Cyclic AMP-mediated Inhibition of Angiotensin II-induced Protein Synthesis Is Associated with Suppression of Tyrosine Phosphorylation Signaling in Vascular Smooth Muscle Cells
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.26879
ISSN1083-351X
AutoresEdith Giasson, Marc J. Servant, Sylvain Meloche,
Tópico(s)Mitochondrial Function and Pathology
ResumoIn the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types. In the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types. Cyclic AMP (cAMP) is a pleiotropic second messenger that has been implicated as a modulator of cell proliferation in several cell types. Intriguingly, depending on the cellular origin and the differentiation state of the cell, cAMP is found to cause either growth inhibition or growth stimulation. For example, elevation of intracellular cAMP stimulates the proliferation of thyrocytes, keratinocytes, epithelial cells, hepatocytes, and Swiss 3T3 cells. On the contrary, elevated cAMP inhibits cell proliferation in fibroblasts, SMC, 1The abbreviations used are: SMC, smooth muscle cell(s); PKA, cAMP-dependent protein kinase; MAP, mitogen-activated protein; Cdk, cyclin-dependent kinase; Ang II, angiotensin II; InsP3, inositol 1,4,5-trisphosphate; ERK, extracellular signal-regulated kinase; IBMX, 3-isobutyl-1-methylxanthine; mAb, monoclonal antibody; MEK, MAP kinase/ERK kinase; kb, kilobase. lymphoid cells, and many tumor cells (for review, see Refs. 1Pastan I.H. Johnson G.S. Anderson W.B. Annu. Rev. Biochem. 1975; 44: 491-522Crossref PubMed Scopus (546) Google Scholar, 2Boynton A.L. Whitfield J.F. Adv. Cyclic Nucleotide Res. 1983; 15: 193-294Google Scholar, 3Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar, 4Dumont J.E. Jauniaux J.C. Roger P.P. Trends Biochem. Sci. 1989; 14: 67-71Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 5Cho-Chung Y.S. Cancer Res. 1990; 50: 7093-7100PubMed Google Scholar). In these cells, cAMP interferes with the mitogenic response to growth factors acting on both receptor tyrosine kinases and G protein-coupled receptors (6Magnaldo I. Pouysségur J. Paris S. FEBS Lett. 1989; 245: 65-69Crossref PubMed Scopus (64) Google Scholar). In addition to their effect on cell proliferation, cAMP analogs can also partially reverse the phenotype of transformed fibroblasts as well as other cancer cells (5Cho-Chung Y.S. Cancer Res. 1990; 50: 7093-7100PubMed Google Scholar, 7Pastan I. Willingham M. Nature. 1978; 274: 645-650Crossref PubMed Scopus (105) Google Scholar). The regulatory effects of cAMP are mediated through activation of the multifunctional cAMP-dependent protein kinase (protein kinase A or PKA) (8Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (959) Google Scholar). These effects are exerted both at the post-translational level and at the transcriptional level through phosphorylation of cAMP-responsive element-binding proteins (CREB/ATF family) (9Roesler W.J. Vandenbark G.R. Hanson R.W. J. Biol. Chem. 1988; 263: 9063-9066Abstract Full Text PDF PubMed Google Scholar, 10Lalli E. Sassone-Corsi P. J. Biol. Chem. 1994; 269: 17359-17362Abstract Full Text PDF PubMed Google Scholar). Although considerable progress has been made in understanding the mechanism of gene regulation by cAMP, little is known about the molecular mechanisms by which the nucleotide modulates cell growth. A number of studies have proposed that cAMP might inhibit cell proliferation by interfering with Ras-dependent activation of MAP kinases (11Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (824) Google Scholar, 12Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 13Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. MacDonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar, 14Sevetson B.R. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10305-10309Crossref PubMed Scopus (345) Google Scholar, 15Burgering B.M.T. Pronk G.J. van Weeren P.C. Charding P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar). Biochemical analysis of the various intermediates in the signaling cascade indicated that cAMP inhibits signal transmission by preventing Ras-dependent activation of the serine/threonine kinase Raf-1 (11Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (824) Google Scholar, 12Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 15Burgering B.M.T. Pronk G.J. van Weeren P.C. Charding P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar). This inhibitory effect of cAMP was mediated by PKA because it was not observed in mutant cells that express a PKA resistant to activation by cAMP (14Sevetson B.R. