Cyclic AMP-mediated Inhibition of Angiotensin II-induced Protein Synthesis Is Associated with Suppression of Tyrosine Phosphorylation Signaling in Vascular Smooth Muscle Cells*

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.

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 AT 1 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-1methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT 1 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, 1 lymphoid cells, and many tumor cells (for review, see Refs. [1][2][3][4][5]. In these cells, cAMP interferes with the mitogenic response to growth factors acting on both receptor tyrosine kinases and G protein-coupled receptors (6). 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 (5,7).
The regulatory effects of cAMP are mediated through activation of the multifunctional cAMP-dependent protein kinase (protein kinase A or PKA) (8). 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) (9,10). 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 (11)(12)(13)(14)(15). 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 (11,12,15). 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 (14). However, treatment of CCL39 fibroblasts (16) or interleukin-2-dependent T lymphocytes (17) 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 (18). cAMP was also shown to reduce the accumulation of c-myc mRNA in various cell lines (19 -22). 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 (23,24). 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. 25 and 26). In cultured aortic SMC, Ang II induces cellular hypertrophy as a result of increased protein synthesis but not cell proliferation (27)(28)(29)(30). The growth-promoting effects of the peptide are mediated by the AT 1 receptor subtype, a member of the superfamily of G protein-coupled receptors. Agonist binding to the AT 1 receptor stimulates the activity of phospholipase C, to generate the second messengers InsP 3 and diacylglycerol, and inhibits the activity of adenylyl cyclase (for review, see Refs. 31 and 32). One of the immediate consequences of these early * This work was supported by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  signals is activation of the MAP kinases ERK1/ERK2 (33)(34)(35) and the 70/85-kDa S6 protein kinases (referred to as p70 S6K ) (30). Activation of the AT 1 receptor also leads to increased tyrosine phosphorylation of multiple proteins in target cells (36 -40). Despite these observations, the nature of the signaling mechanisms coupling the AT 1 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.

EXPERIMENTAL PROCEDURES
Materials and Antibodies-125 I-Labeled [Sar 1 ,Ile 8 ]Ang II (sarile) was prepared by radioiodination of sarile using a solid phase method as described (41). 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 M solution in 10% ascorbic acid. The source of other materials has been described (30).
Antisera SM1 and ␣IIcp42 have been described and specifically immunoprecipitate the MAP kinases ERK1 and ERK2, respectively (42,43). Antiserum S6-24 was produced in rabbits against a synthetic peptide corresponding to amino acids 2-30 of rat p70 S6K (Quality Controlled Biochemicals). The anti-p125 FAK 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.
Cell Culture-Rat aortic SMC were cultured and synchronized as described previously (30). Rat1-AT 1 cells are Rat1 fibroblasts stably expressing the human Ang II AT 1 receptor. 2 Rat1-AT 1 cells were grown in minimum essential medium supplemented with 10% 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 mM Hepes (pH 7.4) and 0.1% 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.
Measurement of Cyclic AMP-The intracellular mass of cAMP was determined by a specific protein binding assay (44). 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 ϫ 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).
Receptor Binding Assay-Membranes from rat aortic SMC were prepared as described (45). Competition binding studies were carried out by incubating aortic SMC membranes (50 g) for 1 h at 25°C with 0.2 nM 125 I-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 MgCl 2 , and 0.1% heat-inactivated bovine serum albumin). Bound 125 I-sarile was separated from free ligand by rapid filtration through GF/B filters presoaked with 0.2% 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 125 I-sarile were used for data analysis. Binding data were analyzed by nonlinear least squares curve fitting using the SCAFIT computer program (46).

Measurement of InsP 3 -
The intracellular mass of InsP 3 was measured by a specific radioreceptor assay as described previously (40). Averages of duplicate determinations of bound [ 3 H]InsP 3 were used for data analysis. The mass of InsP 3 is expressed as pmol of InsP 3 produced/mg of protein.
