(Hypertension. 2000;35:313.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Department of Biochemistry (T.Y., K.N., Y.Y., S.I., T.I.), Vanderbilt University School of Medicine, Nashville, Tenn; the 3rd Department of Internal Medicine (T.Y., H.S.) and Department of Dermatology (Y.Y.), Yokohama City University School of Medicine, Yokohama, Japan; Neurobiology of Aging Laboratories (S.T.), Mt. Sinai School of Medicine, New York, NY; and the Department of Anatomy and Physiology (E.D.M.), Meharry Medical College, Nashville, Tenn.
Correspondence to Dr Tadashi Yamakawa, 3rd Department of Internal Medicine, Yokohama City University School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama, 236-0004, Japan. E-mail Yamakat{at}med.yokohama-cu.ac.jp
| Abstract |
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Key Words: angiotensin II hypertrophy G proteins
| Introduction |
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The low-molecular-weight G protein Rho is a member of the Rho family of small GTPases that also includes Rac and Cdc42. These GTPases act as molecular switches to regulate cellular functions, the best characterized of which are changes in the actin cytoskeleton. An increasing body of evidence has revealed that some heterotrimeric G-proteincoupled receptors (GPCRs) signal through small G proteins, such as Rho. GPCR agonists, such as lysophosphatidic acid7 and carbachol,8 have been shown to increase levels of membrane-associated Rho or decrease cytosolic Rho, which is indicative of Rho activation. Very recently, it has been reported that Rho and Rho-kinase mediate thrombin-stimulated VSMC DNA synthesis and migration.9 The AT1 receptor is one of the GPCRs whose activation of the AT1 receptor is also known to activate Rho. However, the roles of downstream signaling and of Rho and Rho-kinase on Ang IIinduced vascular hypertrophy remain to be determined.
To gain insight into the mechanism of vascular hypertrophy induced by Ang II, we examined the roles of Rho and Rho-kinase on Ang IIinduced leucine uptake in VSMCs.
| Methods |
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Cell Culture
VSMCs were prepared from the thoracic aorta of 12-week-old Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Mass) by the explant method as previously described.10 Subcultured VSMCs from passages 3 to 15, used in the experiments, showed >99% positive immunostaining against smooth muscle
-actin antibodies and were negative for mycoplasma infection. For subsequent experiments, cells at
80% confluence in culture wells were used 1 day after serum depletion.
Subcellular Fractionation
Cell-free lysates were prepared by adding 100 µL hypotonic lysis buffer (per 60-mm dish) containing 20 mmol/L Tris (pH 8.0), 3 mmol/L MgCl2, 0.4 mmol/L AEBSF, 5 µg/mL aprotinin, 2 µg/mL trypsin inhibitor, and 20 µmol/mL leupeptin. After 3 cycles of freeze and thaw, samples were centrifuged at 100 000g at 4°C for 60 minutes. The supernatant was saved as a "soluble" fraction. Pellets were washed twice with the same lysis buffer and resuspended in 100 µL of the lysis buffer supplemented with 1% Triton X-100 and 0.1% SDS. Cell debris was separated by centrifugation at 14 000 rpm for 20 minutes at 4°C, and supernatant was saved as a "particulate" fraction. The protein content of each fraction was determined by the Lowry method.
Preparation of Cell Extracts and Western Blotting
VSMCs were stimulated with agonists for a specified duration. Then cells were lysed with ice-cold lysis buffer, pH 7.4, containing 500 mmol/L HEPES, 5 mmol/L EDTA, 50 mmol/L NaCl, 1% Triton X-100, a mixture of protease inhibitors, and 1 mmol/L sodium orthovanadate. Solubilized proteins were centrifuged at 14 000g for 30 minutes, and supernatants were stored at -80°C. Proteins (25 µg) were separated by SDS-PAGE, followed by Western blot analysis with the use of indicated antibodies and the ECL detection system (Amersham).
[3H]Leucine Incorporation
Subconfluent cells in 12-well plates were incubated for 24 hours in serum-free DMEM in the absence or presence of the stimuli. During the last 6 hours of incubation, [3H]leucine at 2 µCi per well was added. Thereafter, cells were washed twice with ice-cold PBS and incubated in 1 mL of 5% trichloroacetic acid for 30 minutes at 4°C. Cells were washed twice with 5% trichloroacetic acid and solubilized in 1 mol/L NaOH for 30 minutes at 37°C. After neutralization, solubilized proteins were counted by a scintillation counter.