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10305-10309Crossref PubMed Scopus (345) Google Scholar). However, treatment of CCL39 fibroblasts (16Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar) or interleukin-2-dependent T lymphocytes (17Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (158) Google Scholar) with cAMP-raising agents was found to block cell proliferation completely without affecting growth factor-induced MAP kinase activation. Another study reported that treatment of murine macrophages with analogs of cAMP raises the overall amount of the inhibitor p27 kip1, thereby increasing its association with cyclin D-Cdk4 and preventing the activation of Cdk4 (18Kato J.Y. Matsuoka M. Polyak K. Massagné J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). cAMP was also shown to reduce the accumulation of c-myc mRNA in various cell lines (19Blomhoff H.K. Smeland E.B. Beiske K. Blomhoff R. Ruud E. Bjoro T. Pfeifer-Ohlsson S. Watt R. Funderud S. Godal T. Ohlsson R. J. Cell. Physiol. 1987; 131: 426-433Crossref PubMed Scopus (72) Google Scholar, 20Trepel J.B. Colamonici O.R. Kelly K. Schwab G. Watt R.A. Sausville E.A. Jaffe E.S. Neckers L.M. Mol. Cell. Biol. 1987; 7: 2644-2648Crossref PubMed Scopus (45) Google Scholar, 21Heldin N.R. Paulsson Y. Forsberg K. Heldin C.H. Westermark B. J. Cell. Physiol. 1989; 138: 17-23Crossref PubMed Scopus (68) Google Scholar, 22Rock C.O. Cleveland J.L. Jackowski S. Mol. Cell. Biol. 1992; 12: 2351-2358Crossref PubMed Scopus (53) Google Scholar). In the yeast Saccharomyces cerevisiae, PKA exercises regulatory control on both growth and division, suggesting a role for cAMP in the homeostatic integration of these two processes (23Baroni M.D. Monti P. Alberghina L. Nature. 1994; 371: 339-342Crossref PubMed Scopus (131) Google Scholar, 24Tokiwa G. Tyers M. Volpe T. Futcher B. Nature. 1994; 371: 342-345Crossref PubMed Scopus (164) Google Scholar). It is not known whether cAMP exerts similar control on the overall rate of protein synthesis in mammalian cells under conditions of growth factor stimulation or cellular stress. Ang II is a growth factor for a number of cell types, including adrenocortical cells, proximal tubular cells, vascular SMC, cardiac myocytes, and cardiac fibroblasts (for review, see Refs. 25Schelling P. Fischer H. Ganten D. J. Hypertens. 1991; 9: 3-15Crossref PubMed Google Scholar and 26Pratt R.E. Dzau V.J. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Inc., Boca Raton, FL1993: 471-483Google Scholar). In cultured aortic SMC, Ang II induces cellular hypertrophy as a result of increased protein synthesis but not cell proliferation (27Geisterfer A.A.T. Peach M.J. Owens G.K. Circ. Res. 1988; 62: 749-756Crossref PubMed Scopus (1012) Google Scholar, 28Berk B.C. Vekshtein V. Gordon H.M. Tsuda T. Hypertension. 1989; 13: 305-314Crossref PubMed Scopus (518) Google Scholar, 29Chiu A.T. Roscoe W.A. McCall D.E. Timmermans P.B.M.W.M. Receptor. 1991; 1: 133-140PubMed Google Scholar, 30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The growth-promoting effects of the peptide are mediated by the AT1 receptor subtype, a member of the superfamily of G protein-coupled receptors. Agonist binding to the AT1receptor stimulates the activity of phospholipase C, to generate the second messengers InsP3 and diacylglycerol, and inhibits the activity of adenylyl cyclase (for review, see Refs. 31Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar and 32Catt K.J. Sandberg K. Balla T. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Inc., Boca Raton, FL1993: 307-356Google Scholar). One of the immediate consequences of these early signals is activation of the MAP kinases ERK1/ERK2 (33Servant M.J. Giasson E. Meloche S. J. Biol. Chem. 1996; 271: 16047-16052Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 34Tsuda T. Kawahara Y. Ishida Y. Koide M. Shii K. Yokoyama M. Circ. Res. 1992; 71: 620-630Crossref PubMed Scopus (120) Google Scholar, 35Duff J.L. Berk B.C. Corson M.A. Biochem. Biophys. Res. Commun. 