Protein Kinase Assays-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 (p70 S6K assays). The enzymatic activity of ERK isoforms was measured by specific immune complex kinase assays using myelin basic protein as substrate as described (30,42). The phosphotransferase activity of p70 S6K was measured by an immune complex kinase assay using the S6 peptide RRRLSSLRA (Upstate Biotechnology) as substrate (30). 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 (33).
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 mouse fosB cDNA (provided by Dr. Rodrigo Bravo, Bristol-Myers Squibb), a 0.7-kb HindIII fragment of mouse egr-1 cDNA (provided by Dr. Trang Hoang, University of Montreal), a 1.8-kb Hin-dIII fragment of mouse c-myc cDNA (provided by Dr. Alain Nepveu, McGill University), and a 1.2-kb XbaI-PstI fragment of rat glyceraldehyde-3-phosphate dehydrogenase cDNA. All of the probes were labeled by random priming.
Analysis of Tyrosine-phosphorylated Proteins-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 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 10 Ϫ4 M phenylmethylsulfonyl fluoride, 10 Ϫ6 M leupeptin, 10 Ϫ6 M pepstatin A, 1% Triton X-100) for 30 min at 4°C. Cell lysates were clarified by centrifugation at 13,000 ϫ 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-p125 FAK , 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 7.5% acrylamide gels and electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham) in 25 mM Tris, 192 mM glycine. After fixation for 15 min in 40% methanol, 7% acetic acid, 3% glycerol, the membrane was blocked for 1 h at 37°C in Tris-buffered saline containing 0.1% Tween 20 and 1% 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, 0.1% Tween 20 prior to incubation for 1 h with horseradish peroxidase-conjugated anti-IgG diluted 1:10,000 in Tris-buffered saline containing 0.1% Tween 20 and 3% 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 7.5% 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, 0.1% Tween 20 for 1 h. Immunoreactive bands were visualized by chemiluminescence.
Protein Synthesis Measurements-Quiescent aortic SMC or Rat1-AT 1 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 [ 3 H]leucine. After 24 h of stimulation, the medium was aspirated, and the cells were incubated for a minimum of 30 min in cold 5% 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.
Other Methods-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 (48).

Increased cAMP Inhibits Ang II-stimulated Protein Synthesis
in Aortic SMC-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 (27)(28)(29)(30). 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 [ 3 H]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 70%, 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 Ϯ 0.2 M forskolin and 79 Ϯ 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 dem-onstrate 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.
Elevation of cAMP Does Not Interfere with Ang II Early Signaling Events-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 recep- tor-mediated responses. Ang II has been shown to exert its hypertrophic effect through activation of the AT 1 receptor subtype, a member of the superfamily of G protein-coupled receptors (29,30). We first tested the effect of increased cAMP levels on the agonist binding properties of the AT 1 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 AT 1 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 ϭ 2.1 nM; forskolin, K d ϭ 1.1 nM; IBMX, K d ϭ 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 AT 1 receptor with G proteins.
We next analyzed the effect of cAMP on phospholipase C activation by measuring the intracellular mass of InsP 3 . Ang II binding to the AT 1 receptor has been shown to stimulate the activity of phospholipase C in aortic SMC and in many other target cells rapidly (31,32). Pretreatment of the cells with either forskolin or IBMX did not prevent the rapid increase in the production of InsP 3 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.
Elevation of cAMP Does Not Inhibit Ang II-dependent Activation of ERK1/ERK2 and p70 S6K -In common with growth factors, Ang II potently stimulates the enzymatic activity of the MAP kinase isoforms ERK1/ERK2 in vascular SMC (30,(33)(34)(35). In view of the demonstration that PKA antagonizes growth factor-induced Ras-dependent activation of MAP kinases in a number of cell types (11)(12)(13)(14)(15), we sought to determine if the inhibitory effect of cAMP on Ang II-induced protein synthesis resulted from a negative regulatory effect on the MAP kinases ERK1/ERK2. Quiescent aortic SMC were treated with forskolin or IBMX prior to Ang II stimulation, and the activity of ERK isoforms was determined by specific immune complex kinase assays. In contrast to the above mentioned results, elevation of cAMP levels did not affect Ang II-dependent activation of ERK1 and ERK2 isoforms in rat aortic SMC (Fig. 6). Detailed kinetic analysis also confirmed that cAMP does not alter the time course of ERK1/ERK2 activation by Ang II (data not shown). We also examined the effect of forskolin and IBMX on the activity of MEK isoforms in Ang II-stimulated cells. Ang II has been shown to activate both MEK1 and MEK2 isoforms in rat aortic SMC (33). As expected, the response to Ang II was not altered in cells treated with cAMP-raising agents (data not shown).