Isolation of Total RNA and Northern Blot Hybridization
Northern blot analysis was performed as described previously.10 VSMCs were stimulated with agonists for the indicated duration, and total RNA was isolated by a 1-step preparation. Total RNA (20 µg) was size-separated by electrophoresis on 1% agarose/formaldehyde gels and then transferred to Hybond-N membranes (Amersham). Hybridization was carried out at 65°C overnight with the solution containing 1 mol/L NaCl, 10% dextran, 1% SDS, 0.1 mg/mL denatured salmon sperm DNA, and a
-32Plabeled denatured c-fos probe. The membranes were washed and then exposed to x-ray film.
Statistics
Data are given as mean±SE. Statistical analyses were performed by ANOVA. A post hoc test was performed by the method of Bonferroni (Glantz 11 ). Significance was accepted at the P<0.05 level.
| Results |
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Effect of C3 Exoenzyme on Ang IIInduced Vascular Hypertrophy
Exoenzyme C3 has been a useful and well-established tool in studying the function of Rho, because it causes ADP-ribosylation at Asn41 of Rho, which results in specific inactivation of Rho.13 To clarify the cellular mechanism by which Ang II induces vascular hypertrophy, we investigated the possible involvement of RhoA cascades because they are known to be activated by Ang II in cardiac myocytes.14 VSMCs were treated with 4 µg/mL of C3 exoenzyme for 48 hours. Treatment with C3 exoenzyme significantly attenuated the leucine incorporation stimulated by Ang II (Figure 1B), suggesting that the small G protein, RhoA, is involved in the vascular hypertrophy induced by Ang II.
Involvement of Rho-Kinase on Ang IIInduced Leucine Uptake
Recently, Uehata et al15 have developed the specific inhibitor of Rho-kinase, Y-27632. To investigate the targets of Rho in protein synthesis induced by Ang II, we examined the effect of 0.5 to 10 µmol/L Y-27632 on Ang IIinduced leucine uptake in VSMCs. Pretreatment of the cells with Y-27632 dose-dependently suppressed the leucine incorporation induced by Ang II (Figure 1C), although Y-27632 itself had no effect on the number of the cells (data not shown).
Effect of the Rho-Kinase Inhibitor on c-fos mRNA Expression
Rapid induction of nuclear proto-oncogenes, such as c-fos, is one of the earliest transcriptional events and has been associated with cellular proliferation, differentiation, and hypertrophy.16 To gain insight into Ang IIinduced proto-oncogene c-fos mRNA expression, we extracted mRNA from VSMCs stimulated by Ang II with or without pretreatment with Y-27632. As shown in Figure 2, Ang II induced a c-fos transcription, which is not expressed in quiescent cells. Ang IIinduced c-fos mRNA expression was slightly, but statistically significantly, suppressed by Y-27632.
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Effect of Y-27632 on ERK1/2 and the p70 S6 Kinase Activation Pathway
To delineate the role of Rho-kinase on Ang IIinduced leucine incorporation, we examined the effect of Y-27632 on ERK1/2 and p70 S6 kinase, which are reported to be involved in vascular hypertrophy. As previously described, Ang IIinduced ERK1/2 phosphorylation peaked at 7 minutes,17 and p70 S6 kinase phosphorylation peaked at 20 minutes (data not shown). Both of them rapidly declined to the basal level. Therefore, after pretreatment with Y-27632, we determined the levels of phosphorylation of ERK1/2 and p70 S6 kinase induced by Ang II only at peak points. Y-27632 had no effect on the ERK1/2 and p70 S6 kinase phosphorylations induced by Ang II (Figure 3).
