scholarly journals Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors

2003 ◽  
Vol 284 (6) ◽  
pp. C1387-C1396 ◽  
Author(s):  
Asad Zeidan ◽  
Ina Nordström ◽  
Sebastian Albinsson ◽  
Ulf Malmqvist ◽  
Karl Swärd ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Latrunculin B ◽  
Rhoa Activity ◽  
Mechanical Compliance ◽  
Tissue Compliance ◽  
Portal Veins ◽  
Endogenous Release

Signaling mechanisms for stretch-dependent growth and differentiation of vascular smooth muscle were investigated in mechanically loaded rat portal veins in organ culture. Stretch-dependent protein synthesis was found to depend on endogenous release of angiotensin II. Autoradiography after [35S]methionine incorporation revealed stretch-dependent synthesis of several proteins, of which SM22 and actin were particularly prominent. Inhibition of RhoA activity by cell-permeant C3 toxin increased tissue mechanical compliance and reduced stretch-dependent extracellular signal-regulated kinase (ERK)1/2 activation, growth, and synthesis of actin and SM22, suggesting a role of the actin cytoskeleton. In contrast, inhibition of Rho-associated kinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance but still inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin B concentration-dependently reduced actin and SM22 synthesis, cytochalasin D did so at low (10−8M) but not at high (10−6M) concentration. The results show that stretch stabilizes the contractile smooth muscle phenotype. Stretch-dependent differentiation marker expression requires an intact cytoskeleton for stretch sensing, control of protein expression via the level of unpolymerized G-actin, or both.

Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle

2007 ◽  
Vol 293 (2) ◽  
pp. C772-C782 ◽  
Author(s):  
Sebastian Albinsson ◽  
Per Hellstrand
Keyword(s):  
Protein Synthesis ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Mechanical Load ◽  
Mammalian Target Of Rapamycin ◽  
Focal Adhesions ◽  
Signal Pathways ◽  
Erk Phosphorylation ◽  
Portal Veins

The vascular smooth muscle phenotype is regulated by environmental factors, such as mechanical forces, that exert effects on signaling to differentiation and growth. We used the mouse portal vein in organ culture to investigate stretch-dependent activation of Akt, ERK, and focal adhesion kinase (FAK), which have been suggested to be involved in the regulation of stretch-dependent protein synthesis. The role of actin polymerization in these signaling events was examined using the actin-stabilizing agent jasplakinolide. Stretch caused a biphasic activation of FAK at 5–15 min and 24–72 h, which may reflect first a direct phosphorylation of preexisting focal adhesions followed by a rearrangement of focal adhesions to accommodate for the increased mechanical load. Phosphorylation of ERK was increased by acute stretch but then decreased, and Akt did not have a distinct peak in stretch-induced phosphorylation. Inhibition of ERK, phosphatidylinositol 3-kinase, or mammalian target of rapamycin reduced global but not contractile protein synthesis with maintained stretch sensitivity. Stabilization of actin filaments with jasplakinolide, in unstretched portal veins, resulted in increased ERK phosphorylation and global protein synthesis as well as the synthesis of contractile proteins. In contrast, stretch during culture with jasplakinolide did not affect FAK phosphorylation or contractility. Therefore, remodeling of smooth muscle cells to adapt to stretch requires a dynamic cytoskeleton.

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction

2005 ◽  
Vol 288 (2) ◽  
pp. H660-H669 ◽  
Author(s):  
Nicholas A. Flavahan ◽  
Simon R. Bailey ◽  
William A. Flavahan ◽  
Srabani Mitra ◽  
Sheila Flavahan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Actin Cytoskeleton ◽  
Vascular Smooth Muscle Cells ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Cytochalasin D ◽  
Cell Periphery

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The α1-adrenergic agonist phenylephrine (0.1–1 μmol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10–60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 μmol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 μmol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.

scholarly journals A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle

2010 ◽  
Vol 299 (5) ◽  
pp. C988-C993 ◽  
Author(s):  
Hak Rim Kim ◽  
Paul C. Leavis ◽  
Philip Graceffa ◽  
Cynthia Gallant ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Direct Detection ◽  
New Method ◽  
Actin Dynamics ◽  
Tat Peptide ◽  
Isolated Cells ◽  
Intact Cells ◽  
Permeabilized Cells

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.

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.