1992; 188: 257-264Crossref PubMed Scopus (197) Google Scholar) and the 70/85-kDa S6 protein kinases (referred to as p70S6K) (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Activation of the AT1 receptor also leads to increased tyrosine phosphorylation of multiple proteins in target cells (36Force T. Kyriakis J.M. Avruch J. Bonventre V. J. Biol. Chem. 1991; 266: 6650-6656Abstract Full Text PDF PubMed Google Scholar, 37Huckle W.R. Prokop C.A. Dy R.C. Herman B. Earp S. Mol. Cell. Biol. 1990; 10: 6290-6298Crossref PubMed Scopus (87) Google Scholar, 38Molloy C.J. Taylor D.S. Weber H. J. Biol. Chem. 1993; 268: 7338-7345Abstract Full Text PDF PubMed Google Scholar, 39Schorb W. Peeler T.C. Madigan N.N. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 19626-19632Abstract Full Text PDF PubMed Google Scholar, 40Leduc I. Haddad P. Giasson E. Meloche S. Mol. Pharmacol. 1995; 48: 582-592PubMed Google Scholar). Despite these observations, the nature of the signaling mechanisms coupling the AT1 receptor to the hypertrophic response remains poorly understood. The aim of this study was to evaluate the effect of increased intracellular levels of cAMP on Ang II-stimulated protein synthesis in vascular SMC. We show that cAMP-raising agents potently inhibit the hypertrophic effect of Ang II. In addition, we demonstrate that increased cAMP selectively antagonizes the stimulatory effect of Ang II on protein tyrosine phosphorylation in these cells. 125I-Labeled [Sar1,Ile8]Ang II (sarile) was prepared by radioiodination of sarile using a solid phase method as described (41Lambert C. Massillon Y. Meloche S. Circ. Res. 1995; 77: 1001-1007Crossref PubMed Scopus (43) Google Scholar). Forskolin, IBMX, Vibrio cholerae toxin and 8-bromo-cAMP were obtained from Calbiochem. Forskolin and IBMX were dissolved in dimethyl sulfoxide to give stock solutions of 100 mm and 500 mm, respectively. Cholera toxin was dissolved in water at a concentration of 1 mg/ml, and 8-bromo-cAMP was dissolved in 10 mm Tris-HCl (pH 7.0) at a concentration of 100 mm. Isoproterenol was a gift of Dr. Michel Bouvier (University of Montreal) and was prepared as a 0.5 msolution in 107 ascorbic acid. The source of other materials has been described (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Antisera SM1 and αIIcp42 have been described and specifically immunoprecipitate the MAP kinases ERK1 and ERK2, respectively (42Meloche S. J. Cell. Physiol. 1995; 163: 577-588Crossref PubMed Scopus (77) Google Scholar, 43Wang Y. Simonson M.S. Pouysségur J. Dunn M.J. Biochem. J. 1992; 287: 589-594Crossref PubMed Scopus (161) Google Scholar). Antiserum S6-24 was produced in rabbits against a synthetic peptide corresponding to amino acids 2–30 of rat p70S6K (Quality Controlled Biochemicals). The anti-p125FAK mAb 2A7 was generously provided by Dr. Thomas Parsons (University of Virginia). The anti-Shc serum was provided by Dr. Louise Larose (McGill University). The anti-paxillin and anti-Pyk2 mAbs were purchased from Transduction Laboratories. The anti-phosphotyrosine mAb 4G10 and anti-Tyk2 polyclonal antibody were obtained from Upstate Biotechnology and Santa-Cruz Biotechnology, respectively. Rat aortic SMC were cultured and synchronized as described previously (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Rat1-AT1 cells are Rat1 fibroblasts stably expressing the human Ang II AT1receptor. 2S. Meloche, M. J. Servant, I. Leduc, and J. Pellerin, submitted for publication. Rat1-AT1 cells were grown in minimum essential medium supplemented with 107 calf serum, 2 mm glutamine, antibiotics, and 0.4 mg/ml Geneticin. They were made quiescent by incubating confluent cell cultures in serum-free Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 15 mmHepes (pH 7.4) and 0.17 bovine serum albumin for 24 h. For experiments with cAMP-raising agents, the cells were treated with vehicle alone or with the indicated concentrations of agents for 30 min before the addition of Ang II. The intracellular mass of cAMP was determined by a specific protein binding assay (44Tovey K.C. Oldham K.G. Whelan J.A.M. Clin. Chim. Acta. 1974; 56: 221-234Crossref PubMed Scopus (468) Google Scholar). Quiescent cells in 35-mm Petri dishes were incubated with the indicated agents for 20 min at 37 °C. After incubation, the medium was removed and the cells washed twice with 1 ml of ice-cold phosphate-buffered saline. The cells were then scraped into 500–1,000 ॖl of cold 50 mm Tris-HCl (pH 7.5), 4 mm EDTA, boiled for 5 min, and centrifuged at 13,000 × g for 5 min at 4 °C. An aliquot of 50–100 ॖl of cell extract was analyzed for cAMP content using a competitive protein binding assay kit as recommended by the manufacturer (Diagnostic Products Corporation). Membranes from rat aortic SMC were prepared as described (45Meloche S. Ong H. Cantin M. De Léan A. Mol. Pharmacol. 