Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Protein tyrosine phosphorylation, stimulated by mitogens such as peptide growth factors, plays an important role in regulation of development, growth, differentiation, and other biological functions in many cells including vascular smooth muscle cells (VSMCs; Srivastava, 1995; Seger and Krebs, 1995). Signaling pathways involved in sequential activation of Ras, Raf-1, and ultimately the protein kinase cascade termed mitogen-activated protein kinase (MAPK) have been shown to be very important in mediating receptor tyrosine kinase (RTK) regulation of cell growth and differentiation (Schlessinger and Ullrich, 1992). Recent work suggests that tyrosine kinase-Ras-MAPK signaling pathways may be also utilized by G protein-coupled receptors (GPCRs), including those for angiotensin II, endothelin, thrombin, and others (for review, see Malarkey et al., 1995; Post and Brown, 1996). Modulation of activity of multiple protein kinases is a key control step in regulation of pathophysiological processes of blood vessels such as growth and vascular remodeling.

₁-Adrenergic receptors (ARs) are members of the class of GPCRs and mediate many of the important physiological effects of catecholamines such as epinephrine. ₁-ARs play a particularly important role in control of cardiovascular responses such as regulation of blood pressure via activation of smooth muscle contraction (Graham et al., 1996). Activation of ₁-AR also stimulates cardiac and vascular smooth muscle growth and hypertrophy (Jackson and Schwartz, 1992; Milano et al., 1994). Human vascular smooth muscle cells (HVSMCs) and cardiac myocytes express at least three subtypes of ₁-ARs, namely, _1A, _1B, and _1D receptors (Price et al., 1994; Hieble et al., 1995). It has been generally accepted that activation of all three subtypes of ₁ receptors increases hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP₃) and diacylglycerol via Gq, a family of pertussis toxin-insensitive G proteins leading to activation of protein kinase C (PKC) and raising intracellular Ca²⁺. Recent evidence demonstrates that ₁ receptors also stimulate production of IP₃ and diacylglycerol via activation of phospholipase C via subunits released from G proteins (Muller and Lohse, 1995). Furthermore, ₁ receptors activate phospholipase D in brain and promote the release of arachidonic acid by activation of phospholipase A2 via pertussis toxin-sensitive G proteins (Perez et al., 1993).

Traditionally, ₁-ARs have been thought of as mainly utilizing PKC and Ca²⁺ to mediate their effects in cells. Indeed, substantial evidence indicates that activation of PKC is involved in ₁-AR induction of mitogenic effects in cardiac myocytes and VSMCs (Kariya et al., 1994). On the other hand, although Ca²⁺ plays an important role in the regulation of smooth muscle contraction, there is little evidence demonstrating a mitogenic role of intracellular Ca²⁺. Additionally, it has become clear in the past several years that activation of PKC or/and raising Ca²⁺ are not sufficient to initiate cell cycle progression (Malarkey et al., 1995; Nishizuka, 1995). Consequently, there likely exist additional signaling mechanisms that contribute to ₁-AR stimulation of growth-related nuclear events. Recent evidence suggests that ₁-AR-stimulated mitogenic responses in neonatal myocytes involve activation of tyrosine protein kinases (TPKs) and activation of the MAPK pathway because inhibitors of tyrosine kinase and MAPK block ₁-AR agonist stimulation of myocyte hypertrophy (Thorburn et al., 1994). However, it is not clear how activation of TPKs leads to MAPK activation and mitogenic responses in these cells. Also, to what extent tyrosine kinases and Ras-MAPK signaling pathways are utilized to mediate ₁-AR effects in VSMCs is largely unknown.

In the present study, we have found ₁-AR activated mitogenic responses such as DNA synthesis and increased cell proliferation; these effects were attenuated by inhibitors of TPKs and MAPK, suggesting that activation of ₁-AR in HVSMCs activates protein kinase cascades, leading to promotion of HVSMC growth. Further experiments demonstrated that ₁-AR agonists directly stimulate phosphorylation and increase in activities of TPKs and MAPKs in these cells. Interestingly, we found that intracellular calcium and calcium-linked TPKs play a critical role in mediating catecholamine stimulation of MAPK signaling pathways.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Control antiserum (anti-rabbit serum and anti-IgG), genistein, H7, myelin basic protein (MBP), norepinephrine, and 4-phorbol 12,13-dibutyrate (PDBu) were purchased from Sigma (St. Louis, MO); [-³²P]ATP (2000 Ci/mmol), [³²P]orthophosphate (370 MBq/ml), and an enhanced chemiluminescence Western Detection System were obtained from Amersham Corp. (Arlington, IL). Immobilon-P transfer membranes were purchased from Millipore Corp. (Bedford, MA); cell culture medium, fetal bovine serum, and human recumbent PDGF-AB and -BB were obtained from Gibco-BRL (Grand Island, NY). Wortmannin was purchased from Worthington Biochemical Co. (Freehold, NJ); antibodies against TPKs, ERK1/2, PLC1, Shc, TPK substrates, and PKC/PKA inhibitors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated MAPK and anti-ERK antibodies were obtained from New England BioLabs, Inc. (Beverly, MA). All other chemicals were reagent or molecular biology grade and were obtained from standard commercial sources.

