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Adrenergic Receptors Activate Phosphatidylinositol 3-Kinase in Human Vascular Smooth Muscle Cells

ROLE IN MITOGENESIS (∗)
  • Zhuo-Wei Hu
    Correspondence
    To whom correspondence should be addressed: VA Medical Center, GRECC 182B, 3801 Miranda Ave., Palo Alto, CA 94304. Tel.: 415-858-3933; Fax: 415-855-9437
    Affiliations
    Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 and Veterans Affairs Medical Center, Palo Alto, California 94304
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  • Xiao-You Shi
    Affiliations
    Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 and Veterans Affairs Medical Center, Palo Alto, California 94304
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  • Richard Z. Lin
    Footnotes
    Affiliations
    Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 and Veterans Affairs Medical Center, Palo Alto, California 94304
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  • Brian B. Hoffman
    Affiliations
    Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 and Veterans Affairs Medical Center, Palo Alto, California 94304
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  • Author Footnotes
    ∗ This work was supported in part by National Institutes of Health Grant HL41315 and by a preclinical award from Pfizer Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    Supported by a Pharmaceutical Research and Manufacturers of America Foundation Fellowship for Careers in Clinical Pharmacology.
Open AccessPublished:April 12, 1996DOI:https://doi.org/10.1074/jbc.271.15.8977
      Activation of α adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of α receptors may induce mitogenesis in HVSMCs, we have found that α receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of α receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85α subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a β adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of α receptors and by pertussis toxin, suggesting that α receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of α receptors activates PI 3-kinase in HVSMCs and that α receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to α receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.

      INTRODUCTION

      α adrenergic receptors are members of the superfamily of G protein-coupled membrane receptors; these pathways mediate many of the important physiological effects of catecholamines such as noradrenaline and adrenaline. α adrenergic receptors play a particularly important role in control of blood pressure via induction of vascular smooth muscle contraction (Minneman and Esbenshade, 1994). Also, activation of α adrenergic receptors stimulates cardiac and vascular smooth muscle growth and hypertrophy (Milano et al., 1994; Nakafuku et al., 1990; and Okazaki et al., 1994). However, signaling pathways utilized by α receptors in promoting mitogenic effects, such as growth-related gene expression and DNA synthesis, are unclear.
      It is generally accepted that activation of α receptors stimulates phospholipase C, leading to increased hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Both inositol 1,4,5-trisphosphate and 1,2-diacylglycerol play important roles as intracellular second messengers that increase intracellular Ca concentrations and activate various isoforms of protein kinase C, respectively. These coupling mechanisms are typically mediated by pertussis toxin-insensitive G proteins, likely in the Gq/11 family (Perez et al., 1993; Schwinn et al., 1995). Additionally, stimulation of α receptors activates phospholipase D and phospholipase A2 via pertussis toxin-insensitive/sensitive G proteins (Minneman and Esbenshade, 1994; Perez et al., 1993).
      Although this predominant view of α receptor signaling provides substantial insight into α receptor-mediated responses in various cells, there are clear indications that these mechanisms may not explain all aspects of α receptor signaling. For example, recent evidence demonstrates that α receptor-stimulated mitogenic responses in myocytes may be due to activation of tyrosine protein kinases (TPKs)
      The abbreviations used are: TPK
      tyrosine protein kinase
      IGF-I
      insulin-like growth factor I
      MAP kinase
      mitogen-activated protein kinase
      PI 3-kinase
      phosphatidylinositol 3-kinase
      SH2
      SRC homology 2
      TLC
      thin layer chromatography
      HVSMC
      human vascular smooth muscle cell.
      and MAP kinases (Thorburn et al., 1994), suggesting that α adrenergic receptors may share common signal pathways with tyrosine kinase receptors in the stimulation of mitogenesis.
      There has been considerable recent interest in lipid kinases that phosphorylate the 3-position of the inositol ring of inositol phospholipids; this has led to the identification of the enzyme PI 3-kinase (for reviews see Carpenter and Cantley(1990), Divecha and Irvine(1995), Fry(1994), and Valius and Kazlauskas(1993). PI 3-kinase is a lipid kinase that has been implicated in the regulation of cell growth and proliferation by receptor tyrosine kinases (Ruderman et al., 1990; Valius and Kazlauskas, 1993), nonreceptor tyrosine kinases (Ding et al., 1995), cytokine receptors (Karnitz et al., 1995) and oncogene products (Fukui et al., 1991). Stimulation of cells with mitogens such as platelet-derived growth factor and many other peptide growth factors leads to accumulation of the lipid products phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (Carpenter and Cantley, 1990; Divecha et al., 1995). Although the function of these lipids has not yet been determined, increasing evidence suggests that they may serve as intracellular second messengers. PI 3-kinase is a heterodimer consisting of a p85 regulatory subunit with SRC homology domains (SH2 and SH3) and a p110 catalytic subunit. A major mode of activation by growth factors likely involves docking of PI 3-kinase through SH2 domains of the p85 subunit to phosphorylated tyrosine residues(s) of tyrosine kinase receptors (Rordorf-Nikolic et al., 1994). Moreover, activation of PI 3-kinase by growth factors may occur via either Ras-dependent (Kodaki et al., 1994) or Ras-independent (Rodriguez-Viciana et al., 1994) pathways. In either situation, activation of Ras is sufficient to activate mitogenic responses in a variety of cells. In other important cases, such as G protein-coupled receptors, PI 3-kinase has been shown to be directly activated by β subunits released from activated G proteins (Zhang et al., 1995).
      In the present study, we have found that α adrenergic receptor-stimulated mitogenic responses, such as DNA synthesis and activation of MAP kinase in HVSMCs, are inhibited by wortmannin, a specific inhibitor of PI 3-kinase. This result suggests that activation of PI 3-kinase is associated with α adrenergic receptor-stimulated responses in HVSMCs. We further demonstrate directly that stimulation of α receptors activates PI 3-kinase via a pertussis toxin-sensitive G protein pathway. Moreover, noradrenaline-stimulated PI 3-kinase is associated with activation of Ras proteins and tyrosine protein kinases.