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Effect of Y-27632 on Ang IIInduced PHAS-I Phosphorylation
PHAS-I generally appears as 3 migrating bands when separated by SDS-PAGE and analyzed by immunoblotting. The nonphosphorylated
form migrates most rapidly. Two others are designated as ß and
.18 19 Increases in phosphorylation of the intermediate ß form to the most highly phosphorylated
form slow migration of PHAS-I proportionally when separated by SDS-PAGE. In unstimulated conditions, the nonphosphorylated
form of PHAS-I in VSMCs is barely detectable. Figure 4A shows a rapid increase in the phosphorylation of PHAS-I after the addition of 100 nmol/L Ang II in quiescent rat cultured VSMCs, where maximal phosphorylation occurred 10 to 20 minutes after Ang II addition and then gradually decreased to basal levels. Figure 4B shows that the phosphorylation of PHAS-I after 10 and 20 minutes of exposure to Ang II was not inhibited by pretreatment with 10 µmol/LY-27632
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| Discussion |
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Because Rho is one of the small GTP-binding proteins, activation of Rho could be reflected in increased binding of GTP. However, neither guanine nucleotide binding assays nor determinations of the guanine nucleotide exchange activity for Rho have been technically feasible to date because the available antibodies are not suitable for immunoprecipitating the native guanine nucleotide bound form of Rho20 in the presence of Mg2+. Because Mg2+ is essential for maintaining the guanine nucleotide binding of Rho,21 it is difficult to immunoprecipitate Rho while preserving its guanine nucleotide binding. It was recently shown that several growth factors, including norepinephrine,22 lysophosphatidic acid, endothelin,7 and insulin,23 cause translocation of a fraction of Rho from the cytoplasmic to the particulate fractions in various cell types, leading to a 1.5- to 2-fold increase in the RhoA content in the particulate fraction. Ang II was also reported to induce RhoA translocation in cardiac myocytes.14 In the present study, we found that Ang II induced RhoA translocation in VSMCs, suggesting that Ang II activates Rho A in VSMCs.
Pretreatment with exoenzyme C3 significantly attenuated the leucine incorporation stimulated with Ang II (Figure 1B), suggesting that the small G protein, RhoA, was involved in the vascular hypertrophy induced by Ang II. Although Ang II induced the translocation of RhoA (Figure 1A), the downstream signaling pathway connecting Rho to protein synthesis remains unclear. Recently, several targets of Rho have been identified, including protein kinase N,24 Rho-kinase,25 and citron-kinase.26 However, precise roles of these proteins are not yet known. Thus, it is interesting to determine which target proteins are related to Ang IIinduced protein synthesis in VSMCs. By using Y-27632, which specifically suppresses Rho-kinase,15 we showed that Rho-kinase plays a role in Ang IIinduced leucine uptake in VSMCs. Figure 1C suggested that Rho-kinase is involved in Ang IIinduced vascular hypertrophy. Rho-kinase may mediate plural pathways from Rho and may function in cooperation with other Rho targets. The identification of physiological substrates for Rho-kinase is expected to shed light on the molecular mechanism of Ang IIinduced vascular hypertrophy. To date, several proteins, such as the myosin-binding subunit of myosin phosphatase, myosin light chain,27 and glial fibrillary acidic protein,28 have been identified as physiological substrates for Rho-kinase. Further studies are necessary to identify Rho-kinase substrates that might be associated with vascular hypertrophy.
Several potential mechanisms for the regulation of Ang IIinduced leucine uptake were investigated in the present study. Ang II activates multiple second-messenger systems in VSMCs,29 and each signaling molecule seems to mediate distinct hypertrophic responses. ERK1/2 and p70 S6 kinase have been reported to be closely related to protein synthesis in VSMCs. However, the relation between these kinases and Rho-kinase has not been clearly identified. Recently, we reported that Rho-kinase was involved in mechanical stressinduced ERK1/2 activation in VSMCs.30 Because Ang II activates the mitogen-activated protein kinase pathways in VSMCs31 and because ERK1/2 is regarded as a critical mediator for cell growth and vascular hypertrophy, we examined whether Rho-kinase might be involved in Ang IIinduced ERK1/2 activation. Y-27632 had no effect on Ang IIinduced ERK1/2 phosphorylation. In addition, Y-27632 had no effect on Ang IIinduced p70 S6 kinase phosphorylation. These observations suggest that Rho and Rho-kinase regulate Ang IIinduced protein synthesis mediated by a pathway different from ERK1/2 or p70 S6 kinase.