Preparation of Cultured Human Aortic Smooth Muscle Cells. Human aortic VSMCs were purchased from Clonetics Corp. (San Diego, CA). Cells were grown in smooth muscle growth medium-2 with 5% fetal bovine serum obtained from Clonetics Corp. or maintained in Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO₂/95% 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 to 90% confluence being reached about 10 days later. The medium was replaced every 2 days. Cells were generally used for studies at 8 to 10 days after seeding. To examine effects of norepinephrine-stimulated changes, cells were incubated with DMEM without serum for the indicated time after achieving confluence. Throughout the course of these experiments, cells from the fifth through seventh passage were used. The cells were treated with agonists or vehicle solution (as control) starting from the longest time point and the cells were harvested at the same time.

₁ Agonist-Induced DNA Synthesis and Cell Proliferation. Stimulation of ₁ receptors induces DNA synthesis (Nakaki et al., 1990) and cell proliferation (Kuriyama et al., 1988). HVSMCs were cultured in DMEM containing 5% fetal bovine serum to near confluence. The medium was replaced with medium containing 0.4% serum for 48 h. Norepinephrine (1 µM) plus the -AR antagonist timolol (1 µM) and ₂-AR antagonist idazoxan (1 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 ₁-receptor agonist-induced increases in [³H]thymidine incorporation occurred via activation of TPKs and MAPKs or through pertussis toxin-sensitive G proteins, cells were preincubated with inhibitors of TPKs and MAPKs for 2 h or with pertussis toxin for 12 h before each experiment.

Immunoprecipitation and Immunodetection. Cultures on 100-mm plates were rinsed with ice-cold PBS containing 1 mM sodium orthovanadate. Cells were incubated with cell lysis buffer [1% nonide 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, and 10 µg/ml of aprotinin, leupeptin] for 10 to 15 min on ice. Insoluble material was removed by centrifugation at 12,100g for 20 min. The amount of cell lysate was normalized by protein content in each experiment. Lysates were incubated with various antibodies as described for 2 h and then with 20 µl of protein A/G plus agarose for 1 h. The beads containing the immunoprecipitates were washed 3 times with cell lysis buffer, once with washing buffer (0.1 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5), once with kinase buffer, and subjected to MAPK assays. For immunodetection, immunoprecipitates were washed three times with lysis buffer, twice with distilled water, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Resolved proteins were transferred to membrane and detected using the enhanced chemiluminescence western blotting detection system (Amersham) with the indicated primary antibody and an appropriate horseradish peroxidase conjugated secondary antibody.

Analysis of Phosphotyrosine Phosphorylation. Analysis of the phosphorylation of phosphotyrosine proteins followed a method described by Siegel (1994). Confluent cultures of cells were serum-starved for 12 h and then labeled with 0.1 mCi/(1 Ci = 37.5 GBq)/ml of [³²P]P_i in phosphate-free DMEM for 12 h. Cells were then stimulated with or without norepinephrine, phenylephrine, or PDGF for the indicated times. In experiments using ₁ antagonists or inhibitors of PKC and tyrosine kinases, the respective compounds were added to cells 60 min before stimulation with agonists and growth factors. After indicated times (1-30 min), cells were washed with ice-cold PBS buffer and lysed on ice in 0.5 ml of cell lysis buffer. Equal protein aliquots (1 mg) were precipitated with 2 µg/mg antiphosphotyrosine antibodies and washed as described above. Immunoprecipitates were solubilized in SDS-PAGE sample buffer and subsequently heated to 95°C for 5 min. Supernatants were resolved by 8% SDS-PAGE. Gels were dried and followed by autoradiography using a PhosphorImager or exposed to Kodak XAR-5 film at 70°C with an intensifying screen for the indicated times.

