Introduction

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Three genes encoding unique ₁-AR subtypes, _1A-, _1B-, or _1D-AR, have been cloned and pharmacologically characterized (Cotecchia et al., 1988; Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1991; Hieble et al., 1995). All three ₁-AR subtypes exhibit similar affinity for endogenous catecholamines (Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1991); however, the cellular functions of these receptors have not been adequately defined. Our previous work showed that although all receptor subtypes are expressed in peripheral arteries, the _1A-AR or _1D-AR couple agonist binding to smooth muscle contraction in a given vessel (Guarino et al., 1996; Hrometz et al., 1999; Piascik and Perez, 2001). In addition to the acute regulation of blood pressure, catecholamines induce vascular smooth muscle cell growth (Johnson et al., 1983; deBlois et al., 1996; Fingerle et al., 1991; van Kleef et al., 1992; Chen et al., 1995). These observations suggest that expressed ₁-AR subtypes may differentially activate signaling pathways and physiological responses.

Mitogen-activated protein kinases (MAPKs) are important mediators of cell growth, proliferation, differentiation, and survival. There are three major MAPK subtypes: the extracellular signal-regulated kinases (ERK1/2), c-Jun NH₂-terminal kinases (JNKs), and p38 kinases (Widmann et al., 1999). Activated MAPKs translocate to the nucleus and phosphorylate multiple transcription factors to increase transcriptional activity (Widmann et al., 1999). ERK1/2 and p38 kinase also phosphorylate cytoplasmic substrates, including those involved in protein biosynthesis (Widmann et al., 1999). Recent studies suggest that ₁-AR subtypes differentially activate MAPK family members and that the profile of MAPK activation uniquely impacts on cellular phenotype (Alexandrov et al., 1999; Zhong and Minneman, 1999; Keffel et al., 2000). For example, in PC12 cells, inducible expression and activation of the _1A-AR, but not the _1B-AR or the _1D-AR subtypes, leads to activation of ERK1/2, JNK, and p38 kinase and promotes neurite outgrowth (Zhong and Minneman, 1999).

Discernment of the role of ₁-AR subtypes in long-term growth responses in tissues that express multiple receptor subtypes is hindered by the lack of ₁-AR subtype-selective agonists. An added complexity in studying the specific role of ₁-AR subtypes in long-term responses is the observation that endogenous ₁-ARs are differentially regulated by chronic agonist exposure in myocardial and vascular smooth muscle cells (Chen et al., 1995; Rokosh et al., 1996). As an alternative approach to study the regulatory roles of ₁-ARs, several laboratories have compared signaling properties of ₁-AR receptor subtypes in heterologous expression systems. These studies have demonstrated intrinsic differences between the ₁-AR subtypes within a cell line and cellular responses for a given ₁-AR expressed in different host cell lines.

Endogenously expressed _1A-ARs mediate hypertrophic growth of myocardial cells (Varma and Deng, 2000). Furthermore, accumulating evidence suggests that _1A-ARs, and to a lesser extent _1D-ARs, regulate arterial blood pressure (Piascik and Perez, 2001). The functional roles of the _1B-AR and _1D-AR in cardiovascular tissues are not well understood. We previously reported divergent regulation of subcellular localization and acute signaling events by _1B-AR and _1D-AR expressed in Rat 1 fibroblasts (McCune et al., 2000). The _1B-AR exhibits properties of a typical G protein-coupled receptor because it is expressed primarily on the cell surface and demonstrates agonist-dependent internalization and ERK1/2 activation (McCune et al., 2000). In contrast, the _1D-AR localizes primarily in internalized subcellular compartments and shows evidence of enhanced ERK1/2 and phospholipase activity in the absence of agonist (McCune et al., 2000). In this report, we examined JNK and p38 kinase activation, cellular proliferation, and protein biosynthesis mediated by these receptor subtypes. Both receptors inhibited DNA synthesis and increased protein biosynthesis and ERK1/2 activity. Interestingly, ERK1/2 and protein biosynthesis were agonist-dependent for _1B-AR and agonist-independent for the _1D-AR, suggesting constitutive ERK1/2 activity was coupled to protein biosynthesis. We also demonstrated divergent regulation of JNK and p38 kinase by these receptor subtypes. For example, the _1B-AR activated p38 kinase but not JNK, whereas the _1D-AR was coupled to JNK but not p38 kinase activation. Finally, we show that the _1B-AR, but not _1D-AR, mediated growth effects through a p38 kinase-dependent pathway. Therefore, although the _1B-AR and _1D-AR mediate similar effects on growth responses, they exhibit different requirements for agonist activation and MAPK isoforms.

