stimulate ERK1/2 phosphorylation. This mutant is not coupled to Gαq, but it is weakly coupled to Gαi. Despite its coupling modification this Receptor was able to stimulate ERK1/2 phosphorylation. Again, M3-short greatly reduced the ability of M3/M2(16aa) to activate ERK1/2 in co-transfected cells. Similar results were obtained in stable-transfected Chinese hamster ovary (CHO) cells lines. In CHO M3 cells carbachol induced a biphasic increase of ERK1/2 phosphorylation; a first increase at doses as low as 0.1 μm and a second increase starting from 10 μm. In CHO M3-short and in double-transfected CHO M3/M3-short cells we observed only the lower doses increase of ERK1/2 phosphorylation; no further increase was observed up to 1 mm carbachol. This suggests that in double-transfected CHO cells M3-short prevents the effect of the higher doses of carbachol on the M3 Receptor. In a final experiment we tested the ability of co-transfected chimeric α2/M3 and M3/α2 Receptors to activate the ERK1/2 pathway. When given alone, carbachol and, to a lesser extent, clonidine, stimulated the coupling of the co-transfected chimeric Receptors to the phospholipase C second messenger pathway, but they were unable to stimulate ERK1/2 phosphorylation. On the contrary, a strong stimulation of ERK1/2 phosphorylation was observed when the two agonists were given together despite the fact that the overall increase in phosphatidylinositol hydrolysis was not dissimilar from that observed in cells treated with carbachol alone. Our data suggest that the activation of the ERK1/2 pathway requires the coincident activation of the two components of a Receptor dimer.