      EXPERIMENTAL PROCEDURES

      Materials

      Noradrenaline, H7, myelin basic protein, and 4β-phorbol 12,13-dibutyrate were purchased from Sigma; [P]ATP (2000 Ci/mmol), [P]orthophosphate, [3H]thymidine, and ECL Western blotting detection kit were from Amersham Corp.; phosphatidylinositol was obtained from Avanti Polar Lipids Inc. (Alabaster, Alabama); human recombinant IGF-I, cell culture medium, and fetal bovine serum were from Life Technologies, Inc.; wortmannin was from Worthington Biochemical Co.; antibodies against PI 3-kinase p85α, p110, phosphotyrosine, Ha-Ras, and protein A/G-agarose were from Santa Cruz Biotechnology. All other chemicals were reagents of molecular biology grade and were obtained from standard commercial sources.

      Preparation of Cultured Human Aortic Smooth Muscle Cells

      Human aortic vascular smooth muscle cells were purchased from Clonetics Corp. (San Diego, CA). Cells were grown in smooth muscle growth medium-2 with 5% fetal bovine serum from Clonetics Corp. at 37°C in a humidified atmosphere of 5% CO, 95% air. The cells were harvested for passaging at confluence with trypsin-EDTA and plated in 100-mm dishes at a density about 5 × 105, with a 80-90% confluence being reached about 10 days later. The medium was replaced every 2 days. Cells were generally used for studies at 8-10 days after seeding. To examine the effects of agonist-stimulated changes, cells were incubated with Dulbecco's modified Eagle's medium without serum for the indicated times after achieving confluence. Throughout these experiments, cells from the fifth through seventh passage were utilized.

