scholarly journals Vinculin Phosphorylation at Tyr1065Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

2013 ◽  
Vol 289 (6) ◽  
pp. 3677-3688 ◽  
Author(s):  
Youliang Huang ◽  
Richard N. Day ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Tension Development ◽  
Muscle Tissues

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

scholarly journals Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue

2011 ◽  
Vol 301 (3) ◽  
pp. L275-L284 ◽  
Author(s):  
Leena P. Desai ◽  
Yidi Wu ◽  
Robert S. Tepper ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Airway Smooth Muscle ◽  
Heavy Chain ◽  
High Load ◽  
Phenotypic Marker ◽  
Akt Activation ◽  
Muscle Tissues ◽  
Low Load ◽  
Muscle Myosin

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1–2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.

Effects of Bronchodilators Combined With Oscillations on the Contracted Airway Smooth Muscle

2010 ◽  
Author(s):  
M. Mathur ◽  
A. M. Al-Jumaily ◽  
G. Ijpma ◽  
R. Alany
Keyword(s):  
Smooth Muscle ◽  
Side Effects ◽  
Airway Smooth Muscle ◽  
Muscle Relaxants ◽  
Combined Effects ◽  
In Vitro Experiments ◽  
Current Asthma ◽  
Cardiovascular Side Effects ◽  
Muscle Tissues

Current asthma treatments using anti-inflammatory agents and airway smooth muscle (ASM) relaxants are expensive, variable in effectiveness and are associated with several cardiovascular side effects. Previous in vitro experiments conducted on ASM tissues suggest that oscillations applied to contracted muscle result in a reduction in the contractile ability of the tissue. This study focuses on investigating the combined effects of muscle relaxants (bronchodilators) and length oscillations on the dynamics of contracted ASM. Isolated porcine tracheal smooth muscle tissues are contracted using Acetylcholine. Isoproterenol (Iso), a β-agonist, is used as a bronchodilator to relax the contracted ASM. Our results suggest that the combined effect of Iso and breathing oscillations is noted to be greater than the added effects of Iso and breathing alone. It can be proposed that breathing oscillations aid the relaxation of ASM by Isoproterenol.

The Proprotein Convertase Furin inhibits IL-13 Induced Inflammation in Airway Smooth Muscle by Regulating Integrin Associated Signaling Complexes

2021 ◽  
Author(s):  
Yidi Wu ◽  
Youliang Huang ◽  
Wenwu Zhang ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Proprotein Convertase ◽  
Akt Activation ◽  
Inflammatory Conditions ◽  
Muscle Tissues ◽  
Muscle Myosin

Furin is a proprotein convertase that regulates the activation and inactivation of multiple proteins including matrix metalloproteinases, integrins and cytokines. It is a serine endoprotease that localizes to the plasma membrane and can be secreted into the extracellular space. The role of furin in regulating inflammation in isolated canine airway smooth muscle tissues was investigated. The treatment of airway tissues with recombinant furin (rFurin) inhibited the activation of Akt and eotaxin secretion induced by IL-13, and it prevented the IL-13 induced suppression of smooth muscle myosin heavy chain expression. rFurin promoted a differentiated phenotype by activating β1 integrin proteins and stimulating the activation of the adhesome proteins vinculin and paxillin by talin. Activated paxillin induced the binding of Akt to β-parvin IPP (ILK, PINCH, parvin) complexes, which inhibits Akt activation. Treatment of tissues with a furin inhibitor or the depletion of endogenous furin using shRNA resulted in Akt activation and inflammatory responses similar to those induced by IL-13. Furin inactivation or IL-13 caused talin cleavage and integrin inactivation, resulting in the inactivation of vinculin and paxillin. Paxillin inactivation resulted in the coupling of Akt to α-parvin IPP complexes, which catalyze Akt activation and an inflammatory response. The results demonstrate that furin inhibits inflammation in airway smooth muscle induced by IL-13, and that the anti-inflammatory effects of furin are mediated by activating integrin proteins and integrin-associated signaling complexes that regulate Akt-mediated pathways to the nucleus. Furin may have therapeutic potential for the treatment of inflammatory conditions of the lungs and airways.

