Abstract 1170: SHP2-dependent Dephosphorylation of p190A Rho GAP Induces RhoA Activation by Angiotensin II in Vascular Smooth Muscle Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Malvyne Rolli-Derkinderen ◽  
Jérémy Brégeon ◽  
Sarah J Parsons ◽  
Pierre Pacaud ◽  
Gervaise Loirand
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Ang Ii ◽  
Rhoa Activation ◽  
Kinase Pathway

Angiotensin II (Ang II) is a major regulator of blood pressure, that essentially acts through activation of Ang II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R receptor activates numerous intracellular signaling pathways, including the small G protein RhoA known to control VSMC proliferation, migration, differentiation and contraction. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor, that replaces bound GDP by GTP and/or inhibition of Rho GTPase-activating-protein (GAP) that hydrolyzes GTP to GDP. Here we assess the involvement of the p190A Rho GAP in RhoA activation induced by Ang II. The introduction of small interfering RNA (siRNA) targeting p190A in VSMC from rat aorta increased basal RhoA-Rho kinase pathway activity (790 ± 11% of control, n = 3). Moreover p190A-siRNA abolished the early activation of RhoA-Rho kinase pathway induced after 5 min of AngII (0.1 μM) stimulation but not the delayed RhoA activation induced after 60 min. We then measured p190A tyrosine phosphorylation known to reflect its activity. In resting VSMC, p190A was basally phosphorylated. In the presence of the AT2R inhibitor PD123319 (1 μM), activation of AT1R induced p190A dephosphorylation that was maximal at 5 min of Ang II stimulation (26 ± 5% of control, n = 4). The activation of AT2R by Ang II in the presence of losartan (1 μM) had no effect, neither on RhoA activation nor on p190A phosphorylation. Expression of a p190A phosphomimetic mutant decreased the basal activity of RhoA-Rho kinase pathway. In contrast expression of catalytically inactive or phosphoresistant p190A mutants increased the basal activity of RhoA-Rho kinase pathway and inhibited RhoA activation by Ang II. Moreover, using siRNA, we show that the tyrosine phosphatase SHP2, known to be activated by AT1R stimulation, was necessary for Ang II-mediated p190A dephosphorylation and RhoA activation. Our work demonstrates that RhoA/Rho kinase activity is controlled by the p190A Rho GAP in VSMC. Activity of p190A is basally high and maintains a low level of RhoA-Rho kinase activity in resting VSMC. Dephosphorylation of p190A through SHP2-dependent process is required for AT1R-induced RhoA activation.

Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells

2009 ◽  
Vol 297 (5) ◽  
pp. C1062-C1070 ◽  
Author(s):  
Jeremy Bregeon ◽  
Gervaise Loirand ◽  
Pierre Pacaud ◽  
Malvyne Rolli-Derkinderen
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Ang Ii ◽  
Rhoa Activation ◽  
Kinase Pathway

Angiotensin II (ANG II) is a major regulator of blood pressure that essentially acts through activation of ANG II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R activates numerous intracellular signaling pathways, including the small G protein RhoA known to control several VSMC functions. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor and/or inhibition of Rho GTPase-activating protein (GAP). Here we hypothesize that a RhoGAP could participate to RhoA activation induced by ANG II in rat aortic VSMC. The knockdown of the RhoGAP p190A by small interfering RNA (siRNA) abolishes the activation of RhoA-Rho kinase pathway induced after 5 min of ANG II (0.1 μM) stimulation in rat aortic VSMC. We then show that AT1R activation induces p190A dephosphorylation and inactivation. In addition, expression of catalytically inactive or phosphoresistant p190A mutants increases the basal activity of RhoA-Rho kinase pathway, whereas phosphomimetic mutant inhibits early RhoA activation by ANG II. Using siRNA and mutant overexpression, we then demonstrate that the tyrosine phosphatase SHP2 is necessary for 1) maintaining p190A basally phosphorylated and activated by the tyrosine kinase c-Abl, and 2) inducing p190A dephosphorylation and RhoA activation in response to AT1R activation. Our work then defines p190A as a new mediator of RhoA activation by ANG II in VSMC.

SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension

2004 ◽  
Vol 286 (5) ◽  
pp. H1954-H1962 ◽  
Author(s):  
Mohammed El Mabrouk ◽  
Quy N. Diep ◽  
Karim Benkirane ◽  
Rhian M. Touyz ◽  
Ernesto L. Schiffrin
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Kinase Activity ◽  
Muscle Cells ◽  
Regulatory Subunit ◽  
Ang Ii ◽  
Wistar Kyoto

We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85α and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85α and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85α-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR.