      Agonist-stimulated DNA Synthesis

      Analysis of agonist-induced DNA synthesis was performed as described previously (Nakaki et al., 1990). HVSMCs were cultured in smooth muscle growth medium-2 containing 5% fetal bovine serum to near confluence. The medium was replaced with Dulbecco's modified Eagle's medium containing 0.4% serum for 48 h. Noradrenaline (1 μM) plus the β receptor blocker timolol (0.5 μM) were added to the medium, and 20 h later [3H]thymidine (0.1 μCi/dish) was added. The incorporation of [3H]thymidine was determined 4 h later. To examine if the α receptor agonist-induced increase in [3H]thymidine incorporation required activation of PI 3-kinase or a pertussis toxin-sensitive G protein, cells were preincubated with wortmannin (2 h) or with pertussis toxin (12 h), respectively.

      Immunoprecipitation and Immunodetection

      After treatment, cultures on 100-mm plates were rinsed with ice-cold phosphate-buffered saline containing 1 mM sodium orthovanadate. Cells were incubated with cell lysis buffer (1% Nonidet P-40, 25 mM Hepes (pH 7.5), 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic acid, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml antipain, aprotinin, and leupeptin) for 10-15 min on the ice. Insoluble material was removed by centrifugation at 12,100 × g for 20 min. The amount of cell lysate was normalized by protein content in each experiment using a kit from Bio-Rad. The lysate was incubated with an appropriate amount of antibody with constant agitation for 2 h and then further incubated with 20-30 μl of protein A/G plus-agarose with agitation for 1 h. The beads containing the immunoprecipitates were washed 4 times with lysis buffer, washed once with washing buffer (0.1 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5), and subjected to PI 3-kinase and MAP kinase assays or analysis of Ras-bound GTP. For immunodetection, immunoprecipitates were washed 3 times with lysis buffer and twice with distilled water, and analyzed by SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred to membrane and detected using the ECL Western blotting detection system (Amersham) with the indicated primary antibody and an appropriate horseradish peroxidase-conjugated secondary antibody.

      In Vitro Assay of MAP Kinase Activity

      Assay of MAP kinase activity was performed following a method described previously (Greenberg et al., 1994). Confluent cells were incubated without serum overnight and treated with noradrenaline or other agonists for various times. The cells were lysed in 0.4 ml of lysis buffer as described above. For MAP kinase activity assay, cell lysate (400 μg of protein) was incubated with antibody against ERK1 (2 μg/mg protein) and washed as above. The washed immunocomplexes were resuspended in 50 μl of kinase buffer (25 mM Hepes, pH 7.5, 10 mM MgCl, 1 mM dithiothreitol, 0.5 mM EGTA, 40 μM ATP, 1 μCi of [P]ATP, and myelin basic protein) (1 mg/ml) as a substrate. The reaction mixture was incubated for 10 min at 30°C because preliminary experiments suggested that noradrenaline-induced myelin basic protein phosphorylation is linear for 20-30 min. The reaction was stopped by spotting 10 μl of reaction mixture onto p-81 phosphocellulose paper (Whatman), which was then washed in 75 mM phosphoric acid with constant stirring for 1 h and transferred to another washing overnight. The papers were washed with acetone for 5 min and dried. P, which represented the phosphorylation of myelin basic protein by MAP kinase, was measured by scintillation spectrophotometry.

      In Vitro Assays of PI 3-Kinase

      For measurement of PI 3-kinase activity, cell lysate (0.5-1 mg of protein) was incubated with antibody against the p85α subunit of PI 3-kinase (2 μg/mg of protein) as described above. Assay of PI 3-kinase activity was conducted as described by Nakafuku et al.(1992). Briefly, the washed pellets were resuspended in 50 μl of kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA) and incubated at 25°C for 10 min after the addition of 0.5 μl of 20 mg/ml phosphatidylinositol dissolved in chloroform to make micelles of PI. Assays were initiated with the addition of 5 μl of ATP solution (0.4 M ATP, 0.1 M MgCl, and 1 μCi/ml [-P]ATP) and incubated at room temperature for 30 min. During this time, the formation of phosphatidylinositol phosphate was linear (data not shown). The reaction was stopped after the addition of 100 μl of chloroform, methanol, 11.6 N HCl (100:200:2). After centrifugation, the lower organic phase was taken for thin layer chromatography (TLC) on Silica Gel plates (Merck) to be developed in chloroform, methanol, 25% ammonium hydroxide, water (43:38:5:7). The plates were exposed to Kodak XAR-5 film at −70°C with an intensifying screen for 16-24 h or were visualized after development with a PhosphorImager System (Molecular Dynamics).