1986; 30: 537-543PubMed Google Scholar). Competition binding studies were carried out by incubating aortic SMC membranes (50 ॖg) for 1 h at 25 °C with 0.2 nm125I-sarile and varying concentrations of Ang II in a total volume of 250 ॖl of binding buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1 mm EDTA, 1 mm MgCl2, and 0.17 heat-inactivated bovine serum albumin). Bound 125I-sarile was separated from free ligand by rapid filtration through GF/B filters presoaked with 0.27 bovine serum albumin, followed by washing with 50 mm Tris-HCl (pH 7.4), 150 mm NaCl. The filters were counted for radioactivity. Averages of duplicate determinations of bound 125I-sarile were used for data analysis. Binding data were analyzed by nonlinear least squares curve fitting using the SCAFIT computer program (46De Léan A. Hancock A.A. Lefkowitz R.J. Mol. Pharmacol. 1982; 21: 5-16PubMed Google Scholar). The intracellular mass of InsP3 was measured by a specific radioreceptor assay as described previously (40Leduc I. Haddad P. Giasson E. Meloche S. Mol. Pharmacol. 1995; 48: 582-592PubMed Google Scholar). Averages of duplicate determinations of bound [3H]InsP3 were used for data analysis. The mass of InsP3 is expressed as pmol of InsP3produced/mg of protein. Quiescent aortic SMC in 60-mm Petri dishes were stimulated with 10 nm Ang II for either 5 min (ERK assays), 3 min (MEK assays), or 15 min (p70S6Kassays). The enzymatic activity of ERK isoforms was measured by specific immune complex kinase assays using myelin basic protein as substrate as described (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 42Meloche S. J. Cell. Physiol. 1995; 163: 577-588Crossref PubMed Scopus (77) Google Scholar). The phosphotransferase activity of p70S6K was measured by an immune complex kinase assay using the S6 peptide RRRLSSLRA (Upstate Biotechnology) as substrate (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The enzymatic activity of MEK1 and MEK2 was assayed by measuring their ability to increase the myelin basic protein kinase activity of recombinant ERK1 in vitro (33Servant M.J. Giasson E. Meloche S. J. Biol. Chem. 1996; 271: 16047-16052Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Quiescent aortic SMC in 100-mm Petri dishes were stimulated with 10 nm Ang II for the indicated times at 37 °C. Total RNA was extracted with guanidinium thiocyanate as described (47Chomczinski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159PubMed Google Scholar). Equal amounts of total RNA (10–20 ॖg) were denatured by heating for 15 min at 65 °C in 2.2 mformaldehyde and 507 formamide and resolved by electrophoresis in a 17 agarose gel containing 1.87 formaldehyde. The RNA was transferred to Hybond-N (Amersham) nylon membranes by vacuum blotting, fixed, and hybridized with 32P-labeled probes. Hybridization was carried out in hybridization medium (5 × SSC (1 × SSC = 150 mm NaCl, 15 mm sodium citrate), 0.17 SDS, 5 × Denhardt's solution (1 × Denhardt's = 0.027 Ficoll 400, 0.027 polyvinylpyrrolidone, and 0.027 bovine serum albumin), 507 formamide, and 100 ॖg/ml herring sperm DNA) containing the labeled probe (1–2 × 106 cpm/ml) for 16 h at 42 °C. The membranes were washed twice at 25 °C for 15 min in 2 × SSC, 0.17 SDS, and twice at 60 °C for 30 min in 0.5 × SSC, 0.17 SDS. The extent of hybridization was analyzed with a PhosphorImager apparatus (Molecular Dynamics). The results were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. The probes used were a 0.9-kb PstI fragment of mouse c-fos cDNA (provided by Dr. Mona Nemer, University of Montreal), a 1.1-kb PstI-EcoRI fragment of mousefosB cDNA (provided by Dr. Rodrigo Bravo, Bristol-Myers Squibb), a 0.7-kb HindIII fragment of mouse egr-1cDNA (provided by Dr. Trang Hoang, University of