It was reported that the Ang IIinduced expression of the c-fos gene was mediated through activation of ERK1/2.32 However, recent studies have demonstrated that Rho proteins are involved in transcriptional activation of the c-fos gene by regulating serum response factor (SRF) in several cell types, including cardiac myocytes, and that the activation of the SRF-linked signaling pathway is not correlated with the activation of ERK1/2.33 34 In practice, Ang IIinduced c-fos mRNA expression in VSMCs was not suppressed completely by the treatment with the MEK-1, a direct upstream of ERK1/2, inhibitor PD98059.35 Thus, it is important to define the c-fos activation mechanism by Ang II and, in particular, to determine whether RhoA and Rho-kinase play roles in this signaling pathway. In the present study, we showed that Rho-kinase is partially involved in Ang IIinduced expressions of the c-fos gene, although Rho-kinase was not involved in Ang IIinduced ERK1/2 phosphorylation (Figure 3A). It is possible that Rho-kinase activation by Ang II affects the protein levels of SRF or SRF binding activity to the c-fos SRE. Further studies are needed to examine the effect of Y-27632 on the levels of SRF and binding activity of SRF to the c-fos SRE in VSMCs. On the basis of the present results, we submit that the Ang IIinduced expression of the c-fos gene might be partially mediated through activation of Rho, Rho-kinase, and SRF, but not ERK1/2, in VSMCs.
The initiation phase of mRNA translation is generally rate limiting for protein synthesis.36 37 Initiation is mediated in part by the eIF-4F complex, which is composed of 3 subunits, eIF-4
, eIF-4A, and eIF-4E.37 The initiation factor, eIF-4E, is the least abundant component of the eIF-4F subunits, and it is generally believed that the amount of eIF-4E is limiting for translation initiation. The availability of eIF-4E is regulated by PHAS-I, initially identified in rat adipocytes.38 When phosphorylated in the appropriate sites after exposure of responsive cells to insulin, PHAS-I dissociates from eIF-4E, thus allowing eIF-4E to participate in translation initiation. Ang II induces phosphorylation of eucaryotic protein synthesis initiation factor 4E in VSMCs.39 In the present study, we tried to determine whether Rho-kinase is involved in Ang IIinduced PHAS-I phosphorylation. Pretreatment of VSMCs with a high concentration of Y-27632 had no effect on Ang IIinduced PHAS-I phosphorylation, suggesting that the role of Rho-kinase in Ang IIinduced protein synthesis might not be due to the modulation of PHAS-I phosphorylation in VSMCs.
Details of the mechanisms of Rho-kinase in Ang IIinduced vascular hypertrophy remain unclear. The present study provides the first evidence that Rho and Rho-kinase may play an important role in Ang IIinduced vascular hypertrophy. We need to investigate the upstream and downstream Rho and Rho-kinase pathways in VSMCs.
| Acknowledgments |
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Received September 14, 1999; first decision October 29, 1999; accepted November 15, 1999.
| References |
|---|
|
|
|---|
2.
Owens GK, Schwartz SM. Alterations in vascular smooth muscle mass in the spontaneously hypertensive rat: role of cellular hypertrophy, hyperploidy, and hyperplasia. Circ Res. 1982;51:280289.
3.
Griffin SA, Brown WC, MacPherson F, McGrath JC, Wilson VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II causes vascular hypertrophy in part by a nonpressor mechanism. Hypertension. 1991;17:626635.
4. Azuma H, Niimi Y, Hamasaki H. Alpha 1-adrenoceptor antagonist activity of novel pyrimidine derivatives (SHI437 and IK29) in rabbit aorta and trigone of the bladder. Br J Pharmacol. 1989;106:665671.[Medline] [Order article via Infotrieve]
5.
Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988;62:749756.
6.
Berk BC, Vekshtein V, Gordon H, Tsuda T. Angiotensin IIstimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305314.
7.
Fleming IN, Elliott CM, Exton JH. Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem. 1996;271:3306733073.
8. Keller J, Schmidt M, Hussein B, Rumenapp U, Jakobs KH. Muscarinic receptor-stimulated cytosol-membrane translocation of Rho-A. FEBS Lett.. 1997;403:299302.[Medline] [Order article via Infotrieve]
9.
