Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

2000 ◽  
Vol 279 (2) ◽  
pp. C308-C318 ◽  
Author(s):  
Dolly Mehta ◽  
Dale D. Tang ◽  
Ming-Fang Wu ◽  
Simon Atkinson ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Phosphorylation ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Cytochalasin D ◽  
Cytoskeletal Proteins ◽  
Tracheal Smooth Muscle ◽  
Contractile Activation ◽  
Mlc Phosphorylation

We investigated whether Rho activation is required for Ca2+-insensitive paxillin phosphorylation, myosin light chain (MLC) phosphorylation, and contraction in tracheal muscle. Tyrosine-phosphorylated proteins have been implicated in the Ca2+-insensitive contractile activation of smooth muscle tissues. The contractile activation of tracheal smooth muscle increases tyrosine phosphorylation of the cytoskeletal proteins paxillin and focal adhesion kinase. Paxillin is implicated in integrin-mediated signal transduction pathways that regulate cytoskeletal organization and cell motility. In fibroblasts and other nonmuscle cells, paxillin tyrosine phosphorylation depends on the activation of Rho and is inhibited by cytochalasin, an inhibitor of actin polymerization. In permeabilized muscle strips, we found that ACh induced Ca2+-insensitive contraction, MLC phosphorylation, and paxillin tyrosine phosphorylation. Ca2+-insensitive contraction and MLC phosphorylation induced by ACh were inhibited by C3 transferase, an inhibitor of Rho activation; however, C3 transferase did not inhibit paxillin tyrosine phosphorylation. Ca2+-insensitive paxillin tyrosine phosphorylation was also not inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D, or by the inhibition of MLC phosphorylation. We conclude that, in tracheal smooth muscle, Rho mediates Ca2+-insensitive contraction and MLC phosphorylation but that Rho is not required for Ca2+-insensitive paxillin tyrosine phosphorylation. Paxillin phosphorylation also does not require actomyosin activation, nor is it inhibited by the actin filament capping agent cytochalasin D.

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

2006 ◽  
Vol 290 (2) ◽  
pp. C463-C471 ◽  
Author(s):  
Shutang Zhou ◽  
Bradley A. Webb ◽  
Robert Eves ◽  
Alan S. Mak
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tyrosine Phosphorylation ◽  
Phorbol Ester ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Phosphorylation Sites ◽  
A7r5 Cells ◽  
Sirna Knockdown

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKCα inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKCα acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation.

Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle

1998 ◽  
Vol 274 (3) ◽  
pp. C741-C747 ◽  
Author(s):  
Dolly Mehta ◽  
Zhonglin Wang ◽  
Ming-Fang Wu ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Phosphorylation ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Maximal Increase ◽  
Force Development ◽  
Muscarinic Stimulation ◽  
Contractile Activation ◽  
Mlc Phosphorylation ◽  
Stimulation Of

The tyrosine phosphorylation of paxillin increases in association with force development during tracheal smooth muscle contraction, suggesting that paxillin plays a role in the contractile activation of smooth muscle [Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am. J. Physiol. 271 ( Cell Physiol. 40): C1594–C1602, 1996]. We compared the Ca2+ sensitivity of the tyrosine phosphorylation of paxillin and myosin light chain (MLC) phosphorylation in tracheal muscle and evaluated whether MLC phosphorylation is necessary to induce paxillin phosphorylation. Ca2+-depleted muscle strips were stimulated with 10−7–10−4M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca2+. In the absence of extracellular Ca2+, 10−4 M ACh induced a maximal increase in paxillin phosphorylation without increasing MLC phosphorylation or force. Increases in extracellular Ca2+ concentration did not further increase paxillin phosphorylation. However, during stimulation with 10−6 M ACh, paxillin phosphorylation increased with increases in extracellular Ca2+ concentration. We conclude that the tyrosine phosphorylation of paxillin can be stimulated by signaling pathways that do not depend on Ca2+ mobilization and that the activation of contractile proteins is not required to elicit paxillin phosphorylation.

scholarly journals Selected Contribution: Roles of focal adhesion kinase and paxillin in the mechanosensitive regulation of myosin phosphorylation in smooth muscle

2001 ◽  
Vol 91 (3) ◽  
pp. 1452-1459 ◽  
Author(s):  
Dale D. Tang ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion Kinase ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Muscle Length ◽  
Cytoskeletal Proteins ◽  
Tracheal Smooth Muscle ◽  
Active Tension ◽  
Intracellular Ca ◽  
Mlc Phosphorylation

The increase in intracellular Ca2+ and myosin light chain (MLC) phosphorylation in response to the contractile activation of tracheal smooth muscle is greater at longer muscle lengths (21). However, MLC phosphorylation can also be stimulated by Ca2+-insensitive signaling pathways (19). The cytoskeletal proteins paxillin and focal adhesion kinase (FAK) mediate a Ca2+-independent length-sensitive signaling pathway in tracheal smooth muscle (30). We used α-toxin-permeabilized tracheal smooth muscle strips to determine whether the length sensitivity of MLC phosphorylation can be regulated by a Ca2+-insensitive signaling pathway and whether the length sensitivity of active tension depends on the length sensitivity of myosin activation. Although active tension remained length sensitive, ACh-induced MLC phosphorylation was the same at optimal muscle length ( L o) and 0.5 L o when intracellular Ca2+ was maintained at pCa 7. MLC phosphorylation was also the same at L o and 0.5 L o in strips stimulated with 10 μM Ca2+. In contrast, the Ca2+-insensitive tyrosine phosphorylation of FAK and paxillin stimulated by ACh was higher at L o than at 0.5 L o. We conclude that the length-sensitivity of MLC phosphorylation depends on length-dependent changes in intracellular Ca2+ but that length-dependent changes in MLC phosphorylation are not the primary mechanism for the length sensitivity of active tension.