Montreal), a 1.8-kb HindIII fragment of mouse c-myc cDNA (provided by Dr. Alain Nepveu, McGill University), and a 1.2-kbXbaI-PstI fragment of rat glyceraldehyde-3-phosphate dehydrogenase cDNA. All of the probes were labeled by random priming. Quiescent aortic SMC were stimulated with 10 nm Ang II for the indicated times at 37 °C. The cells were then washed twice in ice-cold phosphate-buffered saline and lysed in Triton X-100 lysis buffer (50 mm Tris-HCl (pH 7.4), 100 mm NaCl, 50 mm NaF, 5 mm EDTA, 40 mmॆ-glycerophosphate, 1 mm sodium orthovanadate, 10−4m phenylmethylsulfonyl fluoride, 10−6m leupeptin, 10−6m pepstatin A, 17 Triton X-100) for 30 min at 4 °C. Cell lysates were clarified by centrifugation at 13,000 ×g for 10 min, and normalized amounts of lysate proteins (500–600 ॖg) were incubated for 4 h at 4 °C with the following antibodies preadsorbed to protein A-Sepharose beads: 10 ॖl of anti-paxillin, 2.5 ॖl of anti-Shc, 5 ॖl of anti-p125FAK, 4 ॖl of anti-Pyk2, or 10 ॖl of anti-Tyk2. Immune complexes were washed three times with Triton X-100 lysis buffer, and the eluted proteins were separated by SDS-gel electrophoresis on 7.57 acrylamide gels and electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham) in 25 mm Tris, 192 mm glycine. After fixation for 15 min in 407 methanol, 77 acetic acid, 37 glycerol, the membrane was blocked for 1 h at 37 °C in Tris-buffered saline containing 0.17 Tween 20 and 17 bovine serum albumin and then incubated for 2 h at 25 °C with mAb 4G10 (1:3,000) in blocking solution. The membrane was washed five times in Tris-buffered saline, 0.17 Tween 20 prior to incubation for 1 h with horseradish peroxidase-conjugated anti-IgG diluted 1:10,000 in Tris-buffered saline containing 0.17 Tween 20 and 37 non-fat dry milk. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham). For immunoblotting of total phosphotyrosyl proteins, equal amounts of lysate proteins (100 ॖg) were resolved on 7.57 acrylamide gels and transferred to nitrocellulose membranes as described above. The membrane was incubated for 2 h at 25 °C with mAb 4G10 (1:3,000) in blocking solution. After washing, the membrane was incubated with horseradish peroxidase-conjugated protein A (1:3,000) in Tris-buffered saline, 0.17 Tween 20 for 1 h. Immunoreactive bands were visualized by chemiluminescence. Quiescent aortic SMC or Rat1-AT1 cells in triplicate wells of 24-well plates were stimulated with the indicated concentrations of Ang II in serum-free medium containing 0.5 ॖCi/ml [3H]leucine. After 24 h of stimulation, the medium was aspirated, and the cells were incubated for a minimum of 30 min in cold 57 trichloroacetic acid. The wells were then washed once with trichloroacetic acid and three times with tap water. The radioactivity incorporated into trichloroacetic acid-precipitable material was measured by liquid scintillation counting after solubilization in 0.1 m NaOH. Where indicated, the cells were stimulated for 24 h with Ang II in the continuous presence of cAMP-elevating agents. Protein concentrations were measured using the BCA protein assay kit (Pierce) with bovine serum albumin as standard. Dose-response curves were analyzed according to a four-parameter logistic equation using the ALLFIT computer program (48De Léan A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar). Ang II is a hypertrophic factor that potently stimulates protein synthesis in rat aortic SMC but has no effect on DNA synthesis or cell proliferation (27Geisterfer A.A.T. Peach M.J. Owens G.K. Circ. Res. 1988; 62: 749-756Crossref PubMed Scopus (1012) Google Scholar, 28Berk B.C. Vekshtein V. Gordon H.M. Tsuda T. Hypertension. 1989; 13: 305-314Crossref PubMed Scopus (518) Google Scholar, 29Chiu A.T. Roscoe W.A. McCall D.E. Timmermans P.B.M.W.M. Receptor. 