Autophosphorylation of TPKs. Quiescent HVSMCs were stimulated with the indicated agents for the indicated times, and whole cell lysates were prepared as described above. Equal protein aliquots (1 mg) were immunoprecipitated for 2 h at 4°C using antibodies against phosphotyrosine proteins (2 µg/mg protein) in the absence or presence of phosphotyrosine (0.2 mM). Immune complexes were recovered as described above and subjected to an additional final rinse with tyrosine kinase buffer (50 mM HEPES, pH 7.4, 10 mM MnCl₂, 1 mM ATP). Immune complexes were resuspended in 25 to 50 µl of kinase buffer containing 50 µCi of [-³²P]ATP. Protein kinase reactions were carried out for 15 min at 37°C. Reactions were stopped by addition of SDS-PAGE sample buffer and subsequently heated to 95°C for 5 min. Labeled phosphoproteins were resolved by 10% SDS-PAGE and visualized by autoradiography as described previously (Burkhardt, 1994).

In Vitro Assays of TPK Activity. For assay of TPK activity, whole cell lysates (400 mg) were immunoprecipitated with antiphosphotyrosine antibody (2 µg/mg protein) as described above. The washed immunocomplexes were resuspended in 50 µl of kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 0.5 mM EGTA, 1 mM dithiothreitol, 40 nM ATP, 1 µCi of [-³²P]ATP, and 10 µg of TPK peptide substrate specific for Src-family kinases (cdc2:8-20; Santa Cruz Biotechnology) (Burkhardt, 1994). The reaction mixture was incubated for 10 min at 30°C because preliminary experiments suggested that the TPK activity is linear for at least 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 for 1 h and transferred to another washing overnight. The papers were washed with acetone for 5 min and dried. ³²P was quantitated by scintillation counting.

In Vitro Assay of MAPK Phosphorylation and Activity. To determine phosphorylation of MAPK, cells were incubated in the absence of serum for 18 h and cells were treated with norepinephrine or other agonists for various times. The cells were lysed in 0.4 ml of lysis buffer. After a 30-min centrifugation (500g) at 4°C, cell lysates (100 µg of protein) were loaded on 12% SDS-PAGE and transferred to membranes as described above for Western blotting. The membrane was probed with an antiphosphorylated MAPK antibody or with an anti-p44^ERK1 as control.

[Ca²⁺]_i Measurement. HVSMCs were plated on coverslips to form a monolayer and loaded with 1.5 µM Fura-2 pentaacetoxymethyl in HBSS containing 0.1% BSA at room temperature for 30 min. Cytosolic free Ca²⁺([Ca²⁺]_i) was determined as described previously (Chen and Giri, 1997). Cell Ca²⁺ responses are expressed as the ratio (F340/F380) of fluorescence intensity at excitation of 340 and 380 nm. For testing chelation of BATPA, the cells were pretreated with 10 mM BAPTA/AM or vehicle DMSO for 30 min at 37°C before fluorescence measurement.

Data Analysis. Data are presented as mean ± S.E.M., and treatment effects were compared by one-way ANOVA or Student's paired t test (two-tailed). P < .05 was used as level of significance.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Inhibitors of TPKs Block Norepinephrine-Induced DNA Synthesis and Cell Proliferation in Human Smooth Muscle Cells. Activation of ₁-AR in rat or human myocytes and VSMCs stimulates DNA synthesis, protein synthesis, and growth-related gene expression (Nakaki et al., 1990; Okazaki et al., 1994; Chen et al., 1996). We wondered if norepinephrine-stimulated DNA synthesis and cell proliferation could be blocked by inhibitors of TPKs and MAPK. Stimulation of HVSMCs with norepinephrine (1 µM) resulted a 90% increase in DNA synthesis (Fig. 1A). This effect of norepinephrine was blocked by ₁-AR antagonist terazosin (1 µM) but not by -AR antagonist timolol (1 µM) or by ₂-AR antagonist idazoxan (1 µM), suggesting that norepinephrine stimulated DNA synthesis via activation of ₁-AR (Fig. 1A). The TPK inhibitor genistein, the MAPK inhibitor 2-aminopurine (2-AP), and the PKC inhibitor H7, completely or partially blocked norepinephrine-stimulated DNA synthesis (Fig. 1A). To investigate potential actions of these inhibitors in norepinephrine stimulation of cell proliferation, partial confluent HVSMCs (70%) were treated with norepinephrine for 3 days in the presence of a -AR antagonist timolol (1 µM) and an ₂-AR antagonist idazoxan (1 µM). Norepinephrine treatment resulted a 50% increase in cell number; TPK, PKC, and MAPK inhibitors, and ₁-AR antagonist terazosin blocked norepinephrine stimulation of cell proliferation (Fig. 1B).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of inhibitors on DNA synthesis and cell proliferation. A, HVSMCs were grown to near confluence in 24-well dishes. The quiescent cells were incubated with DMEM containing 0.4% fetal calf serum plus norepinephrine (1 µM) for 20 h in the presence of timolol (1 µM) and idazoxan (1 µM). Cells were then incubated with [3H]thymidine (0.1 µCi) for another 4 h. Inhibitors were added to cell dishes 1 h before addition of norepinephrine. The inhibitor H7 (1 µM), 2-AP (1 mM), or genistein (0.1 µM) was added as indicated. Incorporation of [3H]thymidine into cells was performed as described in Experimental Procedures. The data are average ± S.E.M of three experiments performed in triplicate. *, comparison to control, p < .05. B, HVSMCs were grown to 70% confluence and incubated with 0.4% fetal calf serum for 48 h. Then medium was changed to DMEM containing 0.4% fetal calf serum plus norepinephrine (1 µM) or PDBu (50 nM), and cells were incubated for 72 h in the presence of timolol (1 µM) and idazoxan (1 µM). Genistein (0.1 µM), 2-AP (1 mM), and H7 (1 µM) were added as indicated. These antagonists were added to cell dishes 1 h before the addition of agonists. At the end of incubation, cells were counted as described in Experimental Procedures. The data are average ± S.E.M of three experiments performed in triplicate. *, comparison to control, p < .05.