Pentobarbital and contraction of vascular smooth muscle

1975 ◽  
Vol 229 (6) ◽  
pp. 1635-1640 ◽  
Author(s):  
BT Altura ◽  
BM Altura
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Dose Response ◽  
Mechanical Activity ◽  
Response Curves ◽  
Aortic Smooth Muscle ◽  
Dose Response Curves ◽  
Portal Veins ◽  
The Right ◽  
Dose Dependent

This study, with isolated rat aortic strips and portal veins, was undertaken to : 1) study the effects, if any, of pentobarbital Na (PTB) (5 x 10(-5) to 2 X 10(-3) M) on reactivity to epinephrine, serotonin, and KCl; 2) determine whether certain concentrations of PTB induce direct actions on aortic strips and portal veins; and 3) gain some insight into how these effects are brought about. The results indicate that PTB can: a) inhibit development of spontaneous mechanical activity in these vessels in anesthetic concentrations; b) dose-dependently attenuate contractions induced by epinephrine, serotonin, and KC1; c) cause a noncompetitive type displacement of the dose response curves of these vasoactive agents; d) attenuate Ca2+- induced contractions of potassium-depolarized aortic strips and portal veins concomitant with a dose-dependent displacement of these dose-response curves to the right; and e) rapidly relax drug as well as Ca2+ -induced contractions of aortas and portal veins. In addition, the data indicate that rat portal venous smooth muscle is more sensitive to the inhibitory actions of PTB than rat aortic smooth muscle. Overall, these data suggest that concentrations of PTB used to induce surgical anesthesia can exert profound depressant effects on at least two different types of vascular smooth muscle that may be related to actions on movement and/or translocation of Ca2+.

scholarly journals Abl knockout differentially affects p130 Crk-associated substrate, vinculin, and paxillin in blood vessels of mice

2009 ◽  
Vol 297 (2) ◽  
pp. H533-H539 ◽  
Author(s):  
Shu Chen ◽  
Ruping Wang ◽  
Qing-Fen Li ◽  
Dale D. Tang
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Contraction ◽  
Knockout Mice ◽  
Actin Polymerization ◽  
Fluorescent Microscopy ◽  
Smooth Muscle Contraction ◽  
Cellular Process ◽  
Actin Dynamics

Actin polymerization has recently emerged as an important cellular process that regulates smooth muscle contraction. Abelson tyrosine kinase (Abl) has been implicated in the regulation of actin dynamics and force development in vascular smooth muscle. In the present study, the systolic blood pressure was lower in Abl−/− knockout mice compared with wild-type mice. The knockout of Abl diminished the tyrosine phosphorylation of p130 Crk-associated substrate (CAS, an adapter protein associated with smooth muscle contraction) in resistance arteries upon stimulation with phenylephrine or angiotensin II. The agonist-elicited enhancement of F-actin-to-G-actin ratios in arteries assessed by fluorescent microscopy was also reduced in Abl−/− mice. It has been known that vinculin is a structural protein that links actin filaments to extracellular matrix via transmembrane integrins, whereas paxillin is a signaling protein associated with focal contacts mediating actin cytoskeleton remodeling. The expression of vinculin and paxillin at protein and messenger levels was lower in arterial vessels from Abl knockout mice. However, the agonist-induced increase in myosin phosphorylation was not attenuated in arteries from Abl knockout mice. These results indicate that Abl differentially regulates Crk-associated substrate, vinculin, and paxillin in arterial vessels. The Abl-regulated cellular process and blood pressure are independent of myosin activation in vascular smooth muscle.

Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells

2000 ◽  
Vol 279 (2) ◽  
pp. C480-C487 ◽  
Author(s):  
Mariko Nakamura ◽  
Masanori Sunagawa ◽  
Tadayoshi Kosugi ◽  
Nicholas Sperelakis
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Cell Voltage ◽  
Cytochalasin D ◽  
Channel Current ◽  
A7r5 Cell ◽  
Tubulin Antibodies ◽  
A7r5 Cell Line

To clarify interactions between the cytoskeleton and activity of L-type Ca2+ (CaL) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca2+ current [ I Ba(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-α-actin and anti-α-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I Ba(L) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I Ba(L) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I Ba(L) due to another unknown mechanism, e.g., a direct block of CaLchannels. In summary, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not.

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