      Analysis of Ras-bound GTP and GDP

      Agonist-stimulated change in Ras-bound GTP was performed following the methods described previously (Nakafuku et al., 1992; Satoh et al., 1990). Quiescent cells were labeled with 0.1 mCi/ml of [P]orthophosphate in phosphate-free Dulbecco's modified Eagle's medium for 12 h. After stimulation with agonists for the indicated times, cells were washed with ice-cold phosphate-buffered saline 3 times and cells lysed as described above. Ras proteins were recovered by immunoprecipitation with an anti-Ras polyclonal antibody. After extensively washing as described above, the immunoprecipitates were suspended in 20 μl of reaction mixture containing 20 mM Hepes, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP. The suspension was heated at 90°C for 3 min and centrifuged for 5 min. The bound nucleotides were separated by TLC on polyethyleneimine-cellulose plate with 0.75 M KHPO for development. GDP and GTP were visualized using unlabeled standards. Ras-associated GTP was calculated from the ratio of GTP to (GDP + GTP). The radioactivity was measured with a PhosphorImager System (Molecular Dynamics).

      Data Analysis

      Data are presented as mean ± S.E., and treatment effects were compared by one-way analysis of variance or Student's paired t test (two-tailed). p < 0.05 was taken as the level of significance.

      RESULTS AND DISCUSSION

      To characterize signaling pathways involved in α receptor stimulation of mitogenic responses in vascular smooth muscle cells, we tested the capacity of wortmannin, a specific inhibitor of PI 3-kinase, to inhibit DNA synthesis. Wortmannin (10 nM) completely blocked noradrenaline-stimulated DNA synthesis as well as platelet-derived growth factor-induced DNA synthesis (Fig. 1A). Noradrenaline's action was also blocked by the α receptor-selective antagonist terazosin. To determine whether the α receptor-mediated increase in [3H]thymidine incorporation was mediated via pertussis toxin-sensitive G proteins, cells were preincubated with pertussis toxin (100 ng/ml) for 12 h before stimulation with noradrenaline. Noradrenaline induced an 89% increase of [3H]thymidine incorporation under control conditions; preincubation with pertussis toxin markedly inhibited the noradrenaline-induced increase in DNA synthesis in these cells (Fig. 1A). Since MAP kinase has been postulated to play a key role in mediating mitogenic responses of many receptors, including α adrenergic receptors in myocytes, we also examined α adrenergic receptor-mediated activation of MAP kinase in HVSMCs (Fig. 1B). Noradrenaline (1 μM) stimulated an approximately 2-fold increase in MAP kinase activity in the presence of a β receptor antagonist timolol. The α receptor antagonist terazosin (1 μM) blocked noradrenaline-activated MAP kinase activity (Fig. 1B). Noradrenaline-stimulated activation of MAP kinase was significantly attenuated by a 12-h preincubation of cells with pertussis toxin (100 ng/ml) (about 30% increase over basal) and partially blocked by inhibitor of PI 3-kinase wortmannin (about 43% increase over basal) (Fig. 1B). Increased MAP kinase activity was not inhibited by the protein kinase C inhibitor H7. These results suggest that activation of PI 3-kinase as well as activation of MAP kinases are involved in mediating catecholamine-induced mitogenesis in human vascular smooth muscle cells. In addition, we found that IGF-I increased MAP kinase activity in these cells and that this response was inhibited by wortmannin but not by pertussis toxin; this result suggests that pertussis toxin was not having nonspecific effects (Fig. 1B).
      Figure thumbnail gr1