₁ Receptors Stimulate Phosphorylation of Tyrosine Proteins. To determine whether incubation of HVSMCs with norepinephrine stimulated phosphorylation of tyrosine proteins, cells were metabolically labeled with ³²P_i for 12 h and stimulated with norepinephrine (10 µM) for the indicated times in the presence of timolol and idazoxan as illustrated in Fig. 2. Norepinephrine rapidly stimulated a time-dependent phosphorylation of several tyrosine proteins, including p145, p125, p85, p75, and p52 (Fig. 2A). Increased phosphorylation of tyrosine proteins occurred at 2 min and was sustained for about 60 min. Norepinephrine induced a similar tyrosine phosphorylation of several protein molecules as platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor I (IGF-I). Figure 2B illustrates the dose-response for norepinephrine-induced phosphorylation of tyrosine proteins. Concentrations of norepinephrine as low as 100 nM stimulated tyrosine phosphorylation of several protein molecules. Figure 2C illustrates the results of inhibitory effects of a ₁ receptor antagonist, inhibitors of TPKs, PKC, and an L-type Ca²⁺ channel blocker. Terazosin, an antagonist of ₁-ARs, inhibited norepinephrine-induced phosphorylation of tyrosine proteins in these cells. Genistein, a TPK inhibitor, partially inhibited norepinephrine-stimulated phosphorylation of some of the TPKs. However, H7, a PKC inhibitor and nifedipine, an L-type Ca²⁺ channel blocker, had little if any effect on norepinephrine-stimulated phosphorylation of tyrosine proteins. This conclusion was further supported by experiments demonstrating that down-regulating PKC by preincubation cells with 4-phorbol 12-myristate 13-acetate (10 µM) for 24 h did not inhibit norepinephrine-stimulated tyrosine phosphorylation (data not shown). Interestingly, a membrane-permeable Ca²⁺ chelator [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA-AM) (10 µM) completely inhibited norepinephrine-induced tyrosine phosphorylation of multiple protein molecules (Fig. 2C), suggesting that the rise in intracellular [Ca²⁺] stimulated by norepinephrine is critical for ₁-AR-mediated tyrosine protein phosphorylation in HVSMCs. Specificity of the antiphosphotyrosine protein antibody was confirmed by experiments in which tyrosine-phosphorylated proteins were not precipitated by control antiserum such as anti-rabbit serum or anti-IgG in the ³²P-labeled HVSMCs (Fig. 2D).