Seasholtz TM, Majumdar M, Kaplan DD, Brown JH. Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res. 1999;84:11861193.
10. Yamakawa T, Eguchi S, Yamakawa Y, Motley ED, Numaguchi K, Utsunomiya H, Inagami T. Lysophosphatidylcholine stimulates MAP kinase activity in rat vascular smooth muscle cells. Hypertension. 1998;31(pt 2):248253.
11. Glantz S. How to use t tests to isolate differences between groups in analysis of variance. In: Primer of Biostatistics. New York, NY: McGraw-Hill;1987:8889.
12.
Bokoch GM, Bohl BP, Chuang TH. Guanine nucleotide exchange regulates membrane translocation of Rac/Rho GTP-binding proteins. J Biol Chem. 1994;269:3167431679.
13.
Sekine A, Fujiwara M, Narumiya S. Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem. 1989;264:86028605.
14.
Aoki H, Izumo S, Sadoshima J. Angiotensin II activates RhoA in cardiac myocytes a critical role of RhoA in angiotensin IIinduced premyofibril formation. Circ Res. 1998;82:666676.
15. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389:990994.[Medline] [Order article via Infotrieve]
16. Verma IM, Sassone-Corsi J. Proto-oncogene fos: complex but versatile regulation. Cell. 1987;51:513514.[Medline] [Order article via Infotrieve]
17.
Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. J Biol Chem. 1996;271:1416914175.
18.
Lin TA, Kong X, Saltiel AR, Blackshear PJ, Lawrence JJ. Control of PHAS-I by insulin in 3T3L1 adipocytes: synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway. J Biol Chem.. 1995;270:1853118538.
19. Beretta L, Gingras AC, Svitkin YV, Hall MN, Sonenberg N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 1996;15:658664.[Medline] [Order article via Infotrieve]
20. Lang P, Gesbert F, Delespine CM, Stancou R, Pouchelet M, Bertoglio J. Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J. 1996;15:510519.[Medline] [Order article via Infotrieve]
21. Aktories K, Just I. In vitro ADP-ribosylation of Rho by bacterial ADP-ribosyltransferases. Methods Enzymol. 1995;256:184195.[Medline] [Order article via Infotrieve]
22.
Gong MC, Fujihara H, Somlyo AV, Somlyo AP. Translocation of rhoA associated with Ca2+ sensitization of smooth muscle. J Biol Chem. 1997;272:1070410709.
23.
Karnam P, Standaert ML, Galloway L, Farese RV. Activation and translocation of Rho (and ADP ribosylation factor) by insulin in rat adipocytes: apparent involvement of phosphatidylinositol 3-kinase. J Biol Chem. 1997;272:61366140.
24. Amano M, Mukai H, Ono Y, Chihara K, Matsui T, Hamajima Y, Okawa K, Iwamatsu A, Kaibuchi K. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N. Science. 1996;271:648650.[Abstract]
25. Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 1996;15:22082216.[Medline] [Order article via Infotrieve]
26. Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H, Ishizaki T, Narumiya S. Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature. 1998;394:491494.[Medline] [Order article via Infotrieve]
27.
Chihara K, Amano M, Nakamura N, Yano T, Shibata M, Tokui T, Ichikawa H, Ikebe R, Ikebe M, Kaibuchi K. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase. J Biol Chem. 1997;272:2512125127.
28.
Kosako H, Amano M, Yanagida M, Tanabe K, Nishi Y, Kaibuchi K, Inagaki M. Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase. J Biol Chem. 1997;272:1033310336.
29. Inagami T, Eguchi S, Numaguchi K, Motley ED, Tang H, Matsumoto T, Yamakawa T. Cross-talk between angiotensin II receptors and the tyrosine kinases and phosphatases. J Am Soc Nephrol. 1999;10:S57S61.
30.
Numaguchi K, Eguchi S, Yamakawa T, Motley ED, Inagami T. Mechanotransduction of rat vascular smooth muscle cells requires RhoA and intact actin filaments. Circ Res. 1999;85:511.
31.
Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem. 1998;273:88908896.