      Figure 1α adrenoreceptor stimulation of DNA synthesis and activation of MAP kinase: effects of inhibitors. A, HVSMCs purchased from Clonetics Corp. (San Diego, CA) were grown to near confluence in a series of 35-mm2 dishes. Quiescent cells were incubated with Dulbecco's modified Eagle's medium containing 0.4% fetal calf serum plus noradrenaline (1 μM) for 20 h in the presence of a β adrenoreceptor antagonist timolol (1 μM) (some cells had been preincubated with 100 ng/ml pertussis toxin (PTx) for 12 h). Cells were then incubated with [3H]thymidine (0.1 μCi) for another 4 h. Potential inhibitors were added to the cell dishes 1 h before the addition of noradrenaline (Nor) as indicated. Incorporation of [3H]thymidine into cells was performed as described under “Experimental Procedures.” The data are an average ± S.E. of three experiments. B, the near confluent HVSMCs were incubated with serum-free Dulbecco's modified Eagle's medium for 18 h. Inhibitors were added into cell dishes 1 h before the addition of agonists. After noradrenaline or vehicle treatment for 10 min, cells were harvested for assay of MAP kinase activity as described under “Experimental Procedures.” The data are average ± S.E. of three experiments. ∗, different from control, p < 0.05;∗∗, different from control, p < 0.01
      Several lines of evidence indicate that PI 3-kinase plays an important role in growth regulation and transformation. Analysis of mutations in the binding site for PI 3-kinase on the polyoma virus middle T antigen, which leads to either failure to associate with PI 3-kinase or impaired capacity to elevate the concentrations of PI 3-kinase products, have been found to result in a transformation-defective phenotype (Fantl et al., 1992). Similarly, point mutations in the PI 3-kinase binding sites of platelet-derived growth factor receptors impair this receptor's ability to stimulate DNA synthesis (Valius and Kazlauskas, 1993). Roche et al.(1994) have shown that microinjection of antibodies specific for the p110 subunit of the PI 3-kinase into quiescent fibroblasts inhibited platelet-derived growth factor-induced DNA synthesis. Finally, a number of studies have demonstrated that inhibition of PI 3-kinase by wortmannin, a specific PI 3-kinase inhibitor, results in blockage of growth factors or serum-induced cell proliferation (Panayotou and Waterfield, 1993; Varticovski et al., 1994; Vemuri and Rittenhouse, 1994), inhibition of protein kinase cascades (Ding et al., 1995), and suppression of growth factor-induced blockade of apoptosis (Yao and Cooper, 1995). Consequently, the present results demonstrate that wortmannin can block noradrenaline-stimulated DNA synthesis and activation of MAP kinase, suggesting that PI 3-kinase plays an important role in α receptor-mediated mitogenesis in HVSMCs.
      The capacity of a whole range of tyrosine kinase receptors or TPKs (Cantley et al., 1991; Rordorf-Nikolic et al., 1995; Van der Geer and Hunter, 1991) to activate PI 3-kinase has been extensively studied; the activated PI 3-kinase is believed to play an important role in signal transduction pathways of peptide growth factors (Fry, 1994). On the other hand, there is an increasing experimental evidence indicating that PI 3-kinase may also be involved in signaling pathways of G protein-coupling receptors; most of this evidence has been derived from investigations of thrombin receptors in platelets (Stephens et al., 1993; Zhang et al., 1995) and chemoattractant receptors in neutrophils (Bokoch, 1995; Varticovski et al., 1994). To determine directly if activation of α receptors expressed in HVSMCs activated PI 3-kinase, we implemented an in vitro assay of PI 3-kinase activity in immunocomplexes precipitated by an antibody against the p85α subunit of PI 3-kinase (Yano et al., 1993). The results demonstrated that noradrenaline stimulated a time-dependent activation of PI 3-kinase at concentration of 10 μM in the presence of a β adrenergic receptor antagonist timolol (1 μM) (Fig. 2A and Table 1). Noradrenaline stimulated a very rapid and significant activation of PI 3-kinase that occurred as early as 1 min after activation of α receptors, with the peak activity at 5 min. The activity of PI 3-kinase declined to basal values 30 min after continued exposure to noradrenaline. Noradrenaline-stimulated activation of PI 3-kinase was also concentration-dependent (Fig. 2B and Table 1). 