β-Catenin regulates airway smooth muscle contraction

2010 ◽  
Vol 299 (2) ◽  
pp. L204-L214 ◽  
Author(s):  
Sepp R. Jansen ◽  
Anna M. Van Ziel ◽  
Hoeke A. Baarsma ◽  
Reinoud Gosens
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Airway Smooth Muscle ◽  
Glycogen Synthase ◽  
Force Production ◽  
Smooth Muscle Contraction ◽  
Cell Contact ◽  
Force Transmission ◽  
Tension Development ◽  
Cell Cell

β-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for β-catenin in the regulation of smooth muscle force production. β-Catenin colocalized with smooth muscle α-actin (sm-α-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-α-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-α-actin and β-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological β-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced β-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-α-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of β-catenin in BTSM strips reduced β-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-α-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of β-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that β-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

scholarly journals A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction

2015 ◽  
Vol 93 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Wenwu Zhang ◽  
Youliang Huang ◽  
Yidi Wu ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Smooth Muscle Contraction ◽  
Tension Development ◽  
Rhoa Gtpase ◽  
Tension Generation ◽  
P21 Activated Kinase ◽  
Signaling Modules ◽  
Mlc Phosphorylation

Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) – p21-activated kinase (PAK) – PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

2004 ◽  
Vol 286 (2) ◽  
pp. C433-C447 ◽  
Author(s):  
Anabelle Opazo Saez ◽  
Wenwu Zhang ◽  
Yidi Wu ◽  
Christopher E. Turner ◽  
Dale D. Tang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesions ◽  
Smooth Muscle Contraction ◽  
Cytoskeletal Proteins ◽  
Tracheal Smooth Muscle ◽  
Muscle Membrane ◽  
Tension Development ◽  
Muscle Tissues ◽  
Primary Determinant ◽  
Smooth Muscle Membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca2+ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction.

Studies of the mechanism of passive anaphylaxis in human airway smooth muscle

1983 ◽  
Vol 61 (7) ◽  
pp. 705-713 ◽  
Author(s):  
C. Davis ◽  
T. R. Jones ◽  
E. E. Daniel
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Immunoglobulin E ◽  
Specific Antigen ◽  
Maximal Response ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Muscle Tissues ◽  
Acid Metabolites

This investigation was carried out to study allergic contraction of passively sensitized human airway smooth muscle in response to specific antigen challenge. We attempted to determine the role played by histamine, slow reaction substances (SRSs), and cyclooxygenase products in the mediation of this response in tracheal smooth muscle. Tissues were passively sensitized with serum from ragweed-sensitive patients (15 h, 4 °C). Subsequent challenge with ragweed antigen produced a slowly developing contraction. The peak contraction to a dose producing a maximal response was 37 ± 6% of the carbachol maximum. Mepyramine (5 × 10−6 M) did not alter the contraction. Methylprednisolone (2 × 10−5 M) attenuated the response to antigen but had no significant effect on the contractile response to arachidonic acid. Indomethacin (5.6–28 × 10−6 M) enhanced the peak antigen-induced contractions by 25 ± 11% whereas 5, 8, 11, 14-eicosatetraynoic acid (6.4 × 10−5 M) selectively attenuated the antigen-induced contraction by 86 ± 12%. Nordihydroguarietic acid (6–12 × 10−6 M) attenuated both the antigen plus arachidonate induced responses. FPL-55712 (1–2 × 10−6 M) antagonized the contractions to antigen. Compound 48/80 and goat antihuman immunoglobulin E produced similar slowly developing contractions in sensitized and in some nonsensitized tissues. These responses, except for an early component of the response to 48/80, were independent of histamine and were reversed by FPL-55712. These findings suggest that arachidonic acid metabolites mediate (slow reacting substances) and modulate (prostaglandins) allergic contraction of human airway smooth muscle while any histamine released contributes little or nothing to the contraction in the larger airways.

Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle

2005 ◽  
Vol 288 (5) ◽  
pp. C1145-C1160 ◽  
Author(s):  
Wenwu Zhang ◽  
Yidi Wu ◽  
Liping Du ◽  
Dale D. Tang ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Tracheal Smooth Muscle ◽  
Wild Type ◽  
Tension Development ◽  
Muscle Tissues ◽  
Muscarinic Stimulation ◽  
Mlc Phosphorylation

Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation.

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