Another potential candidate for the inhibitory action of cAMP is the serine/threonine kinase p70 S6K , the major S6 protein kinase in vivo. We have demonstrated previously that Ang II stimulates the phosphotransferase activity of p70 S6K in aortic SMC and that inhibition of this enzyme with rapamycin correlates with inhibition of Ang II-induced protein synthesis (30). To determine if cAMP antagonizes Ang II-stimulated p70 S6K activation, cells were treated with forskolin or IBMX, and the activity of p70 S6K was measured by immune complex kinase assay. Fig. 7 shows that treatment with cAMP-raising agents had no effect on Ang II-mediated activation of p70 S6K in rat aortic SMC. These results indicate that cAMP does not interfere with the two major protein kinase signaling cascades to exert its inhibitory action on protein synthesis.
Elevation of cAMP Does Not Prevent Ang II-stimulated Early Growth Response Genes Induction-Ang II has been shown to increase mRNA expression of the c-fos (49 -51), c-jun (52), egr-1 (53), fosB (54), and c-myc (55) genes in vascular SMC. However, the role of these early gene products in the stimulatory effect of Ang II on protein synthesis remains to be established. In this study, we analyzed the effect of raising cAMP levels on mRNA expression of the c-fos, fosB, and egr-1 genes in rat aortic SMC. Treatment of the cells with either forskolin or IBMX had no effect on the peak induction of these genes in response to Ang II stimulation (Fig. 8A). We also examined the effect of cAMP on the induction of the c-myc gene. Previous studies in diverse cell types have shown that elevation of cAMP can inhibit growth factor-dependent expression of c-myc mRNA by downregulating the transcription of the gene (19 -22). Fig. 8B shows that Ang II induced the expression of the c-myc gene in aortic SMC, with a peak induction observed after 2-4 h. Neither forskolin or IBMX prevented the induction of the c-myc gene in response to Ang II. Moreover, incubation with either compound had no effect on the time course of c-myc mRNA expression in Ang II-stimulated cells (data not shown). Together, these data suggest that cAMP does not inhibit the trophic effect of Ang II by blocking early growth response genes induction.
Increased cAMP Inhibits Ang II-stimulated Tyrosine Phos-phorylation of Cellular Proteins-Ang II stimulates tyrosine phosphorylation of multiple proteins in vascular SMC and other target cells (36 -40). This signaling pathway appears to play a critical role in the hypertrophic action of Ang II because treatment of aortic SMC with selective tyrosine kinase inhibitors completely abrogates Ang II-dependent increase in protein synthesis (40). These observations led us to examine the effect of cAMP elevation on Ang II-dependent activation of tyrosine phosphorylation. Preincubation of aortic SMC with forskolin or IBMX resulted in a complete inhibition of Ang II-stimulated tyrosine phosphorylation of the 120 -125-and 65-75-kDa bands, the two major substrates detected in these cells (Fig.  9A). As predicted from the enzymatic assays (see Fig. 6), treatment of cells with either compound had no influence on tyrosine phosphorylation of the MAP kinase isoforms ERK1/ERK2, clearly showing the selectivity of the cAMP inhibitory action (Fig. 9B). We and others have identified the major 65-75-kDa tyrosine phosphorylated substrate in Ang II-treated cells as the focal adhesion-associated protein paxillin (56,57). We therefore examined more specifically the effect of cAMP on tyrosine phosphorylation of paxillin. Addition of Ang II increased tyrosine phosphorylation of paxillin in quiescent aortic SMC, and this response was blocked completely by preincubation with either forskolin or IBMX (Fig. 9C). In contrast, neither compound prevented tyrosine phosphorylation of the adaptor protein