32. Xu Q, Liu Y, Gorospe M, Udelsman R, Holbrook NJ. Acute hypertension activates mitogen-activated protein kinases in arterial wall. J Clin Invest. 1996;97:508514.[Medline] [Order article via Infotrieve]
33. Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell. 1995;81:11591170.[Medline] [Order article via Infotrieve]
34.
Ueyama T, Sakoda T, Kawashima S, Hiraoka E, Hirata K, Akita H, Yokoyama M. Activated RhoA stimulates c-fos gene expression in myocardial cells. Circ Res. 1997;81:672678.
35.
Xi XP, Graf K, Goetze S, Fleck E, Hsueh WA, Law RE. Central role of the MAPK pathway in Ang IImediated DNA synthesis and migration in rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999;19:7382.
36. Morley SJ. Intracellular signalling pathways regulating initiation factor eIF4E phosphorylation during the activation of cell growth. Biochem Soc Trans. 1997;25:503509.[Medline] [Order article via Infotrieve]
37. Proud CG. Protein phosphorylation in translational control. Curr Top Cell Regul. 1992;32:243369.[Medline] [Order article via Infotrieve]
38.
Lin T-A, Kong X, Haystead TAJ, Pause A, Belsham G, Sonenberg N, Lawrence JCJ. PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science. 1994;266:653656.
39.
Rao GN, Griendling KK, Frederickson RM, Sonenberg N, Alexander RW. Angiotensin II induces phosphorylation of eukaryotic protein synthesis initiation factor 4E in vascular smooth muscle cells. J Biol Chem. 1994;269:71807184.
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M. Ruiz-Ortega, M. Ruperez, V. Esteban, J. Rodriguez-Vita, E. Sanchez-Lopez, G. Carvajal, and J. Egido Angiotensin II: a key factor in the inflammatory and fibrotic response in kidney diseases Nephrol. Dial. Transplant., January 1, 2006; 21(1): 16 - 20. [Abstract] [Full Text] [PDF] |
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H. Ohtsu, M. Mifune, G. D. Frank, S. Saito, T. Inagami, S. Kim-Mitsuyama, Y. Takuwa, T. Sasaki, J. D. Rothstein, H. Suzuki, et al. Signal-Crosstalk Between Rho/ROCK and c-Jun NH2-Terminal Kinase Mediates Migration of Vascular Smooth Muscle Cells Stimulated by Angiotensin II Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1831 - 1836. [Abstract] [Full Text] [PDF] |
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H. Shimokawa and A. Takeshita Rho-Kinase Is an Important Therapeutic Target in Cardiovascular Medicine Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1767 - 1775. [Abstract] [Full Text] [PDF] |
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U. G. B. Haider, T. U. Roos, M. I. Kontaridis, B. G. Neel, D. Sorescu, K. K. Griendling, A. M. Vollmar, and V. M. Dirsch Resveratrol Inhibits Angiotensin II- and Epidermal Growth Factor-Mediated Akt Activation: Role of Gab1 and Shp2 Mol. Pharmacol., July 1, 2005; 68(1): 41 - 48. [Abstract] [Full Text] [PDF] |
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K. Budzyn, P. D. Marley, and C. G. Sobey Opposing Roles of Endothelial and Smooth Muscle Phosphatidylinositol 3-Kinase in Vasoconstriction: Effects of Rho-Kinase and Hypertension J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1248 - 1253. [Abstract] [Full Text] [PDF] |
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T. Kishi, Y. Hirooka, A. Masumoto, K. Ito, Y. Kimura, K. Inokuchi, T. Tagawa, H. Shimokawa, A. Takeshita, and K. Sunagawa Rho-Kinase Inhibitor Improves Increased Vascular Resistance and Impaired Vasodilation of the Forearm in Patients With Heart Failure Circulation, May 31, 2005; 111(21): 2741 - 2747. [Abstract] [Full Text] [PDF] |
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G. Loirand, M. Rolli-Derkinderen, and P. Pacaud RhoA and resistance artery remodeling Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1051 - H1056. [Abstract] [Full Text] [PDF] |
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Z. Zhu, S. Zhu, D. Liu, T. Cao, L. Wang, and M. Tepel Thiazide-Like Diuretics Attenuate Agonist-Induced Vasoconstriction by Calcium Desensitization Linked to Rho Kinase Hypertension, February 1, 2005; 45(2): 233 - 239. [Abstract] [Full Text] [PDF] |
