Tyrosine phosphorylation of the dense plaque protein paxillin is regulated during smooth muscle contraction

1996 ◽  
Vol 271 (5) ◽  
pp. C1594-C1602 ◽  
Author(s):  
Z. Wang ◽  
F. M. Pavalko ◽  
S. J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Cell Membrane ◽  
Tyrosine Phosphorylation ◽  
Actin Filaments ◽  
Time Course ◽  
Smooth Muscle Contraction ◽  
External Stress ◽  
Force Development ◽  
Contraction And Relaxation

Regulation of the attachment of actin filaments to the cell membrane at membrane-associated dense plaque (MADP) sites could allow smooth muscle cells to modulate their cytostructure in response to changes in external stress. In this study, changes in the tyrosine phosphorylation of the MADP protein paxillin were measured by Western blot during the contraction and relaxation of tracheal smooth muscle strips. Tyrosine phosphorylation of paxillin increased by three- to fourfold with a time course similar to force development during contractile stimulation with acetylcholine (ACh), 5-hydroxytryptamine, and KCl and decreased during washout of contractile stimuli and during relaxation induced by forskolin. Immunoprecipitation of muscle extracts with multiple rounds of anti-phosphotyrosine antibody removed approximately 20% of the total paxillin in resting muscles and approximately 60% of paxillin in muscles maximally stimulated with ACh. These results provide the first evidence associating the tyrosine phosphorylation of paxillin with the active contraction of smooth muscle or with any functional response of a fully differentiated tissue in vivo. The results are consistent with a role for MADP proteins in the regulation of force development in smooth muscle.

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.

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

2009 ◽  
Vol 296 (5) ◽  
pp. C1024-C1033 ◽  
Author(s):  
Ying-Ming Liou ◽  
Masaru Watanabe ◽  
Masatoshi Yumoto ◽  
Shin'ichi Ishiwata
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Actin Filaments ◽  
Smooth Muscles ◽  
Smooth Muscle Contraction ◽  
Irreversible Damage ◽  
Cross Sectional ◽  
Thin Filaments ◽  
Force Development ◽  
Skinned Smooth Muscle

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca2+-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca2+-dependent force development was partially restored, but the Ca2+-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca2+sensitivity and the reversible Ca2+-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

FRET analysis of actin–myosin interaction in contracting rat aortic smooth muscle

2009 ◽  
Vol 87 (5) ◽  
pp. 327-336 ◽  
Author(s):  
J. Black ◽  
A. Dykes ◽  
S. Thatcher ◽  
D. Brown ◽  
E.C. Bryda ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Visual Evaluation ◽  
Aortic Smooth Muscle ◽  
Force Development

We examined the interaction of smooth muscle myosin with α-actin and β-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from α-actin but not β-actin. Whole-cell FRET analysis confirmed these observations (α-actin–myosin –67%, β-actin–myosin –2%). Time course studies further showed that α-actin–myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in α-actin–myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (–4%). By comparison, β-actin–myosin complex was unchanged except during slow force development, in which the association was significantly decreased (–30%). Similar to that of α-actin–myosin, Alexa 488 – phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of α-actin–myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein–protein associations.

Human vascular smooth muscle responses mediated by α2 mechanisms in vivo and in vitro

1985 ◽  
Vol 68 (s10) ◽  
pp. 147s-150s ◽  
Author(s):  
S. Thom ◽  
J. Calvete ◽  
R. Hayes ◽  
G. Martin ◽  
P. Sever
Keyword(s):  
Smooth Muscle ◽  
Physiological Role ◽  
Smooth Muscle Contraction ◽  
Α1 Receptors ◽  
Dose Dependent ◽  
Higher Doses ◽  
Α2 Receptors ◽  
Autoregulatory Mechanism