Shc, another major target of Ang II signaling (39,58). These observations are consistent with the idea that Ang II activates at least two tyrosine kinase pathways, one of which is sensitive to cAMP. To explore further the mechanism by which cAMP interferes with tyrosine phosphorylation signaling, we examined the effect of cAMP elevation on the tyrosine phosphorylation level of Ang II-regulated cellular tyrosine kinases. Ang II significantly increased the phosphotyrosine content of the focal adhesion-associated kinases p125 FAK and Pyk2 (also known as FIG. 7. Elevation of cAMP does not interfere with Ang II-dependent activation of p70 S6 kinase. Quiescent rat aortic SMC were pretreated for 30 min with vehicle alone or with either 10 M forskolin (Forsk) or 0.1 mM IBMX prior to stimulation with medium (cont) or 10 nM Ang II for 15 min. Cell lysates were prepared and subjected to immunoprecipitation with p70 S6K antiserum preadsorbed to protein A-Sepharose beads. The phosphotransferase activity of p70 S6K was assayed directly on the immune complexes using an S6 peptide as substrate. The enzymatic activities are expressed as pmol of phosphate incorporated into the peptide/min/mg of lysate protein. Each value represents the mean Ϯ S.D. of duplicate determinations. The data presented are representative of four independent experiments with similar results.
FIG. 8. Increased cAMP does not prevent the induction of early growth response genes by Ang II in rat aortic SMC. Quiescent rat aortic SMC were pretreated for 30 min with vehicle alone or with either 10 M forskolin (Forsk) or 0.1 mM IBMX. The cells were then stimulated or not (cont) with 10 nM Ang II for the following times: 30 min (c-fos), 60 min (egr-1 and fosB), or 120 min (c-myc). The specificity of the Ang II response was confirmed by preincubating the cells for 10 min with 10 Ϫ5 M losartan prior to Ang II addition. Total cellular RNA was extracted from the cells and analyzed serially by Northern hybridization using the indicated 32 P-labeled probes as described under "Experimental Procedures." The results were normalized by rehybridization of the blots with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. The extent of hybridization was visualized and quantified by PhosphorImaging analysis. Similar results were obtained in three separate experiments.
CAK beta and RAFTK) and of the Janus kinase Tyk2 in aortic SMC (Fig. 9D). Treatment of the cells with forskolin or IBMX did not affect Ang II-induced tyrosine phosphorylation of p125 FAK and Pyk2, thereby indicating that these tyrosine kinases are not the major paxillin kinases. However, both agents completely blocked the increased tyrosine phosphorylation of Tyk2 in response to Ang II (Fig. 9D). Thus, these results provide strong evidence that raising intracellular cAMP levels interferes with Ang II-stimulated tyrosine phosphorylation in vascular SMC.
Increased cAMP Inhibits Ang II-stimulated Protein Synthesis in a Heterologous Cell Line Expressing the Human AT 1 Receptor-We have established a rat fibroblast cell line expressing a physiological number of human AT 1 receptors which shows an increased rate of protein synthesis in response to Ang II. 2 To determine whether the growth-inhibitory effect of cAMP was specific to the cellular context, we examined the effect of cAMP elevation on Ang II-induced protein synthesis in Rat1-AT 1 cells. As shown in Fig. 10, treatment of Rat1-AT 1 cells with forskolin completely abolished the stimulatory effect of Ang II on protein synthesis. Forskolin was found to be more potent in inhibiting the trophic effect of Ang II in Rat1-AT 1 cells compared with aortic SMC, with half-maximal inhibition observed at 0.044 Ϯ 0.006 M concentration. Notably, forskolin also inhibited Ang II-induced tyrosine phosphorylation in Rat1-AT 1 cells (data not shown).