1 μM of noradrenaline caused a significant increase in PI 3-kinase activity. However, the increased PI 3-kinase activity declined at 100 μM of noradrenaline concentration, indicating that blunted response of the α receptors to their ligand occurred. Noradrenaline-activated PI 3-kinase was specifically mediated by α receptors because the α-selective antagonist terazosin inhibited this activation; on the other hand, the β adrenergic receptor antagonist timolol and α adrenergic receptor antagonist idazoxan did not inhibit activation of PI 3-kinase (Fig. 3, lanes 2-4). Additionally, activation of PI 3-kinase by noradrenaline was inhibited by wortmannin (10 nM) as expected (Fig. 3, lane 6).
      Figure thumbnail gr2
      Figure 2Noradrenaline-stimulated time- and concentrationdependent activation of PI 3-kinase. A, cells were treated with vehicle or noradrenaline (10 μM) for the indicated times. The whole cell lysates (1 mg of protein) were subjected to immunoprecipitation with a rabbit polyclonal antibody against the p85α subunit of PI 3-kinase. PI 3-kinase activity in immunoprecipitates from noradrenaline-treated or control cells was determined as described under “Experimental Procedures.” Change in activity of PI 3-kinase is presented as production of phosphatidylinositol phosphate (PIP). The autoradiogram of TLC of PI 3-kinase was exposed for 20 h. Experiments were repeated 3 times with essentially identical results. B, cells were treated with vehicle or the indicated concentrations of noradrenaline for 5 min, and cell lysates were prepared and immunoprecipitated as described above. The autoradiogram of TLC of PI 3-kinase was exposed for 24 h. Experiments were repeated 3 times with essentially identical results.
      Figure thumbnail gr3
      Figure 3Activation of PI 3-kinase inhibited by α adrenergic receptor antagonists and pertussis toxin. Cells were pretreated with α receptor-selective antagonist terazosin (10 μM) or the α receptor-selective antagonist idazoxan (10 μM), an inhibitor of PI 3-kinase wortmannin (10 nM) for 2 h or with pertussis toxin (PTx; 100 ng/ml) for 12 h. Cells were then treated with vehicle, 10 μM of noradrenaline or 100 ng/ml of IGF-I for 5 min. Cell lysates (1 mg of protein) were prepared, immunoprecipitated with anti-p85α of PI 3-kinase antibody, and subjected to determination of PI 3-kinase as described above. The autoradiogram of TLC of PI 3-kinase was exposed for 24 h. Experiments were repeated 3 times with similar results.
      Thrombin and several chemoattractants stimulate cell responses in several different cell types via activation of pertussis toxin-sensitive G proteins leading to activation of PI 3-kinase. There are several signaling pathways that are utilized by G protein coupling to thrombin and chemoattractant receptors in the activation of PI 3-kinase (Bokoch, 1995; Stephens et al., 1993). One pathway involves pertussis toxin-sensitive G proteins including P21 heterologous small G proteins such as Ras or Rho (Zhang et al., 1995). Another pathway occurs via activation of the traditional TPKs such as SRC kinases (Cantley et al., 1991). For the latter pathway, activation of thrombin and chemoattractant receptors leads to phosphorylation of cytosol TPKs, which in turn may provide phosphorylated site(s) for binding of the p85α subunit of PI 3-kinase through SH2 to TPKs. Additionally, a recent study has identified a novel p110 isoform of the catalytic subunit of PI 3-kinase that is activated without association with the p85 subunit of the originally described PI 3-kinase heterodimer (Stoyanov et al., 1995); interestingly, β subunits released from receptor-activated G proteins directly activate this newly described PI 3-kinase catalytic moiety (Stephens et al., 1993). Although it has been generally accepted that α receptor-stimulated responses are likely predominantly mediated by α subunits released by pertussis toxin-insensitive G proteins, likely in the Gq/11 family (Schwinn et al., 1995), increasing evidence