	Materials and Methods

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Cell Culture. Rat 1 fibroblasts stably transfected with either the cloned human _1B- or _1D-AR (GlaxoSmithKline, Uxbridge, Middlesex, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin mixture (Invitrogen), and Geneticin (250 µg/ml; Invitrogen) at 37°C in 5% CO₂. Twenty-four hours after plating, cells were washed and then serum-deprived for 48 h before the addition of drugs.

[³H]Thymidine Incorporation. Rat 1 fibroblasts plated on 24-well dishes at 1 × 10⁴ cells/well were treated with the indicated drugs for 24 h with 1 µCi [³H]thymidine (PerkinElmer Life Sciences, Boston, MA) included during the last 6 h of incubation. Kinase inhibitors were added 20 min before phenylephrine (PE). Cells were rinsed with phosphate-buffered saline (PBS), fixed for 10 min with ice-cold methanol, washed 3 × 5 min with ice-cold 10% trichloroacetic acid, and dissolved in 1 N sodium hydroxide. [³H]Thymidine incorporation was quantified using liquid scintillation counting and used as an index of DNA synthesis.

[³⁵S]Methionine Incorporation. Incorporation of [³⁵S]methionine in Rat 1 fibroblasts was performed as described by Xin et al. (1997) with modifications (Xin et al., 1997). Serum-deprived Rat 1 fibroblasts plated on 24-well dishes at 1 × 10⁴ cells/well were washed twice with methionine-free DMEM and incubated for 30 min. Cells were then treated with the indicated drugs in low-methionine (2 mg/l) DMEM for 24 h. Kinase inhibitors were added 20 min before PE. [³⁵S]Methionine (1 µCi; PerkinElmer Life Sciences) was added during the last 6 h of incubation. Sample processing was essentially the same as for thymidine incorporation assay. [³⁵S]Methionine incorporation was quantified using liquid scintillation counting and served as an index of protein synthesis.

Preparation of GST-c-Jun(1-135). Recombinant c-Jun [c-Jun(1-135)] was produced in Escherichia coli as a glutathione S-transferase fusion protein expressed from plasmid pGEX-c-Jun (Prasad et al., 1995). GST-c-Jun(1-135) fusion protein was purified by conjugation to glutathione-Sepharose beads (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and eluted with 20 mM glutathione in 100 mM Tris, pH 8. The eluate was dialyzed for 2 h at 4°C to remove glutathione.

ERK1/2 Activity Assay. ERK1/2 activity was detected by the in-gel method as described previously (McCune et al., 2000). Cells were treated with the indicated drugs, washed with ice-cold PBS, and scraped into 1 ml of 250 mM ice-cold buffered sucrose. Cell pellets were resuspended in cold lysis buffer [20 mM Tris-HCl, pH 7.4, 250 mM NaCl, 2.5 mM EDTA, 3 mM EGTA, 20 mM -glycerophosphate, 0.5% (v/v) Nonidet P-40, 100 µM Na₃VO₄, and protease inhibitors (Calbiochem, La Jolla, CA)] for 30 min, centrifuged (15 min; 15,000g; 4°C), and the supernatant collected. Protein was resolved on 10% SDS-polyacrylamide gels containing 0.5 mg/ml myelin basic protein (MBP). After electrophoresis, the gels were washed with 20% 2-propanol in 50 mM HEPES, pH 7.6, and then with 5 mM -mercaptoethanol in HEPES buffer. Proteins were denatured in 6 M urea and gradually renatured in HEPES buffer containing 0.05% (v/v) Tween 20 and 5 mM -mercaptoethanol (renaturation buffer) at 4°C. After overnight incubation in renaturation buffer at 4°C, gels were preincubated in 25 ml of cold kinase buffer (20 mM HEPES, 20 mM MgCl₂, 2 mM dithiothreitol, 5 mM -glycerophosphate, 100 µM Na₃VO₄, pH 7.6) for 30 min. Phosphorylation of MBP was performed in situ by soaking the gel in 25 ml of kinase buffer containing 20 µM ATP and 150 to 160 µCi of [-³²P]ATP (New England Biolabs, Beverly, MA) for 90 to 120 min at 30°C. After extensive washing with 5% trichloroacetic acid/1% sodium pyrophosphate the gels were dried and exposed to film. ³²P incorporation into MBP was determined by densitometric analysis.