1991; 1: 133-140PubMed Google Scholar, 30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To examine the effect of cAMP on the growth response to Ang II, aortic SMC were treated with a variety of agents known to increase intracellular cAMP, and the rate of protein synthesis was determined by [3H]leucine incorporation. As shown in Fig. 1, the addition of cAMP-raising agents strongly inhibited the stimulatory effect of Ang II on protein synthesis, without affecting the basal rate of protein synthesis. All of these agents were found to raise the intracellular concentration of cAMP significantly (data not shown). The growth-inhibitory effect of cAMP was reversible, and no sign of long term cytotoxicity was observed at the concentrations of agents used. The cAMP phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin were the most effective inhibitors, reducing the trophic effect of Ang II by 100 and 707, respectively. These two compounds were therefore used in all subsequent experiments. Pharmacological studies revealed that forskolin and IBMX block Ang II-induced leucine incorporation in a dose-dependent manner. Half-maximal inhibition was observed at a concentration of 0.5 ± 0.2 ॖm forskolin and 79 ± 18 ॖm IBMX (Fig. 2). We also analyzed the effect of the two inhibitors on the rate of protein synthesis at different concentrations of Ang II. Fig.3 shows that treatment with forskolin or IBMX inhibited the induction of protein synthesis by every concentration of the peptide. The half-maximal effect of Ang II on protein synthesis was found to be similar in the absence or in the presence of either forskolin or IBMX. Taken together, these results demonstrate that elevation of intracellular levels of cAMP, through different cellular mechanisms, antagonizes the stimulatory effect of Ang II on protein synthesis. This inhibitory effect of cAMP is presumably mediated by activation of PKA.Figure 3Effect of forskolin and IBMX on the dose dependence of Ang II for the stimulation of protein synthesis.Quiescent rat aortic SMC were stimulated for 24 h with the indicated concentrations of Ang II in the absence (○) or presence of 10 ॖm forskolin (•) or 0.1 mm IBMX (▿). Protein synthesis was measured by [3H]leucine incorporation. Each value represents the mean ± S.E. of triplicate determinations. Similar results were obtained in three different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To explore the mechanism by which cAMP interferes with the activation of protein synthesis by Ang II, we examined the effects of forskolin and IBMX on various receptor-mediated responses. Ang II has been shown to exert its hypertrophic effect through activation of the AT1 receptor subtype, a member of the superfamily of G protein-coupled receptors (29Chiu A.T. Roscoe W.A. McCall D.E. Timmermans P.B.M.W.M. Receptor. 1991; 1: 133-140PubMed Google Scholar, 30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We first tested the effect of increased cAMP levels on the agonist binding properties of the AT1 receptor by performing competition binding studies in membranes derived from either control cells or cells treated with forskolin or IBMX. As shown in Fig. 4, treatment with cAMP-raising agents did not change the total number of membrane AT1 receptor sites. Computer analysis by nonlinear regression revealed that Ang II competition binding data are best explained by a model with two different affinity states of the receptor. The proportion of high affinity sites and the affinity for Ang II (control, K d = 2.1 nm; forskolin,K d = 1.1 nm; IBMX, K d = 1.6 nm) was similar in control cells and cAMP-treated cells, indicating that elevation of cAMP does not interfere with the initial coupling of the AT1 receptor with G proteins. We next analyzed the effect of cAMP on phospholipase C activation by measuring the intracellular mass of InsP3. Ang II binding to the AT1 receptor has been shown to stimulate the activity of phospholipase C in aortic SMC and in many other target cells rapidly (31Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar, 32Catt K.J. Sandberg K. Balla T. Raizada M.K. Phillips M.I. Sumners C. Cellular and Molecular Biology of the Renin-Angiotensin System. CRC Press, Inc., Boca Raton, FL1993: 307-356Google Scholar). Pretreatment of the cells with either forskolin or IBMX did not prevent the rapid increase in the production of InsP3 induced by Ang II (Fig.5). These results indicate that cAMP does not inhibit the growth effect of Ang II by interfering with early receptor-mediated signaling events. In common with growth factors, Ang II potently stimulates the enzymatic activity of the MAP kinase isoforms ERK1/ERK2 in vascular SMC (30Giasson E. Meloche S. J. Biol. Chem. 1995; 270: 5225-5231Abstract Full Text Full Text PDF PubMed Scopus (105) Googl
Referência(s)