scholarly journals Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle

2013 ◽  
Vol 305 (2) ◽  
pp. C215-C227 ◽  
Author(s):  
Ransom H. Poythress ◽  
Cynthia Gallant ◽  
Susanne Vetterkind ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Protein Localization ◽  
Focal Adhesions ◽  
Significant Inhibition ◽  
Adhesion Protein ◽  
Endosomal Recycling ◽  
Focal Adhesion Protein ◽  
And Function

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.

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.

scholarly journals TGF-β suppresses the upregulation of MMP-2 by vascular smooth muscle cells in response to PDGF-BB

2010 ◽  
Vol 298 (1) ◽  
pp. C191-C201 ◽  
Author(s):  
George M. Risinger ◽  
Dawn L. Updike ◽  
Elizabeth C. Bullen ◽  
James J. Tomasek ◽  
Eric W. Howard
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Transforming Growth Factor ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Threshold Level ◽  
Inhibitory Signaling

During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) α-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-β, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-β was able to block or reverse this transition to a noncontractile state. TGF-β did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-β1 levels, it suppressed TGF-β2 and TGF-β3 expression, leading to a net decrease in the total TGF-β pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-β, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.

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.

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

2006 ◽  
Vol 291 (3) ◽  
pp. H1226-H1235 ◽  
Author(s):  
Hui Dong ◽  
Yanfen Jiang ◽  
Chris R. Triggle ◽  
Xiaofang Li ◽  
Jonathan Lytton
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Vascular Tone ◽  
Rt Pcr ◽  
Aortic Smooth Muscle ◽  
Voltage Gated ◽  
Reverse Mode ◽  
And Function ◽  
Contraction And Relaxation

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.

Sign in / Sign up

Export Citation Format

Share Document