Viscoelastic and dynamic nonlinear properties of airway smooth muscle tissue: roles of mechanical force and the cytoskeleton

2006 ◽  
Vol 290 (6) ◽  
pp. L1227-L1237 ◽  
Author(s):  
Satoru Ito ◽  
Arnab Majumdar ◽  
Hiroaki Kume ◽  
Kaoru Shimokata ◽  
Keiji Naruse ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cultured Cells ◽  
Kinase Inhibitor ◽  
Viscoelastic Model ◽  
Harmonic Distortion ◽  
Cytochalasin D ◽  
Tracheal Smooth Muscle ◽  
Smooth Muscle Tissue ◽  
Nonlinear Properties ◽  
Dynamic Nonlinearity

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G′) and loss (G") moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G′ spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion ( Kd), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased Kd. Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased Kd. Following cytochalasin D, G, H, and Kd were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.

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.

scholarly journals The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

2010 ◽  
Vol 299 (2) ◽  
pp. C298-C306 ◽  
Author(s):  
Wenwu Zhang ◽  
Liping Du ◽  
Susan J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Cell Types ◽  
Small Gtpase ◽  
Calyculin A ◽  
Active Tension ◽  
Tension Generation ◽  
Mlc Phosphorylation

The small GTPase RhoA increases the Ca2+ sensitivity of smooth muscle contraction and myosin light chain (MLC) phosphorylation by inhibiting the activity of MLC phosphatase. RhoA is also a known regulator of cytoskeletal dynamics and actin polymerization in many cell types. In airway smooth muscle (ASM), contractile stimulation induces MLC phosphorylation and actin polymerization, which are both required for active tension generation. The objective of this study was to evaluate the primary mechanism by which RhoA regulates active tension generation in intact ASM during stimulation with acetylcholine (ACh). RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, in the intact tissues or by treating them with the cell-permeant RhoA inhibitor, exoenzyme C3 transferase. RhoA inactivation reduced ACh-induced contractile force by ∼60% and completely inhibited ACh-induced actin polymerization but inhibited ACh-induced MLC phosphorylation by only ∼20%. Inactivation of MLC phosphatase with calyculin A reversed the reduction in MLC phosphorylation caused by RhoA inactivation, but calyculin A did not reverse the depression of active tension and actin polymerization caused by RhoA inactivation. The MLC kinase inhibitor, ML-7, inhibited ACh-induced MLC phosphorylation by ∼80% and depressed active force by ∼70% but did not affect ACh-induced actin polymerization, demonstrating that ACh-stimulated actin polymerization occurs independently of MLC phosphorylation. We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation.

scholarly journals Leukotriene C4 enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway

2001 ◽  
Vol 132 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Hidekazu Setoguchi ◽  
Junji Nishimura ◽  
Katsuya Hirano ◽  
Shosuke Takahashi ◽  
Hideo Kanaide
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Tracheal Smooth Muscle ◽  
Leukotriene C4 ◽  
Rho Kinase Inhibitor

scholarly journals Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain

2011 ◽  
Vol 439 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Dean P. Staus ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Actin Polymerization ◽  
Serum Response Factor ◽  
Rho Kinase ◽  
Specific Gene ◽  
Related Transcription Factor ◽  
Inhibitory Domain ◽  
Serum Response ◽  
Acidic Region ◽  
Basic Region

It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr1061 and Ser1070) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.

Abstract 641: Expression Of GLUT4 In Vascular Smooth Muscle Affects Endothelial Function In Hypertension.

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Kevin B Atkins ◽  
Jharna Saha ◽  
Frank C Brosius
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Interesting Phenomenon ◽  
Glut4 Expression ◽  
Endothelial Denudation ◽  
Rho Kinase Inhibitor ◽  
Total Body ◽  
Rats And Mice

Expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice, and total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity. To demonstrate that the effect on vascular response to GLUT4 overexpression is vascular rather than systemic in origin we utilized smooth muscle-specific GLUT4 transgenic mice (SMG4). GLUT4 expression in aortae of SMG4 compared to WT mice was increased 2-3 fold. Adult wild-type (WT) and SMG4 mice were made hypertensive or not through implantation of angiotensin II (AngII; 1.4mg/kg/d for 2 wks) or vehicle containing osmotic mini-pumps. Both WT and SMG4 mice AngII-treated mice exhibited significantly increased systolic blood pressure. In AngII-treated WT mice (WT-AngII) aortic GLUT4 expression was significantly decreased, whereas GLUT4 expression in aortae of AngII-treated SMG4 mice (SMG4-AngII) was maintained. The phosphorylation of ERM and MYPT1(Thr850) were significantly increased in aortae of WT-AngII compared to WT-Sham and SMG4-AngII mice. Responsiveness to the contractile agonists, phenylephrine, 5-HT, and PGF 2 was significantly increased in endothelium-intact aortic rings from WT-AngII mice, but remained normal in aortae of SMG4-AngII mice. Following pretreatment with Rho-kinase inhibitor Y-27632, relative inhibition of contractility to 5-HT was equal in aortae from WT-AngII and SMG4-AngII-treated mice. With endothelial denudation, contractility to 5-HT was equally enhanced in aortae of WT-AngII and SMG4-AngII-treated mice. Interestingly, whereas acetylcholine stimulated relaxation was significantly decreased in aortic rings of WT-AngII mice, relaxation in rings from SMG4-AngII mice was not significantly different from WT or SMG4. These results demonstrate an interesting phenomenon whereby decreased expression of GLUT4 in vascular smooth muscle leads to an endothelial dysfunction that not only impairs relaxation, but also enhances contractility.

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