Actin filament-dependent modulation of translocated RhoA, PKC α and association with HSP27 in the particulate fraction of smooth muscle cells isolated from the rabbit colon

2001 ◽  
Vol 120 (5) ◽  
pp. A327-A327
Author(s):  
K BITAR ◽  
M PAWAR ◽  
P THOMAS
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Actin Filament ◽  
Muscle Cells ◽  
Particulate Fraction ◽  
Rabbit Colon

Agonist-induced association of tropomyosin with protein kinase Cα in colonic smooth muscle

2005 ◽  
Vol 288 (2) ◽  
pp. G268-G276 ◽  
Author(s):  
Sita Somara ◽  
Haiyan Pang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Calcium Dependent

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

Translocation and association of ROCK-II with RhoA and HSP27 during contraction of rabbit colon smooth muscle cells

2004 ◽  
Vol 319 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Suresh B Patil ◽  
Yasuhiro Tsunoda ◽  
Mercy D Pawar ◽  
Khalil N Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Rabbit Colon

RhoA- and PKC-α-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells.

2006 ◽  
Vol 290 (1) ◽  
pp. G83-G95 ◽  
Author(s):  
Suresh B. Patil ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Phosphatase Activity ◽  
Cross Talk ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Selective Inhibition ◽  
Particulate Fraction ◽  
Myosin Phosphatase ◽  
Sustained Phosphorylation

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-α. Presently, we examined the cross talk between RhoA and PKC-α in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-α, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 ± 3.52 and 42.19 ± 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 ± 46.60% and 290.59 ± 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-α by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-α pathways, suggesting cross talk between RhoA and PKC-α resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.

scholarly journals Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction.

1995 ◽  
Vol 130 (1) ◽  
pp. 193-206 ◽  
Author(s):  
K E Bornfeldt ◽  
L M Graves ◽  
E W Raines ◽  
Y Igarashi ◽  
G Wayman ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Actin Filament ◽  
Muscle Cells ◽  
Calcium Mobilization ◽  
Signal Transduction Pathways ◽  
Pdgf Receptor ◽  
Sphingosine 1 Phosphate ◽  
Arterial Smooth Muscle Cells

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.

Agonist-induced activation of Rhoa, Rho-kinase, PKCa and association with Hsp27 in smooth muscle cells from the rabbit colon

2000 ◽  
Vol 118 (4) ◽  
pp. A88
Author(s):  
Adenike I. Ibitayo ◽  
Esli E. Gollapalli ◽  
Chung Owyang ◽  
Yasuhiro Tsunoda ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Rabbit Colon

scholarly journals Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein

2010 ◽  
Vol 298 (3) ◽  
pp. C559-C571 ◽  
Author(s):  
Hak Rim Kim ◽  
Philip Graceffa ◽  
François Ferron ◽  
Cynthia Gallant ◽  
Malgorzata Boczkowska ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Actin Filament ◽  
Hot Spots ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Cell Cortex ◽  
Sites Of Action

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768–778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with α-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with α-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.

The response of smooth muscle cells in the rabbit colon to anal stenosis: A preliminary report

Pathology ◽  
1973 ◽  
Vol 5 (3) ◽  
pp. 209-218 ◽  
Author(s):  
Lourdes Brent
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Preliminary Report ◽  
Muscle Cells ◽  
Anal Stenosis ◽  
Rabbit Colon
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