20-Hydroxyeicosatetraenoic Acid Mediates Angiotensin Ii-Induced Phospholipase D Activation in Vascular Smooth Muscle Cells

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 723-723
Author(s):  
Jean-Hugues Parmentier ◽  
Mubarack M Muthalif
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Map Kinase ◽  
Vascular Smooth Muscle Cells ◽  
Ang Ii ◽  
Hydroxyeicosatetraenoic Acid ◽  
Map Kinase Pathway ◽  
Kinase Pathway

P164 Angiotensin II (Ang II) promotes arachidonic acid (AA) release via activation of cytosolic phospholipase A2 (cPLA 2 ) and D (PLD) in rabbit vascular smooth muscle cells (VSMC). Ang II also stimulates the Ras/MAP kinase pathway, and MAP kinase activates cPLA 2 and PLD in VSMC. The increase in Ras/MAP kinase activity elicited by Ang II in VSMC is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and to a lesser extent by 12(S)-HETE, metabolites of AA generated by cytochrome P450 (CYP) and lipoxygenase (LO), respectively, upon activation of cPLA 2 by calcium calmodulin-dependent kinase II. The purpose of this study was to determine if Ang II-induced PLD activation stimulation in VSMC from rabbit aorta is mediated by the Ras/MAP kinase pathway via AA metabolites generated by cPLA 2 . Ang II (100 nM) increased PLD activity measured as the production of 3 H-phosphatidylethanol from phospholipids in VSMC prelabeled with 3 H-oleic. Inhibitors of PLD (C 2 -ceramide, 10 μM), phosphatidate phosphohydrolase (propranolol, 10 μM) and diacylglycerol lipase (RHC 80267, 10 μM) attenuated Ang II-induced AA release. PLD activity was not altered by propranol and RHC 80267. Therefore, Ang II-induced AA release is partly mediated through PLD activation. Ang II-induced PLD activation was decreased by MAFP (50 μM), a cPLA 2 inhibitor. Inhibitors of lipoxygenase (baicalein, 5 μM) and CYP (ODYA, 5 μM) but not cycloxygenase (indomethacin, 10 μM) attenuated Ang II-induced PLD activation. AA metabolites of CYP, 20-HETE (0.25-1 μM) and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III, 25 μM, 18 hr) and MAP kinase kinase (UO126, 10 μM) significantly attenuated the increase in PLD activity elicited by 20-HETE. These data suggest that the CYP metabolite, 20-HETE, generated from AA following cPLA 2 activation by Ang II, stimulates Ras/MAP kinase pathway, which in turn activates PLD and release additional AA.

Angiotensin II modulates frizzled-2 receptor expression in rat vascular smooth muscle cells

2005 ◽  
Vol 108 (6) ◽  
pp. 523-530 ◽  
Author(s):  
Giovanna CASTOLDI ◽  
Serena REDAELLI ◽  
Willy M. M. van de GREEF ◽  
Cira R. T. di GIOIA ◽  
Giuseppe BUSCA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Ang Ii ◽  
Aortic Smooth Muscle

Ang II (angiotensin II) has multiple effects on vascular smooth muscle cells through the modulation of different classes of genes. Using the mRNA differential-display method to investigate gene expression in rat aortic smooth muscle cells in culture in response to 3 h of Ang II stimulation, we observed that Ang II down-regulated the expression of a member of the family of transmembrane receptors for Wnt proteins that was identified as Fzd2 [Fzd (frizzled)-2 receptor]. Fzds are a class of highly conserved genes playing a fundamental role in the developmental processes. In vitro, time course experiments demonstrated that Ang II induced a significant increase (P<0.05) in Fzd2 expression after 30 min, whereas it caused a significant decrease (P<0.05) in Fzd2 expression at 3 h. A similar rapid up-regulation after Ang II stimulation for 30 min was evident for TGFβ1 (transforming growth factor β1; P<0.05). To investigate whether Ang II also modulated Fzd2 expression in vivo, exogenous Ang II was administered to Sprague–Dawley rats (200 ng·kg−1 of body weight·min−1; subcutaneously) for 1 and 4 weeks. Control rats received normal saline. After treatment, systolic blood pressure was significantly higher (P<0.01), whereas plasma renin activity was suppressed (P<0.01) in Ang II- compared with the saline-treated rats. Ang II administration for 1 week did not modify Fzd2 expression in aorta of Ang II-treated rats, whereas Ang II administration for 4 weeks increased Fzd2 mRNA expression (P<0.05) in the tunica media of the aorta, resulting in a positive immunostaining for fibronectin at this time point. In conclusion, our data demonstrate that Ang II modulates Fzd2 expression in aortic smooth muscle cells both in vitro and in vivo.

K depletion alters angiotensin II receptor expression in vascular smooth muscle cells

1990 ◽  
Vol 258 (5) ◽  
pp. C849-C854 ◽  
Author(s):  
S. L. Linas ◽  
R. Marzec-Calvert ◽  
M. E. Ullian
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Receptor Expression ◽  
Ang Ii ◽  
Surface Receptors ◽  
K Depletion