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K. Lockman, J. S. Hinson, M. D. Medlin, D. Morris, J. M. Taylor, and C. P. Mack Sphingosine 1-Phosphate Stimulates Smooth Muscle Cell Differentiation and Proliferation by Activating Separate Serum Response Factor Co-factors J. Biol. Chem., October 8, 2004; 279(41): 42422 - 42430. [Abstract] [Full Text] [PDF] |
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K. A. Fagan, M. Oka, N. R. Bauer, S. A. Gebb, D. D. Ivy, K. G. Morris, and I. F. McMurtry Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L656 - L664. [Abstract] [Full Text] [PDF] |
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T. Nagaoka, Y. Morio, N. Casanova, N. Bauer, S. Gebb, I. McMurtry, and M. Oka Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L665 - L672. [Abstract] [Full Text] [PDF] |
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D. G. Romero, M. Plonczynski, G. R. Vergara, E. P. Gomez-Sanchez, and C. E. Gomez-Sanchez Angiotensin II early regulated genes in H295R human adrenocortical cells Physiol Genomics, September 16, 2004; 19(1): 106 - 116. [Abstract] [Full Text] [PDF] |
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B.R. Wamhoff, D.K. Bowles, O.G. McDonald, S. Sinha, A.P. Somlyo, A.V. Somlyo, and G.K. Owens L-type Voltage-Gated Ca2+ Channels Modulate Expression of Smooth Muscle Differentiation Marker Genes via a Rho Kinase/Myocardin/SRF-Dependent Mechanism Circ. Res., August 20, 2004; 95(4): 406 - 414. [Abstract] [Full Text] [PDF] |
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K. Budzyn, P. D. Marley, and C. G. Sobey Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R342 - R348. [Abstract] [Full Text] [PDF] |
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T. Yoshida, M. H. Hoofnagle, and G. K. Owens Myocardin and Prx1 Contribute to Angiotensin II-Induced Expression of Smooth Muscle {alpha}-Actin Circ. Res., April 30, 2004; 94(8): 1075 - 1082. [Abstract] [Full Text] [PDF] |
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M. Higashi, H. Shimokawa, T. Hattori, J. Hiroki, Y. Mukai, K. Morikawa, T. Ichiki, S. Takahashi, and A. Takeshita Long-Term Inhibition of Rho-Kinase Suppresses Angiotensin II-Induced Cardiovascular Hypertrophy in Rats In Vivo: Effect on Endothelial NAD(P)H Oxidase System Circ. Res., October 17, 2003; 93(8): 767 - 775. [Abstract] [Full Text] [PDF] |
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A. P. SOMLYO and A. V. SOMLYO Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase Physiol Rev, October 1, 2003; 83(4): 1325 - 1358. [Abstract] [Full Text] [PDF] |
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Y. Gorin, J. M. Ricono, N.-H. Kim, B. Bhandari, G. G. Choudhury, and H. E. Abboud Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells Am J Physiol Renal Physiol, August 1, 2003; 285(2): F219 - F229. [Abstract] [Full Text] [PDF] |
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S. Pelletier, F. Duhamel, P. Coulombe, M. R. Popoff, and S. Meloche Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors Mol. Cell. Biol., February 15, 2003; 23(4): 1316 - 1333. [Abstract] [Full Text] [PDF] |
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C. Yan, D. Kim, T. Aizawa, and B. C. Berk Functional Interplay Between Angiotensin II and Nitric Oxide: Cyclic GMP as a Key Mediator Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 26 - 36. [Abstract] [Full Text] [PDF] |
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N. Kobayashi, S. Horinaka, S.-i. Mita, S. Nakano, T. Honda, K. Yoshida, T. Kobayashi, and H. Matsuoka Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts Cardiovasc Res, September 1, 2002; 55(4): 757 - 767. [Abstract] [Full Text] [PDF] |
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C. Mueller, S. Baudler, H. Welzel, M. Bohm, and G. Nickenig Identification of a Novel Redox-Sensitive Gene, Id3, Which Mediates Angiotensin II-Induced Cell Growth Circulation, May 21, 2002; 105(20): 2423 - 2428. [Abstract] [Full Text] [PDF] |