suggests that pertussis toxin-sensitive G proteins may also be utilized to transduce the signals of α receptor stimulation (Perez et al., 1993). To test if pertussis toxin-sensitive G proteins are involved in α receptor-mediated activation of PI 3-kinase, HVSMCs were preincubated with pertussis toxin (100 ng/ml for 12 h) and then stimulated by noradrenaline; as indicated in Fig. 3, lane 5, pertussis toxin completely blocked activation of PI 3-kinase in these cells. However, IGF-I (100 ng/ml), a well-known activator of a tyrosine kinase receptor, increased PI 3-kinase activity in these cells, but the response was not attenuated by pertussis toxin (Fig. 3, lanes 7 and 8). This result demonstrates the specificity of pertussis toxin in inhibiting activation of PI 3-kinase by α receptors. α receptors are known to activate protein kinase C and increase intracellular concentrations of Ca. We found that the L channel Ca blocker nifedipine did not inhibit noradrenaline-stimulated PI 3-kinase (data not shown); 4β-phorbol 12,13-dibutyrate, an activator of protein kinase C, did not increase PI 3-kinase activity in these cells, suggesting that activation of PI 3-kinase is independent of activation of protein kinase C.
      To determine if there was direct coupling of α receptor to PI 3-kinase activity as has been found for tyrosine kinase receptors, α or α receptors were immunoprecipitated with specific antibodies directed against each of these subtypes. The α or α receptor proteins could be recovered by these antibodies, but no PI 3-kinase activity was detected in immunoprecipitates of noradrenaline-treated cell lysates with either antibody, suggesting that activation of PI 3-kinase by noradrenaline is transduced by downstream mechanisms that do not invoke docking of PI 3-kinase with these receptors (data not shown). To determine if Ras and TPKs are involved in activation of PI 3-kinase by α receptors, cell lysates from control or noradrenaline-treated HVSMCs were immunoprecipitated with anti-Ras or anti-phosphotyrosine; PI 3-kinase activity was measured in the immunoprecipitates (Fig. 4A). There was basal activity of PI 3-kinase detected in Ras or tyrosine protein immunocomplexes from control cells; however, a significant increase in PI 3-kinase activity could be detected in both anti-Ras- or anti-phosphotyrosine immunocomplexes from noradrenaline-stimulated cells (Fig. 4A and Table 2). These results demonstrate that both Ras protein and tyrosine proteins are associated with α receptor-activated PI 3-kinase in HVSMCs. However, these data do not provide the sequence of activation of PI 3-kinase, Ras protein, or tyrosine kinases by α receptors. It is known that PI 3-kinase may act at either downstream (Kodaki et al., 1994) or upstream of Ras protein (Rodriguez-Viciana et al., 1994) in other cells. We further investigated the interaction of PI 3-kinase and Ras protein in noradrenaline-stimulated HVSMCs; as illustrated in Fig. 4B, noradrenaline stimulated a time-dependent increase in Ras-bound GTP in the presence of antagonists of α and β receptors, suggesting that activation of α receptors stimulates an increase in the active Ras-GTP. On analysis of the temporal relationship between activation of PI 3-kinase (Fig. 2A) and increased active Ras protein (Fig. 4B), the active Ras-GTP appeared later than activation of PI 3-kinase, suggesting that Ras protein might function as a target of PI 3-kinase after stimulation of cells with noradrenaline. This possibility was supported by the fact that noradrenaline-stimulated increase in the active Ras-GTP could be partially blocked by the specific inhibitor of PI 3-kinase wortmannin as was terazosin, pertussis toxin, and genistein (Fig. 4C). We postulate that Ras protein is localized downstream of PI 3-kinase and functions as a target of PI 3-kinase. The definite interaction between the two important protein molecules in HVSMCs by activation of α receptors will require further investigation. Since increased activity of PI 3-kinase had been found in