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Norepinephrine stimulated time- and concentration-dependent phosphorylation of protein tyrosine kinases. A, [32P]Pi-labeled cells were treated with vehicle or norepinephrine (10 µM), PDGF-BB (1 nM), or IGF-I (10 nM) for the indicated times. Cell lysates (1 mg of protein) were subjected to immunoprecipitation with a monoclonal antibody against phosphotyrosine proteins. Phosphorylation of tyrosine proteins in immunoprecipitates from control or agonist-treated cells was determined as described in Experimental Procedures. The autoradiogram of film was exposed for 20 h. Experiments were repeated three times with essentially identical results. B, cells were treated with vehicle or indicated concentrations of norepinephrine for 10 min and cell lysates were prepared and immunoprecipitated as described above. The autoradiogram of film was exposed for 24 h. Experiments were repeated three times with essentially identical results. C, cells were metabolically labeled with [32P]Pi and treated with norepinephrine (10 µM) as described above. Various inhibitors including terazosin (1 µM), genistein (50 µM), H7 (10 µM), nifedipine (10 µM), and BAPTA-AM (10 µM) were added to cell dishes 1 h before addition of agonists. Data is a representative of three experiments. D, cell lysate (1 mg of protein) was subjected to immunoprecipitation with an anti-rabbit serum or an anti-IgG antibody respectively. Phosphorylated tyrosine proteins in immunoprecipitates from control or agonist-treated cells were determined. The autoradiogram of film was exposed for 22 h. Experiments were repeated three times with essentially identical results.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Intracellular Ca2+ chelator BAPTA-AM attenuated 1-adrenergic agonist-stimulated Ca2+ mobilization. Fura2-loaded cells were incubated with DMSO control (A) or intracellular Ca2+ chelator BAPTA-AM (10 µM; B) for 30 min at 37°C. A normal Ca2+ response to 10 µM phenylephrine (PE) is shown in the DMSO-pretreated cells with Ca2+ influx after addition of Ca2+ (2 mM). In cells pretreated with BAPTA-AM, PE did not significantly increase in [Ca2+ ]i until ionomycin (Ion) was added, suggesting that pretreatment with BAPTA attenuated PE-stimulated Ca2+ mobilization but did not interfere with the Ca2+/Fura2 signal and cell viability. Data is representative of three experiments.

₁ Receptors Increase Tyrosine Kinase Phosphorylation and Activity. The capability of ₁ receptor activation to induce phosphorylation of TPKs in HVSMCs was investigated by measurements of phosphorylation in vitro of proteins immunoprecipitated by an antiphosphotyrosine antibody in norepinephrine-treated HVSMCs (Fig. 4). In these experiments, HVSMCs were treated with or without norepinephrine (10 µM) for 10 min, cell lysates were immunoprecipitated with antiphosphotyrosine antibody, and in vitro kinase activity was determined as described in Experimental Procedures. The results of these experiments demonstrated that norepinephrine stimulated phosphorylation of some of these proteins (autophosphorylation; Fig. 4). Precipitation of cell lysates with antiphosphotyrosine antibody in the presence of 2 mM tyrosine phosphate significantly inhibited autophosphorylation of these phosphotyrosine proteins except for a protein of p85 kDa (Fig. 4A, lanes 3 and 5), which may relate to serine/threonine phosphorylation of ₁ receptor-stimulated phospholipase A2 (Xing and Insel, 1996) or p85 subunit of phosphatidylinositol 3-kinase (PI-3-kinase; Hu et al., 1996).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Autophosphorylation of norepinephrine-stimulated TPKs. HVSMCs were incubated with serum-free DMEM for 24 h and then treated with vehicle, 10 µM norepinephrine, or 1 nM PDGF-BB for 10 min. Cell lysates (1 mg of protein) were prepared, immunoprecipitated, and subjected to autophosphorylation of TPKs as described in Experimental Procedures. The autoradiogram of film was exposed for 10 h. Experiments were repeated three times with similar results.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Activities of TPKs in norepinephrine-stimulated HVSMCs. Confluent HVSMCs were incubated with serum-free DMEM for 24 h and cells were treated with or without norepinephrine (10 mM) for indicated times in the presence of timolol (1 µM) and idazoxan (1 µM). Cell lysates (400 µg) were subjected to immunoprecipitation with antiphosphotyrosine. TPK activities in the immunocomplexes were determined using a synthetic TPK peptide (10 µg) specific for Src family kinases as kinase substrate as described in Experimental Procedures. Relative kinase activity was calculated using control activity at 0 min as 100%. The data are average ± S.E.M. of four experiments.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Norepinephrine stimulated tyrosine-phosphorylated proteins included PLC1 and adapter protein Shc. Quiescent HVSMCs were treated with or without norepinephrine (10 µM) for indicated times in the presence of timolol (1 µM) and idazoxan (1 µM). Terazosin (1 µM; T) or genistein (50 µM; G) was added into dishes 1 h before norepinephrine treatment. Cell lysates were subjected to immunoprecipitation with an antiphosphotyrosine or antiphospholipase C1 antibodies and Western blotting with anti-phospholipase C1 antibody (A). These results show increased tyrosine phosphorylation of phospholipase C1, inhibited by T and G with similar total amounts of phospholipase C1 in each group of cells. B, illustrates similar experiments involving immunoprecipitation with an antiphosphotyrosine antibody and Western blotting with anti-Shc antibody. The data are a representative of three experiments.

