Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells

2003 ◽  
Vol 81 (3) ◽  
pp. 274-287 ◽  
Author(s):  
G Bkaily ◽  
S Sleiman ◽  
J Stephan ◽  
C Asselin ◽  
S Choufani ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Nuclear Translocation ◽  
De Novo ◽  
Ang Ii ◽  
De Novo Synthesis ◽  
Nuclear Compartments

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R –GFP overexpressing hVSMCs, Ang II (10–9 and 10–4 M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R–GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R–GFP overexpressing hVSMCs, Ang II (10–15 to 10–4 M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 × 10–11 and 5 × 10–9 M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.Key words: angiotensin, calcium, protein synthesis, nucleus, AT1 receptor, nuclear de novo synthesis.

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.

Angiotensin II type 1 receptor-mediated activation of Ras in cultured rat vascular smooth muscle cells

1996 ◽  
Vol 271 (2) ◽  
pp. H595-H601 ◽  
Author(s):  
M. Okuda ◽  
Y. Kawahara ◽  
M. Yokoyama
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Smooth Muscle Cells ◽  
Map Kinase ◽  
Vascular Smooth Muscle Cells ◽  
Ang Ii ◽  
Kinase Activation ◽  
Ras Activation ◽  
Fos Expression

Angiotensin II (ANG II), a potent growth-promoting factor of vascular smooth muscle cells (VSMC), induces activation of mitogen-activated protein (MAP) kinases and subsequent expression of the c-fos protooncogene in VSMC. However, it remains obscure whether ANG II induces activation of the ras protooncogene product (Ras), and if it does, whether Ras is involved in signaling from the ANG II receptor to the MAP kinase pathway in VSMC. In cultured VSMC, ANG II activated Ras comparably to epidermal growth factor. ANG II-induced Ras activation was detectable within 1 min and maximal at 2–5 min. The ANG II type 1 (AT1) receptor antagonist, CV-11974, completely inhibited this reaction. Pertussis toxin treatment of VSMC inhibited ANG II-induced Ras activation by approximately 70% but had no effect on ANG II-induced MAP kinase activation and c-fos expression. These results indicate that ANG II activates Ras via AT1 receptors, which are predominantly linked to a G protein of the Gi subfamily in VSMC1 and suggest that Ras activation may not be a prerequisite for ANG II-induced MAP kinase activation and c-fos expression in this cell type.

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.

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.

Role of oxidative stress in angiotensin II-induced enhanced expression of Giα proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells

2007 ◽  
Vol 292 (4) ◽  
pp. H1922-H1930 ◽  
Author(s):  
Yuan Li ◽  
Georgios Lappas ◽  
Madhu B. Anand-Srivastava
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Adenylyl Cyclase ◽  
Vascular Smooth Muscle Cells ◽  
Ang Ii ◽  
Enhanced Expression

We have previously reported that angiotensin II (ANG II) treatment of A10 vascular smooth muscle cells (VSMCs) increased inhibitory G proteins (Gi protein) expression and associated adenylyl cyclase signaling which was attributed to the enhanced MAP kinase activity. Since ANG II has been shown to increase oxidative stress, we investigated the role of oxidative stress in ANG II-induced enhanced expression of Giα proteins and examined the effects of antioxidants on ANG II-induced enhanced expression of Giα proteins and associated adenylyl cyclase signaling in A10 VSMCs. ANG II treatment of A10 VSMCs enhanced the production of O2− and the expression of Nox4 and P47phox, different subunits of NADPH oxidase, which were attenuated toward control levels by diphenyleneiodonium (DPI). In addition, ANG II augmented the expression of Giα-2 and Giα-3 proteins in a concentration- and time-dependent manner; the maximal increase in the expression of Giα was observed at 1 to 2 h and at 0.1–1.0 μM. The enhanced expression of Giα-2 and Giα-3 proteins was restored to control levels by antioxidants such as N-acetyl-l-cysteine, α-tocopherol, DPI, and apocynin. In addition, ANG II also enhanced the ERK1/2 phosphorylation that was restored to control levels by DPI. Furthermore, the inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of 5′- O-(3-triotriphosphate) (receptor-independent Gi functions) and ANG II-, des(Glu18,Ser19,Glu20,Leu21,Gly22)atrial natriuretic peptide4-23-NH2 (natriuretic peptide receptor-C agonist), and oxotremorine-mediated inhibitions of adenylyl cyclase (receptor-dependent functions) that were augmented in ANG II-treated VSMCs was also restored to control levels by antioxidant treatments. In addition, Gsα-mediated diminished stimulation of adenylyl cyclase by stimulatory hormones in ANG II-treated cells was also restored to control levels by DPI. These results suggest that ANG II-induced enhanced levels of Giα proteins and associated functions in VSMCs may be attributed to the ANG II-induced enhanced oxidative stress, which exerts its effects through mitogen-activated protein kinase signaling pathway.

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