Y-27632, A Rho-Kinase Inhibitor, Inhibits Proliferation and Adrenergic Contraction of Prostatic Smooth Muscle Cells

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated inhibition of MLC20dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5′- O-(3-thiotriphosphate) (GTPγS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPγS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

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Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1and S1P2

AJP Cell Physiology ◽

10.1152/ajpcell.00429.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. C1130-C1138 ◽

Cited By ~ 81

Author(s):

Huiping Zhou ◽

Karnam S. Murthy

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Muscle Cells ◽

Rt Pcr ◽

Sustained Contraction ◽

Gastric Smooth Muscle ◽

Rho Kinase Inhibitor ◽

Sphingosine 1 Phosphate

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC20) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P1and S1P2receptors. S1P activated Gq, G13, and all Giisoforms and stimulated PLC-β1, PLC-β3, and Rho kinase activities. PLC-β activity was partially inhibited by pertussis toxin (PTX), Gβ or Gαqantibody, PLC-β1 or PLC-β3 antibody, and by expression of Gαqor Gαiminigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of Gα13or Gαqminigene and abolished by expression of both. S1P stimulated Ca2+release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC501 nM). Initial contraction and MLC20phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Gαqor Gβ antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC20phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P2and S1P1involving concurrent activation of PLC-β1 and PLC-β3 via Gαqand Gβγi, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca2+release and MLCK-mediated MLC20phosphorylation, and 2) sustained contraction exclusively mediated by S1P2involving activation of RhoA via Gαqand Gα13, resulting in Rho kinase- and PKC-dependent MLC20phosphorylation.

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Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00305.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. G23-G31 ◽

Cited By ~ 52

Author(s):

Jiean Huang ◽

Huiping Zhou ◽

Sunila Mahavadi ◽

Wimolpak Sriwai ◽

Vijay Lyall ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Dominant Negative ◽

Phosphatase Inhibitors ◽

Sustained Contraction ◽

Pi Hydrolysis

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC50 0.7 ± 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated Gq and G13, stimulated Gαq-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, and increased cytosolic free Ca2+. PI hydrolysis was blocked by expression of Gαq minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC50 = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca2+-dependent contraction and myosin light-chain (MLC)20 phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC20 phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of Gαq or Gα13 minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC20 phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a Gαq-mediated cascade involving Ca2+/calmodulin activation of MLC kinase and transient MLC20 phosphorylation and contraction as well as a sustained Gαq- and Gα13-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC20 phosphorylation and contraction.

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Abstract P182: The Novel Perivascular Adipose Tissue Adipokine, Chemerin, Signals Through G i - and Calcium-Dependent Mechanisms

Hypertension ◽

10.1161/hyp.68.suppl_1.p182 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

David J Ferland ◽

Emma S Darios ◽

Richard R Neubig ◽

Benita Sjögren ◽

Nguyen Truong ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Isometric Contraction ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Muscle Cells ◽

Calcium Flux ◽

Western Blots ◽

Perivascular Adipose Tissue ◽

Erk Mapk

Chemerin is an adipokine associated with inflammation, increased blood pressure, and may be a link between the pathologies of obesity and hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via the chemerin receptor and calcium flux in smooth muscle cells. Known mediators of the amplified arterial responsiveness seen in hypertension (L-type Ca 2+ channels, Src, and Rho kinase) were interrogated by isometric contraction of rat aortic rings in parallel with calcium kinetics of rat aortic smooth muscle cells. Western blots were also used to observe phosphorylation of Erk/MAPK. Chemerin-9 (nonapeptide of the chemerin S 157 isoform) caused a concentration-dependent contraction of isolated aorta (EC 50 100 nM) and elicited a concentration-dependent intracellular calcium response (EC 50 10 nM). Both calcium influx and isometric contraction, respectively, were reduced (units of “% of vehicle response”) by Pertussis toxin (G i inhibitor; 0±3% and 23±9%), verapamil (L-type Ca 2+ channel inhibitor; 38±20% and 23±4%), PP1 (Src inhibitor; 43±23% and 15±4%), and Y27632 (Rho Kinase inhibitor; 58±23% and 22±4%) but U73122 (PLC inhibitor) had little to no effect (71±31% and 71±12%). PD098059 (Erk/MAPK inhibitor) did not inhibit chemerin-9 induced contraction (117±19%) and phosphorylation did not change after chemerin-9 stimulation [1.12±0.14 (44 kDa) and 1.11±0.29 (42 kDa) fold-increase with chemerin-9 contraction compared to vehicle, p>0.05]. The chemerin receptor-selective antagonist CCX832 inhibited chemerin-9-induced calcium flux and aortic contraction and calcium flux (0.1±10.3% and 10±7%). These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through the G i -linked chemerin receptor to activate L-type Ca 2+ channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us one step closer to targeting chemerin as a unique form of therapy.

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Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00092.2002 ◽

2003 ◽

Vol 284 (6) ◽

pp. L1020-L1026 ◽

Cited By ~ 34

Author(s):

Stephen M. Carlin ◽

Michael Roth ◽

Judith L. Black

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Airway Smooth Muscle ◽

Kinase Inhibitor ◽

Airway Remodeling ◽

Muscle Cells ◽

Mek Inhibitor ◽

Airway Smooth Muscle Cells ◽

Human Airway Smooth Muscle ◽

Human Airway

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-μm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFABwere all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3′-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00350.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. C463-C471 ◽

Cited By ~ 30

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.

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

Hypertension ◽

10.1161/hyp.62.suppl_1.a641 ◽

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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