₁ Receptor Stimulation of MAPK Partially Involves a Pertussis Toxin-Sensitive G Protein. The MAPK cascade plays an important role in mediating TPK signaling pathways stimulated by many growth factors, as well as ligands for GPCRs (Malarkey et al., 1995). We had found that norepinephrine-stimulated tyrosine phosphorylation included two protein molecules at the position of 44/42 kDa (Fig. 2). Because this doublet is compatible in size with ERK1/2, we further determined the profiles of MAPK activation in norepinephrine-treated HVSMCs. Figure 7 illustrates these results. Norepinephrine (1 µM) stimulated a rapid increase in phosphorylation of p44/42^ERK1/2 (Fig. 7A) without a change in the amount of proteins (Fig. 7B). Also, norepinephrine stimulated an increase in MAPK activity in the presence of timolol and idazoxan (Fig. 7C). Norepinephrine-stimulated increase in MAPK activity was time-dependent and the increased activity was maintained above basal for 30 min (Fig. 7, C and D). The norepinephrine-stimulated increase in activity of MAPK could be almost completely blocked by ₁ receptor antagonist terazosin (1 µM; Fig. 8). Interestingly, pertussis toxin (100 ng/ml) partially inhibited activation of MAPK, suggesting that norepinephrine-stimulated activation of MAPK may involve a pertussis toxin-sensitive G protein. Additionally, norepinephrine-stimulated activation of MAPK could be partially blocked by the PTK inhibitor genistein or by Ca²⁺ chelator BAPTA-AM (10 µM; Fig. 8), suggesting that TPK and the intracellular Ca²⁺ are critical for ₁-AR-mediated MAP activation in HVSMCs. Norepinephrine-stimulated MAPK activity was also partially inhibited by PKC inhibitor H7 (data not shown). IGF-I receptors are RTKs known to activate MAPK (Hansson and Thoren, 1995) and PI-3 kinase (Karenberg et al., 1994). IGF-I-increased MAPK activity in these cells was not inhibited by pertussis toxin (Fig. 8). These results suggest that pertussis toxin was not having nonspecific effects on hormonal activation of MAPK.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Norepinephrine stimulated phosphorylation and increase in activity of MAPK. A, norepinephrine stimulated phosphorylation of p42/44ERK1/2. Quiescent HVSMCs were treated with or without norepinephrine (10 µM) for indicated times in the presence of timolol (1 µM) and idazoxan (1 µM). Cells were lysed by direct addition of SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blotting. The blots were probed with an antiphosphorylated MAPK described in Experimental Procedures. The data is a representative of three experiments. B, same blot was probed with an anti-p44ERK1, indicating that similar amounts of these proteins were in each lane. C, norepinephrine stimulated a time-dependent increase in activity of MAPK. Quiescent HVSMCs were treated with or without norepinephrine (10 µM) for indicated times. Cell lysates (250 µg) were prepared and subjected to immunoprecipitation with an anti-p42ERK2 antibody. Washed immunocomplexes were resuspended in a kinase buffer and subjected to kinase activity assay using MBP as substrate as described in Experimental Procedures. D, data are average ± S.E.M of three experiments.

View larger version (63K): [in this window] [in a new window]   Fig. 8.   Effect of inhibitors on norepinephrine-stimulated activation of MAPK. Cells were treated as described in the legend of Fig. 7. Inhibitors (1 µM terazosin, 50 µM genistein, or 10 µM BAPTA-AM) were added to dishes 1 h before norepinephrine (10 µM) or IGF-I (10 nM) treatment. PTx (100 ng/ml) was added 12 h before agonist treatment. Cell lysates (250 µg) were prepared and subjected to immunoprecipitation with an anti-p42ERK2 antibody. Washed immunocomplexes were resuspended in a kinase buffer and kinase activity was measured using MBP as substrate. Data are average ± S.E.M. of four experiments. *, comparison to control, p < .05.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Regulation of tyrosine protein phosphorylation by various mitogens plays a central role in the control of cell growth and differentiation. In the present study, our results demonstrate that inhibitors of TPKs and MAPK attenuate catecholamine-stimulated DNA synthesis and cell proliferation in HVSMCs. Norepinephrine was found to stimulate time- and concentration-dependent tyrosine phosphorylation of multiple proteins. Tyrosine-phosphorylated proteins had TPK activity in autophosphorylation assays, suggesting that some of these proteins may function as TPKs. This observation was further supported by experiments that directly demonstrated increased TPK activity in norepinephrine-stimulated HVSMCs. These effects of norepinephrine were completely blocked by a TPK inhibitor genistein. A PKC inhibitor and an L-type calcium channel blocker did not attenuate norepinephrine-stimulated phosphorylation of tyrosine proteins. Interestingly, an intracellular calcium chelator, BAPTA-AM, completely attenuated norepinephrine-stimulated phosphorylation of tyrosine proteins, suggesting that intracellular Ca²⁺ plays a critical role in mediating ₁ receptor stimulation of phosphorylation of tyrosine proteins. ₁ receptor stimulation caused a time-dependent increase in MAPK activity due to increased phosphorylation of p42/44^ERK1/2. Our study demonstrates that ₁ receptor-mediated activation of MAPK was also attenuated by TPK inhibitors and the intracellular Ca²⁺ chelator BAPTA-AM.