1. The effects of compounds with α2-agonist and α2-antagonist properties on human forearm blood flow and on isolated human arterial segments have been studied. 2. The findings from these studies in vivo and in vitro did not provide evidence in support of the hypothesis that postsynaptic α2-receptors mediate smooth muscle contraction in the tissues under investigation. 3. The constriction of the forearm vascular bed in response to low intra-arterial doses of idazoxan (RX 781094), an α2-antagonist, provides evidence for a physiological role for a presynaptic α2 autoregulatory mechanism. 4. The variability of the forearm vascular responses to higher doses of idazoxan highlights the pitfalls that may have misled previous authors in their interpretation of the results of similar studies. A U-shaped dose-response curve to compounds with mixed α2-and α1-antagonist properties may be constructed, which emphasizes the importance of the dose-dependent selectivity of these antagonists at α2- and α1-receptors. 5. The effect of idazoxan on the responses of arterial segments in vitro to exogenous catecholamines was dependent on the integrity of the endothelium, and provides evidence that α2-receptors may mediate release of the endothelium-derived relaxing factor.

scholarly journals Abl activation regulates the dissociation of CAS from cytoskeletal vimentin by modulating CAS phosphorylation in smooth muscle

2010 ◽  
Vol 299 (3) ◽  
pp. C630-C637 ◽  
Author(s):  
Li Jia ◽  
Dale D. Tang
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Actin Polymerization ◽  
Spatial Location ◽  
Smooth Muscle Contraction ◽  
Nonreceptor Tyrosine Kinase ◽  
Force Development ◽  
Muscle Inhibition ◽  
A Cell ◽  
Contractile Activation

Abl is a nonreceptor tyrosine kinase that is required for smooth muscle contraction. However, the mechanism by which Abl regulates smooth muscle contraction is not completely understood. In the present study, Abl underwent phosphorylation at Tyr412 (an index of Abl activation) in smooth muscle in response to contractile activation. Treatment with a cell-permeable decoy peptide, but not the control peptide, attenuated Abl phosphorylation during contractile stimulation. Treatment with the decoy peptide did not affect the association of Abl with the cytoskeletal protein vinculin and the spatial location of vinculin in smooth muscle. Inhibition of Abl phosphorylation by the decoy peptide attenuated the agonist-induced phosphorylation of Crk-associated substrate (CAS), an adapter protein participating in the signaling processes that regulate force development in smooth muscle. Additionally, previous studies have shown that contractile stimulation triggers the dissociation of CAS from the vimentin network, which is important for cytoskeletal signaling and contraction in smooth muscle. In this report, the decrease in the amount of CAS in cytoskeletal vimentin in response to contractile activation was reversed by the Abl inhibition with the decoy peptide. Moreover, force development and the enhancement of F-actin-to-G-actin ratios (an indication of actin polymerization) upon contractile activation were also attenuated by the Abl inhibition. However, myosin phosphorylation induced by contractile activation was not affected by the inhibition of Abl. These results suggest that Abl regulates the dissociation of CAS from the vimentin network, actin polymerization, and contraction by modulating CAS phosphorylation in smooth muscle.

scholarly journals MP06-12 GHRELIN-MEDIATED PROMOTION OF PROSTATE GROWTH AND PROSTATE SMOOTH MUSCLE CONTRACTION: EVIDENCE FROM FUNCTIONAL, IN VIVO, AND GENOMIC APPROACHES

2019 ◽  
Vol 201 (Supplement 4) ◽  
Author(s):  
Xiaolong Wang* ◽  
Yiming Wang ◽  
Christian Gratzke ◽  
Bingsheng Li ◽  
Qingfeng Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Smooth Muscle Contraction ◽  
Prostate Growth

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation

2004 ◽  
Vol 287 (3) ◽  
pp. C594-C602 ◽  
Author(s):  
Christopher M. Rembold ◽  
Robert L. Wardle ◽  
Christopher J. Wingard ◽  
Timothy W. Batts ◽  
Elaine F. Etter ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Time Course ◽  
Muscle Force ◽  
Regulatory System ◽  
Force Development ◽  
Cross Bridge ◽  
Cross Bridges ◽  
The Relationship ◽  
Cooperative Activation

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of ∼0.15 mol Pi/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser19-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser19-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

scholarly journals Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs

2002 ◽  
Vol 93 (4) ◽  
pp. 1296-1300 ◽  
Author(s):  
Debra J. Turner ◽  
Peter B. Noble ◽  
Matthew P. Lucas ◽  
Howard W. Mitchell
Keyword(s):  
Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Porcine Model ◽  
Smooth Muscle Contraction ◽  
Airway Wall ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Airway Narrowing

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0–20 cmH2O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls ( P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi ( P < 0.01) and smooth muscle strips ( P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.

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