anti-phosphotyrosine protein immunocomplexes, cells were metabolically labeled with [P]P and stimulated with or without noradrenaline. Cell lysates from control and noradrenaline-treated cells were immunoprecipitated with antibodies directed against the p85 subunit of PI 3-kinase and then resolved by SDS-polyacrylamide gel electrophoresis. Noradrenaline stimulated phosphorylation of the p85 subunit of PI 3-kinase (Fig. 4D, lanes 1-3). To determine if noradrenaline stimulated tyrosine phosphorylation of PI 3-kinase, cell lysates from control and noradrenaline-treated cells were immunoprecipitated with anti-p85 antibody and then detected by anti-phosphotyrosine antibody. Results indicated that noradrenaline stimulated a tyrosine phosphorylation of p85 itself (Fig. 4E, lanes 1-3).
      Figure thumbnail gr4
      Figure 4Noradrenaline-activated PI 3-kinase associates with activated Ras and a tyrosine-phosphorylated protein. A, cells were treated with vehicle or noradrenaline (10 μM) for 5 min. Cell lysates (500 μg of protein) were subjected to immunoprecipitation (IP) with anti-H-Ras (lanes 1 and 2), anti-phosphotyrosine (anti-Tyr) (lanes 3 and 4), or anti-p85 of PI 3-kinase (lanes 5 and 6). PI 3-kinase activity in immunoprecipitates from noradrenaline-treated or control cells was determined as described in . The autoradiogram of TLC of PI 3-kinase was exposed for 16 h. Experiments were repeated twice with essentially identical results. B, HVSMCs were metabolically labeled with [P]P for 12 h and treated with noradrenaline (10 μM) in the presence of timolol (1 μM) and idazoxan (1 μM) for the indicated times. Cell lysates were prepared and immunoprecipitated with anti-Ha-Ras antibody. TLC was used to separate GTP and GDP; autoradiograms of TLC plates were exposed for 10 h. Ras-bound GTP (percentage of GTP + GDP) was calculated with a PhosphorImager system (Molecular Dynamics), and percentages are shown at the bottom. Experiments were repeated 3 times with essentially identical results. C, HVSMCs were labeled as in B. Inhibitors as indicated were added to dishes for 2 or 12 h (pertussis toxin; PTX) before stimulation of cells with noradrenaline for 10 min. Changes in Ras-bound GTP were measured as in B. The autoradiogram of TLC was exposed for 10 h. Experiments were repeated twice with essentially identical results. D, HVSMCs were metabolically labeled with [P]P for 12 h and treated with noradrenaline (10 μM) in the presence of timolol (1 μM) and idazoxan (1 μM) for the indicated times. Cell lysates were prepared and immunoprecipitated with anti-p85 of PI 3-kinase and then resolved by SDS-polyacrylamide gel electrophoresis. The autoradiogram of the film was exposed for 16 h. Experiments were repeated twice with essentially identical results. This result indicates that noradrenaline treatment stimulated a phosphorylation of the p85 of PI 3-kinase as indicated by the arrow. E, cell lysates from control or noradrenaline-treated cells were immunoprecipitated with anti-P85 of PI 3-kinase and resolved by SDS-polyacrylamide gel electrophoresis. Blots were probed with an anti-phosphotyrosine antibody. Experiments were repeated twice with essentially identical results. This result suggests that noradrenaline stimulates tyrosine phosphorylation of p85 as indicated by the arrow.
      In summary, the results demonstrate that α receptors expressed in HVSMCs are coupled to stimulation of PI 3-kinase via pertussis toxin-sensitive G proteins. Activation of PI 3-kinase by noradrenaline leads to association of the kinase with activation of Ras proteins and TPKs. The results highlight the potential importance of α receptors in the activation of PI 3-kinase, particularly concerning activation of mitogenesis in vascular smooth muscle cells. Moreover, these results broaden concepts relating to interaction and cross-talk of α adrenergic receptors with families of tyrosine kinases.

      Acknowledgments

      We thank Robert J. Lefkowitz's laboratory for providing anti-α and α receptor antibodies.