There is substantial evidence indicating that stimulation of ₁ receptors enhances growth-related gene expression and cell growth in cardiac myocytes and VSMCs. In cardiac myocytes, stimulation of ₁ receptors produces long-term changes in the cardiac structure and function, including an increase in cell size and an increase in the expression of the cardiac structural genes such as -myosin heavy chain gene (Simpson et al., 1991; Morgan and Baker, 1991). In vascular smooth muscle, it has long been known that adrenergic agonists lead to the growth of arterial smooth muscle cells in vitro. Catecholamines stimulate the proliferation of rat and bovine smooth muscle cells (Jackson and Schwartz, 1992) in primary culture via activation of ₁ receptors. In intact animals, activation of ₁ receptors may contribute to atherosclerosis by enhancing proliferation of VSMCs (Hauss et al., 1990). Indeed, the ₁ receptor antagonists prazosin and doxazocin inhibits smooth muscle hyperplasia induced in experimental models of endothelial injury (Fingerle et al., 1991; Vashisht et al., 1992). Activation of ₁ receptors on cardiac myocytes and VSMCs leads to stimulation of DNA and protein synthesis, either directly or indirectly via stimulation activation of growth factor expression such as platelet-derived growth factor (Majesky et al., 1990). Moreover, we recently found that ₁ agonists markedly increase expression of early and delayed proto-oncogenes in vitro in intact aorta (Okazaki et al., 1994). ₁ agonist stimulation of mitogenesis in HVSMCs is associated with activation of PI-3 kinase (Hu et al., 1996b). In the present study, we found that mitogenic responses of HVSMCs to ₁ receptor stimulation could be blocked by inhibition of TPKs. Moreover, stimulation of VSMCs with ₁ receptor agonists results in tyrosine phosphorylation of several proteins, suggesting that phosphorylation of tyrosine proteins plays an important role in ₁ receptor signal transduction.

It is not clear how TPKs contribute to the overall signal transduction of specific GPCRs; recent evidence suggests that these kinases play an important role in GPCR signaling in a number of different cells (Malarkey et al., 1995). In contrast to peptide growth factor receptors, which possess endogenous TPK activities, GPCRs have not been demonstrated to posses endogenous TPK activity. Agonist stimulation of these receptors leads to activation of several different G proteins that dissociate into  and subunits. There is evidence indicating that these subunits may activate TPKs, leading to tyrosine phosphorylation of target proteins. The phosphorylated proteins may then serve as links between the receptors or G proteins and various adapter proteins such as Grb2, SOS1, or Shc (Downward, 1994). These adapter proteins all possess SRC homology 2 or 3 (SH2 or SH3) domains and some are TPKs that subsequently dock and activate downstream effectors such as p21^Ras leading to activation of protein kinase cascades (Smithgall, 1995). In the present study, we have demonstrated that activation of ₁ receptors stimulates tyrosine phosphorylation of several protein molecules and activates MAPK. The results suggest that two tyrosine-phosphorylated proteins are phospholipase C1 and adapter protein Shc.

It is unlikely that PKC plays a major role in mediating ₁ receptor activation of TPKs in HVSMCs because neither enzyme inhibition nor down-regulation of PKC inhibited TPK activation. Similarly, an L-type calcium channel blocker did not inhibit ₁ receptor stimulation of tyrosine protein phosphorylation. On the other hand, the intracellular Ca²⁺ chelator BAPTA-AM completely blocked norepinephrine stimulation of tyrosine protein phosphorylation in these cells. Recent studies suggest that an increase in intracellular Ca²⁺ concentration may be an important early event in GPCR-mediated TPK/Ras-MAPK signaling pathways. For example, angiotensin II activation of several TPKs and downstream MAPK in cultured rat neonatal myocytes is Ca²⁺-dependent.