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N. Kobayashi, S. Nakano, S.-i. Mita, T. Kobayashi, T. Honda, Y. Tsubokou, and H. Matsuoka Involvement of Rho-Kinase Pathway for Angiotensin II-Induced Plasminogen Activator Inhibitor-1 Gene Expression and Cardiovascular Remodeling in Hypertensive Rats J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 459 - 466. [Abstract] [Full Text] [PDF] |
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T. Yamakawa, S.-i. Tanaka, Y. Yamakawa, J. Kamei, K. Numaguchi, E. D. Motley, T. Inagami, and S. Eguchi Lysophosphatidylcholine Activates Extracellular Signal-Regulated Kinases 1/2 Through Reactive Oxygen Species in Rat Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 752 - 758. [Abstract] [Full Text] [PDF] |
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K. G. Lamping Enhanced Contractile Mechanisms in Vasospasm: Is Endothelial Dysfunction the Whole Story? Circulation, April 2, 2002; 105(13): 1520 - 1522. [Full Text] [PDF] |
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C. Kataoka, K. Egashira, S. Inoue, M. Takemoto, W. Ni, M. Koyanagi, S. Kitamoto, M. Usui, K. Kaibuchi, H. Shimokawa, et al. Important Role of Rho-kinase in the Pathogenesis of Cardiovascular Inflammation and Remodeling Induced by Long-Term Blockade of Nitric Oxide Synthesis in Rats Hypertension, February 1, 2002; 39(2): 245 - 250. [Abstract] [Full Text] [PDF] |
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M. Meier, G. L. King, A. Clermont, A. Perez, M. Hayashi, and E. P. Feener Angiotensin AT1 Receptor Stimulates Heat Shock Protein 27 Phosphorylation In Vitro and In Vivo Hypertension, December 1, 2001; 38(6): 1260 - 1265. [Abstract] [Full Text] [PDF] |
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K. Matrougui, L. B. Tanko, L. Loufrani, D. Gorny, B. I. Levy, A. Tedgui, and D. Henrion Involvement of Rho-Kinase and the Actin Filament Network in Angiotensin II-Induced Contraction and Extracellular Signal-Regulated Kinase Activity in Intact Rat Mesenteric Resistance Arteries Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1288 - 1293. [Abstract] [Full Text] [PDF] |
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Y. Funakoshi, T. Ichiki, H. Shimokawa, K. Egashira, K. Takeda, K. Kaibuchi, M. Takeya, T. Yoshimura, and A. Takeshita Rho-Kinase Mediates Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cells Hypertension, July 1, 2001; 38(1): 100 - 104. [Abstract] [Full Text] [PDF] |
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L. Hunyady, Z. Gaborik, G. Vauquelin, and K. J Catt Review: Structural requirements for signalling and regulation of AT1-receptors Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S16 - S23. [PDF] |
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H. Shimokawa, K. Morishige, and A. Takeshita C-type natriuretic peptide and vascular remodeling: Reply J. Am. Coll. Cardiol., January 1, 2001; 37(1): 333 - 334. [Full Text] [PDF] |
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R. J. Solaro Myosin Light Chain Phosphatase : A Cinderella of Cellular Signaling Circ. Res., August 4, 2000; 87(3): 173 - 175. [Full Text] [PDF] |
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A. D Hughes AT 1-signalling in vascular smooth muscle Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 125 - 130. [PDF] |
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D. E. Vatner and D. L. Kunze Prologue: low-molecular-weight GTPases in the heart and circulation Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1733 - H1735. [Full Text] [PDF] |
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C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF] |
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W. Ni, K. Egashira, C. Kataoka, S. Kitamoto, M. Koyanagi, S. Inoue, and A. Takeshita Antiinflammatory and Antiarteriosclerotic Actions of HMG-CoA Reductase Inhibitors in a Rat Model of Chronic Inhibition of Nitric Oxide Synthesis Circ. Res., August 31, 2001; 89(5): 415 - 421. [Abstract] [Full Text] [PDF] |
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