JNK Activity Assay. Activities of the 46- and 55-kDa isoforms of JNK were determined by in-gel activity assays essentially as described for ERK1/2, except 0.1 mg/ml GST-c-Jun(1-135) was used as substrate.

p38 Kinase Immunoblotting. Cells were maintained in serum-free media or treated with PE (100 µM) or anisomyosin (50 ng/ml) for 15 min. Cells were washed twice with ice-cold PBS and lysed with 150 µl of SDS-sample buffer. The lysates were sonicated (5 × 2 s), boiled for 5 min, and cooled on ice. Equal volumes of lysate were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Phosphorylated and total p38 kinase was detected by protein immunoblotting with a 1:1000 dilution of rabbit polyclonal phosphospecific (Thr180/Tyr192) or total p38 kinase antibodies (New England Biolabs). Primary antibody was detected with 1:2000 horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham Pharmacia Biotech AB). Bands were visualized by chemiluminescence (ECL+; Amersham Pharmacia Biotech AB) and quantitated by phosphorimaging (Molecular Dynamics, Sunnyvale, CA).

Data Analysis. Differences among treatment groups were detected by one- or two-way analysis of variance with repeated measures followed by Student-Newman-Keuls multiple comparison tests. In the absence of a significant treatment by treatment group interaction, the statistical significance of the main or overall effect of a particular treatment is reported. Differences between the cell lines were detected by unpaired, two-tailed Student's t test. All calculations were performed using the Statistica program (release 5.1; StatSoft, Tulsa, OK). A p < 0.05 was considered significant. Nonlinear regression analyses of concentration-response curves were performed using GraphPad Prism (version 2.01; GraphPad Software, San Diego, CA).

	Results

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Agonist Activation of _1B- and _1D-AR Inhibits DNA Synthesis. The studies presented here extend our previous report demonstrating differences in the regulatory properties of the _1B- and _1D-AR (McCune et al., 2000). To determine the role of _1B-AR and _1D-ARs in cellular proliferation, we examined PE-mediated [³H]thymidine incorporation in Rat 1 fibroblasts stably expressing these receptor subtypes. Cells treated for 24 h with various concentrations of the ₁-AR agonist PE exhibited a concentration-dependent decrease in [³H]thymidine incorporation (Fig. 1). Maximal percentage of inhibition was not different for the two cell lines (_1B, 40 ± 11%; _1D-AR, 19 ± 7%; N.S.). The observed decrease in [³H]thymidine incorporation after prolonged PE treatment was not associated with a loss of cell number or decreased cell viability (data not shown). Levels of [³H]thymidine incorporation were similar in the absence of agonist [_1B, 8.6 ± 3.2 cpm (× 10³); _1D, 11.0 ± 2.7 cpm (× 10³); N.S.] and nonlinear regression revealed similar log IC₅₀ values for both cell lines (_1B, 6.6 ± 0.3; _1D, 7.0 ± 0.2; N.S.).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Phenylephrine inhibits DNA synthesis in Rat 1 fibroblasts expressing the 1B- or 1D-AR subtype. Rat 1 fibroblasts were treated with the indicated concentrations of PE for 24 h. Basal values for [3H]thymidine incorporation were 8600 ± 3200 dpm for the 1B and 11,000 ± 2700 dpm for the 1D (NS). Data points represent the means ± S.E.M. of three experiments performed in triplicate and are expressed as percentage of control for each cell line.