A major question is how intracellular Ca²⁺ participates in activation of TPKs by GPCR agonists such as angiotensin II and norepinephrine. Stimulation of PLC1 by many growth factors increases intracellular Ca²⁺ mobilization via release of IP₃ (Carpenter et al., 1992), indicating that activation of PLC1 may function as an early mediator of GPCR-stimulated TPK/MAPK signaling pathway (Rusanescu et al., 1995). Activation of members of cytosolic TPKs such as the SRC family may also serve as mediator of GPCR-stimulated intracellular Ca²⁺ signaling to activate MAPK (Dikic et al., 1996). Additionally, focal adhesion kinase (p125^FAK) has been suggested to mediate GPCR-stimulated Ca²⁺-dependent signaling. Tyrosine phosphorylation of p125^FAK is dependent on intracellular Ca²⁺ (Shattil et al., 1994). In this context, proline-rich tyrosine kinase 2 (PYK2), a member of the p125^FAK family, has attracted particular attention. PYK2 has been found to be rapidly phosphorylated on tyrosine residues in response to various stimuli including GPCR ligands that elevate intracellular calcium concentrations. Phosphorylated PYK2 then in turn activates Ras-MAPK activity (Lev et al., 1995). In the present study, we have found that activation of ₁ receptors likely stimulates tyrosine phosphorylation of PLC1, which could lead to Ca²⁺ mobilization that may in turn activate the Ras-MAPK cascade. The detailed interactions between PLC1, Ca²⁺, and TPKs such as P125^FAK or PYK2 as mediators of norepinephrine-stimulated MAPK activation requires further detailed study.

It is now known that the MAPK cascade is not restricted to RTK signaling pathways but is also utilized by phorbol esters, heat shock, and GPCR ligands to induce mitogenesis (Malarkey et al., 1995; Post and Brown, 1996). Activation of P21^Ras and then Raf-1 are key steps not only for the understanding of growth factor signal transduction but also for GPCR activation of MAPK pathways. Generally, growth factors activate p21^Ras, which stimulates Raf-1, leading to activation of MAPK via phosphorylated RTKs. On the other hand, GPCR agonists stimulate MAPK via two independent pathways depending on G protein-receptor coupling. For receptors coupled to pertussis toxin-sensitive G proteins such as thrombin and lysophosphatidic acid, activation of MAPK is via activation of p21^Ras (LaMorte et al., 1994). This pathway is PKC-independent. For receptors coupled to pertussis toxin-insensitive G proteins such as the Gq family, stimulation of these receptors leads activation of PKC. Activated PKC in turn directly activates Raf-1 and thereby activates MAPK. Indeed, there is evidence that demonstrates that PKC phosphorylates Raf-1 upon serine residues in vitro (Kolch et al., 1993); _1B receptors activate MAPK via stimulation of pertussis toxin-insensitive G proteins, PKC, and Raf-1 (Hawes et al., 1995). However, muscarinic M1 receptors have been found to activate MAPK via pertussis toxin-insensitive G proteins using Ras-dependent pathways (Crespo et al., 1994), indicating that p21^Ras may also be utilized by pertussis toxin-insensitive GPCRs to activate MAPK. We have recently demonstrated that ₁ receptors activate p21^Ras protein in HVSMCs (Hu et al., 1996). In the present study, we provide evidence to show that ₁ receptor-activated MAPKs are attenuated by pertussis toxin and a TPK inhibitor, suggesting that pertussis toxin-sensitive G protein and TPKs are involved in ₁ receptor-activated MAPK signaling pathways in HVSMCs. These results indicate that TPK/Ras-MAPK signaling pathways are utilized by ₁ receptors to mediate mitogenic actions of catecholamines in HVSMCs.

In summary, we have characterized phosphorylation of tyrosine proteins as an early event in the ₁ receptor-induced protein kinase signaling cascade, which may be responsible for regulation of catecholamine stimulation of mitogenic effects in HVSMCs. Activation of tyrosine phosphorylation by ₁ receptors was independent in the activation of PKC but dependent of the intracellular Ca²⁺ in these cells, suggesting that the intracellular Ca²⁺ plays a critical role in ₁ receptor-signaling pathways not only for smooth muscle contraction but also for cell growth (Scheme 1). HVSMCs provide an important model system for the further elucidation of ₁ adrenergic signaling mechanisms, particularly relating to cell growth and division, which is important for vascular changes in diseases such as hypertension and atherosclerosis.

View larger version (20K): [in this window] [in a new window]   Scheme 1.   Scheme of Ca2+ and Ca2+-dependent protein kinases in 1-AR-mediated mitogenic responses in VSMCs. Stimulation of 1-AR leads to activation of phospholipase C and phospholipase C via G proteins. PLC activation increases intracellular Ca2+, which promotes activation of several TPKs. Activated protein kinases then phosphorylate adapter proteins such as Shc to turn on downstream signal cascades to produce cell mitogenic responses. MAPK is activated via this pathway and in part via 1-AR activation of PKC.