DISCUSSION
The role of cAMP as a regulator of cell growth has received considerable attention since the demonstration that elevation of the intracellular level of the nucleotide can stimulate or inhibit cell proliferation (1)(2)(3)(4)(5). However, until recently, the mechanism by which cAMP negatively affects the proliferation of normal and transformed cells has remained largely unknown. More recent work in various cellular models has led to new hypothesis to explain cAMP action (see below). In particular, cAMP has been shown to induce G 1 arrest in macrophages by interfering with the cell cycle machinery at the level of Cdk4 activation (18). All of these studies have examined the consequences of cAMP elevation on mitogen-induced cell cycle progression and cell division. Here, we have investigated the effect of cAMP on the induction of protein synthesis by the hypertrophic factor Ang II in aortic SMC and in fibroblasts expressing the human AT 1 receptor.
We report that a variety of agents known to elevate the intracellular concentration of cAMP potently inhibit the stimulatory effect of Ang II on protein synthesis (Fig. 1). The inhibition was dose-dependent and was observed at all concentrations of Ang II. Because these agents increase cAMP by different mechanisms of action, the above data indicate that the growth-inhibitory response is mediated by the elevation of intracellular cAMP levels. The effect of cAMP is most presumably dependent on the activity of PKA, as virtually all of the effects of the nucleotide in mammalian cells are attributed to this multifunctional enzyme. Interestingly, we show that the inhibitory effect of cAMP on Ang II-induced protein synthesis is not restricted to vascular SMC but is also observed in heterologous rat fibroblasts expressing the human AT 1 receptor. These data are consistent with the idea that cAMP has a generalized effect on the signaling machinery linking the AT 1 receptor to the regulation of protein synthesis. Indeed, previous studies have shown that cAMP-elevating agents exert similar inhibitory effect on the trophic action of Ang II in epithelial cell lines of renal origin (59,60). Similar to vascular SMC, Ang II was found to stimulate the rate of protein synthesis and to induce cellular hypertrophy in these cells, without increasing DNA content. However, the mechanism by which cAMP interferes with the hypertrophic effect of Ang II was not addressed in the latter studies. Thus, in addition to its well defined modulatory role on cell proliferation and cellular morphology, cAMP also exerts negative regulatory effects on the stimulation of protein synthesis by hypertrophic factors.
The molecular basis of the hypertrophic action of Ang II is still largely undefined. Activation of the AT 1 receptor is known to trigger multiple G protein-mediated signaling pathways, including the inhibition of adenylyl cyclase (31,32). Based on the observation that Ang II decreases the intracellular concentration of cAMP in the renal epithelial cell lines MCT and LLC-PK 1 and that cAMP-raising agents inhibit Ang II-induced protein synthesis, it has been proposed that the hypertrophic action of the hormone may be mediated by the decrease in intracellular cAMP (59,60). However, several lines of evidence do not support this hypothesis but rather indicate that G iregulated pathways are not important for the hypertrophic effect of Ang II. We have demonstrated that pretreatment of aortic SMC or Rat1-AT 1 cells with concentrations of pertussis toxin which completely prevent the decrease in cAMP levels have no effect on Ang II-induced protein synthesis. 2 In addition, Ang II has been shown to increase slightly the cAMP content in cardiac myocytes where it induces cellular hypertrophy potently (61). The physiological significance of the inhibition of adenylyl cyclase remains to be determined.