Dietary K depletion (KD) results in increases in the number of angiotensin II (ANG II) receptors and prevents ANG II-induced downregulation of ANG II receptors in membrane preparations of vessels from KD animals. Because dietary KD results in changes in factors other than K, we K depleted vascular smooth muscle cells (VSMC) in culture to determine the specific effects of KD on ANG II receptor expression and processing. Scatchard analysis of ANG II uptake at 4 degrees C revealed that the number of surface receptors was increased by 37% in cells in which K had been reduced by 45%. This increase also occurred in the presence of cycloheximide. To determine the effect of KD on receptor processing, we measured the number of surface receptors after exposure to ANG II in concentrations sufficient to cause down-regulation. After 30-min exposure to ANG II, the number of surface receptors was reduced by 63% in control cells but only 33% in KD cells. Thirty minutes after withdrawing ANG II, surface binding returned to basal levels in control cells but was still reduced by 20% in KD cells. To determine the functional significance of impaired receptor processing, we measured ANG II uptake at 21 degrees C. Uptake at 21 degrees C depends on the functional number of receptors, i.e., the absolute number of surface receptors and the rate at which receptors are recycled to the surface after ANG II binding. ANG II uptake at 21 degrees C was reduced by 50% in KD cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle

1994 ◽  
Vol 266 (5) ◽  
pp. C1406-C1420 ◽  
Author(s):  
B. S. Dixon ◽  
R. V. Sharma ◽  
T. Dickerson ◽  
J. Fortune
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Ang Ii ◽  
Kinase C ◽  
Membrane Bound

The effects of bradykinin (BK) and angiotensin II (ANG II) were compared in cultured rat mesenteric arterial smooth muscle cells. BK and ANG II activated a phosphoinositide-specific phospholipase C, leading to the rapid release of [3H]inositol phosphates, an increase in intracellular calcium, and formation of sn-1,2-diacylglycerol (DAG). DAG formation was biphasic with a transient peak at 5 s followed by a sustained increase from 60 to 600 s. The BK-mediated increases in inositol triphosphate and DAG were dose dependent with half-maximal increases at concentrations of 5 and 2 nM, respectively. Both hormones were found to activate protein kinase C (PKC) as assessed by phosphorylation of the 68- to 72-kDa intracellular PKC substrate myristoylated alanine-rich C kinase substrate. However, despite similar phosphorylation of this substrate, only ANG II produced a significant increase in membrane-bound PKC activity. The mechanism accounting for the inability of BK to increase membrane-bound PKC activity is unclear. Our studies excluded differential translocation of PKC to the nuclear membrane, production of an inhibitor of membrane-bound PKC activity, and expression of BK and ANG II receptors on different cells as the mechanism. Vascular smooth muscle cells were found to express at least four different PKC isozymes: alpha, delta, zeta, and a faint band for epsilon. All of the isozymes except zeta-PKC were translocated by treatment with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. However, neither ANG II nor BK produced significant translocation of any measured isozyme; therefore, we could not exclude the possibility that ANG II and BK activate different isozymes of PKC. Both hormones were found to have a similar small and inconsistent effect in stimulating [3H]thymidine incorporation. These observations demonstrate that BK and ANG II have similar biochemical effects on vascular smooth muscle cells and imply that, in selected vessels, the vasodilatory effects of BK mediated by the endothelium may be partially counterbalanced by a vasoconstrictor effect on the underlying vascular smooth muscle cells.

Kinin B1 receptor upregulation by angiotensin II and endothelin-1 in rat vascular smooth muscle cells: receptors and mechanisms

2010 ◽  
Vol 299 (5) ◽  
pp. H1625-H1632 ◽  
Author(s):  
Marielle Morand-Contant ◽  
Madhu B. Anand-Srivastava ◽  
Réjean Couture
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Protein Expression ◽  
Smooth Muscle Cells ◽  
Receptor Antagonist ◽  
Vascular Smooth Muscle Cells ◽  
Ang Ii ◽  
Phosphatidylinositol 3 Kinase

Oxidative stress upregulates the kinin B1 receptor (B1R) in diabetes and hypertension. Since angiotensin II (ANG II) and endothelin-1 (ET-1) are increased in these disorders, this study aims at determining the role of these two prooxidative peptides in B1R expression in rat vascular smooth muscle cells (VSMC). In the A10 cell line and aortic VSMC, ANG II enhanced B1R protein expression in a concentration- and time-dependent manner (maximal at 1 μM and 6 h). In A10 cells, ANG II (1 μM) also increased B1R mRNA expression at 3 h and the activation of induced B1R with the agonist [Sar-d-Phe8]-des-Arg9-BK (10 nM, 5 min) significantly enhanced mitogen -activated protein kinase (MAPK1/2) phosphorylation. The enhancing effect of ANG II on B1R protein expression in A10 cells was normalized by the AT1 (losartan) but not by the AT2 (PD123319) receptor antagonist. Furthermore, it was inhibited by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and NF-κB (MG132) but not of MAPK (PD098059). Whereas the ETB receptor antagonist (BQ788) had no effect, the ETA receptor antagonist (BQ123) blocked the effect of ANG II at 6–8 h but not at an early time point. BQ123 and BQ788 also blocked the increasing effect of ET-1 on B1R protein expression. Antioxidants ( N-acetyl-l-cysteine and diphenyleneiodonium) abolished ANG II- and ET-1-increased B1R protein expression. In conclusion, B1R induction is linked to oxidative stress and activation of phosphatidylinositol 3-kinase and NF-κB. The newly synthesized B1R is functional and can activate MAPK signaling in VSMC. The effect of ANG II is mediated by the AT1 receptor and the subsequent activation of ETA through ET-1 release.

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