Inhibition of [³H]Thymidine Incorporation Mediated by _1B- and _1D-AR Is Reversed by Prazosin. To determine whether the effects of PE in _1B- and _1D-AR-expressing cells were mediated through ₁-ARs, we examined the effect of various adrenoceptor antagonists on PE-mediated inhibition of [³H]thymidine incorporation (Table 1). PE (1 µM) reduced DNA synthesis to 44 ± 2% of that observed in unstimulated _1B-AR cells and 17 ± 4% for the _1D-AR. The effect of PE on DNA synthesis was blocked by 1 µM prazosin (nonselective ₁-AR antagonist) in both cell lines and not affected by either 1 µM yohimbine (₂-AR antagonist) or 1 µM propranolol (-AR antagonist), suggesting that agonist-induced inhibition of DNA synthesis was mediated through the expressed ₁-AR.

Coupling of _1B- and _1D-ARs to Protein Biosynthesis. Because ₁-ARs are known to regulate both proliferative and hypertrophic growth responses, we also examined [³⁵S]methionine incorporation as an index of protein biosynthesis. Levels of basal [³⁵S]methionine incorporation were greater in the _1D-AR compared with the _1B-AR [7.8 ± 1.0 cpm (× 10³) for the _1B-AR and 30.8 ± 3.4 cpm (× 10³) for the _1D-AR (p < 0.001)]. PE increased [³⁵S]methionine incorporation in a concentration-dependent manner in _1B- and _1D-AR-expressing fibroblasts (Fig. 2), with a greater maximal effect of PE in the _1B cell line (238 ± 16 versus 162 ± 29%; p < 0.05). Differences in agonist and basal [³⁵S]methionine incorporation between the two cell lines were not due to differences in cell number (data not shown). The log EC₅₀ values for two receptors were similar for the two cell lines (_1B-AR, 7.2 ± 0.3; _1D-AR, 6.9 ± 0.5; N.S.).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Phenylephrine increases [35S]methionine incorporation in Rat 1 fibroblasts expressing the 1B- or 1D-AR subtype. Rat 1 fibroblasts were treated with various concentrations of PE for 24 h. Basal levels of [35S]methionine incorporation were 7800 ± 900 cpm for the 1B and 30,800 ± 3400 cpm for the 1D (p < 0.001). Data shown are the means ± S.E.M. of three to four separate experiments performed in triplicate and are expressed as percentage of control for each cell line (*, maximal PE-induced protein synthesis in 1B-AR- versus 1D-AR-expressing cells; p < 0.05).

Coupling of _1B- and _1D-ARs to ERK1/2. We compared the ability of these receptor subtypes to regulate ERK1/2 activation (Fig. 3). In _1B-AR-expressing cells, PE (100 µM; 5 min) significantly increased ERK1/2 activity over basal levels (*p < 0.05). In contrast, we did not detect PE- or serum-induced increases in ERK1/2 activity for the _1D-AR. Basal ERK1/2 activity was 2-fold greater in the _1D-AR-expressing cells (_1B, 1.7 ± 0.8 IOD; _1D, 3.5 ± 0.9 IOD) and similar in magnitude to the _1B cell line treated with PE (_1B, 4.7 ± 1.8 IOD). We previously showed that 1 µM prazosin inhibited basal ERK1/2 activity by ~50% in _1D-AR-expressing cells (McCune et al., 2000). High basal ERK1/2 activity may have precluded our ability to observe further increases in ERK1/2 activity induced by 10% fetal bovine serum or PE.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Phenylephrine activates ERK1/2 in 1B- but not 1D-AR-expressing Rat 1 fibroblasts. Rat 1 fibroblasts were maintained in serum-free DMEM (control), or treated with DMEM containing PE (100 µM), or 10% fetal bovine serum for 5 min. Cell lysates were prepared and equal amounts of protein were separated by SDS-PAGE containing 0.5 mg/ml myelin basic protein (substrate for ERK1/2) and ERK1/2 activity determined by in-gel assays. Data shown are the means ± S.E.M. from six separate experiments performed in duplicate and are normalized to percentage of control for each cell line (*, PE-induced ERK1/2 in 1B-AR- versus 1D-AR-expressing cells; p < 0.05). Basal levels of ERK activity in the 1D cell line were nearly 2-fold greater than that observed for the 1B cell line (1B, 1.7 ± 0.8 IOD; 1D, 3.5 ± 0.9 IOD).