We have investigated the biochemical mechanism by which cAMP elevation antagonizes Ang II-induced protein synthesis in aortic SMC. We first excluded the possibility that cAMP was acting very early at the level of receptor activation. No significant effect of forskolin or IBMX was found on the total number of AT 1 receptors, the formation of high affinity receptor sites, and the activation of phospholipase C. We then examined the possibility that cAMP elevation might interfere with MAP kinase or p70 S6K signaling cascades. As mentioned, a number of reports have shown that cAMP-raising agents inhibit ERK1/ ERK2 activation in parallel to their antiproliferative effect in several cell types (11)(12)(13)(14)(15). Although these findings clearly demonstrated a functional cross-talk between the MAP kinase and PKA cascades, there is no direct evidence that the inhibition of cell proliferation by cAMP can be attributed to inhibition of early MAP kinase activation. In this regard, treatment of CCL39 fibroblasts (16) or T lymphocytes (17) with concentrations of cAMP-raising agents which completely suppress growth factor-induced DNA synthesis has no effect on ERK1/ ERK2 activation. In the present study, we clearly show that Ang II-dependent activation of ERK or MEK isoforms is unaffected by the elevation of cAMP levels in aortic SMC. The reason for these differential effects of PKA on the ERK path-way is not known, but a possible explanation is that stimulation of MEK activity by distinct growth factors involves distinct MAP kinase kinase kinases that are regulated differently by PKA. Additional studies are clearly necessary to resolve this question. We also establish that increased cAMP does not interfere with the activation of p70 S6K by Ang II. Although much less is known on the regulation of the p70 S6K pathway by cAMP, a recent study has shown that cAMP elevation inhibits interleukin-2-dependent activation of p70 S6K in T lymphocytes (17). In contrast, results from another study in CCL39 fibroblasts showed that addition of prostaglandin E 1 , which augments cAMP levels and inhibits cell proliferation, does not interfere with the activation of p70 S6K (16). Again, these results suggest that different mechanisms of p70 S6K activation might be operating in these cell types or in response to these receptor systems.
We also provide evidence that cAMP does not affect the induction of immediate-early genes in aortic SMC. It is interesting to note that in contrast to the results presented here, increased cAMP has been shown to down-regulate c-myc mRNA expression in several cell types, including normal and neoplastic B cells (19), leukemic cells (20), fibroblasts (21), and a macrophage cell line (22). The cAMP-mediated reduction in c-myc RNA levels results from a decrease in c-myc transcription (20,22). Despite these observations, the significance of c-myc down-regulation is not known because constitutive expression of c-myc was insufficient to override the growth-inhibitory effect of cAMP in murine macrophages (22).
Accumulating evidence suggests that tyrosine phosphorylation may play a significant role in the growth response to G protein-coupled receptor agonists. Numerous studies have shown that growth factors such as thrombin, bombesin, vasopressin, endothelin, and lysophosphatidic acid stimulate tyrosine phosphorylation of multiple substrates in their target cells (36,62,63). The observation that selective tyrosine kinase inhibitors can block the stimulation of DNA synthesis induced by thrombin (64), endothelin (65), and bombesin (66) has provided strong evidence for the importance of this signaling pathway in the mitogenic response. Ang II was also reported to induce tyrosine phosphorylation of multiple proteins (36 -40) and to stimulate the activity of cytosolic tyrosine kinases (39,(67)(68)(69) in target cells. Most importantly, treatment of aortic SMC with the tyrosine kinase inhibitors genistein and herbimycin A was found to abolish completely the stimulatory effect of Ang II on protein synthesis (40). In this study, we demonstrate that cAMP elevation interferes with tyrosine phosphorylation signaling as revealed by inhibition of Ang II-dependent tyrosine phosphorylation of the Janus kinase Tyk2 and of the focal adhesion protein paxillin. Interestingly, the effect of cAMP is selective with regard to the tyrosine kinase or the target substrate, clearly indicating that Ang II activates more than one tyrosine kinase pathway in vascular SMC. Thus, agents that raise cAMP levels may provide very useful tools to dissect the individual roles of Ang II-regulated tyrosine kinases in the induction of protein synthesis. In this regard, we are currently investigating the specific role of Janus kinases in the nuclear and cytoplasmic events controlling the rate of protein synthesis. Preliminary pharmacological data suggest that activation of the Janus kinase/Stat pathway is an essential component of Ang II hypertrophic action. 3 In summary, our results reveal a functional cross-talk between tyrosine phosphorylation, and more specifically Tyk2, and the cAMP signaling system. During the course of this work, two other groups reported that activation of PKA inhibits cytokine-dependent activation of the Janus kinase/Stat pathway in the myeloma cell line U266 (70) and in monocytes (71). Most importantly, these observations identify a novel mechanism by which cAMP may exert growth-inhibitory effects in specific cell types.