Coupling of _1B- and _1D-ARs to JNK. We examined the ability of PE and anisomyosin (positive control) to activate JNK in _1B-AR- and _1D-AR-expressing fibroblasts (Fig. 4). Although anisomyosin (50 ng/ml) stimulated JNK activity to a similar extent in both cell lines, PE (100 µM; 20 min) increased JNK activity only in _1D-AR fibroblasts (*p < 0.01). Basal JNK activity was similar in both _1B-AR- and _1D-AR-expressing fibroblasts (average basal activity of _1B relative to _1D was 75 ± 30%; n = 4; N.S.).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Phenylephrine activates c-Jun NH2-terminal kinase in 1D- but not 1B-expressing Rat 1 fibroblasts. Cells were untreated (control), or treated with PE (100 µM) or anisomycin (50 ng/ml) for 20 min. Lysates were collected and equal amounts of protein were separated by SDS-PAGE containing 0.1 mg/ml GST-cJun fusion protein. The gels were dried and subjected to autoradiography followed by densitometric analysis. The average basal activity of 1B relative to 1D was 75 ± 30% (N.S.). Data shown are the means ± S.E.M. from four separate experiments performed in duplicate and are normalized to percentage of control for each cell line (*, PE-induced JNK activity in 1B-AR- versus 1D-AR-expressing fibroblasts; p < 0.01). A representative autoradiograph is shown.

Coupling of _1B- and _1D-ARs to p38 Kinase. We compared the ability of PE to activate p38 kinase in both cell lines by immunoblotting for phospho- and total p38 kinase in unstimulated, PE-, or anisomyosin-treated cell lines (Fig. 5). PE (100 µM; 15 min) induced p38 kinase activity in _1B-AR, but not in _1D-AR-expressing fibroblasts (*p < 0.05).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Phenylephrine activates p38 kinase in 1B- but not 1D-expressing Rat 1 fibroblasts. Cells were untreated (C), or incubated with PE (100 µM) or anisomycin (50 ng/ml; An) for 15 min. Equal amounts of protein were separated by 10% SDS-PAGE and subjected to immunoblotting as described under Materials and Methods. Columns represent means ± S.E.M. of four to five separate experiments (*, PE-induced p38 activity in 1B-AR- versus 1D-AR-expressing fibroblasts; p < 0.05.) Representative autoradiographs of phospho- and total p38 kinase are shown.

PD98059 Blocks PE-Induced ERK1/2 Activation in _1B-AR Cells and Decreases Basal ERK1/2 Activity in _1D-AR Cells. Activation of MAPK family members requires phosphorylation on both threonine and tyrosine by dual specificity kinases (Widmann et al., 1999). To assess the role of ERK1/2 and p38 kinase in ₁-AR-mediated DNA and protein biosynthesis we used selective cell-permeable inhibitors of these MAPK isoforms. Rat 1 fibroblasts were pretreated for 20 min with DMSO (0.1% v/v; control), 10 µM PD98059, a selective inhibitor of ERK1/2 activation (Dudley et al., 1995), or the p38 kinase inhibitor SB203580. After 5 min of PE or 10% FBS (control for ERK1/2 activation) treatment, cell lysates were prepared and ERK1/2 activity was determined using in-gel kinase assays. As shown in Fig. 6, PE-induced ERK1/2 activation in _1B-AR-expressing cells was blocked by PD98059. Unlike a previous report demonstrating inhibition of ERK1/2 activity by p38 kinase in Rat 1 fibroblasts (Alexandrov et al., 1999), we did not detect differences in either basal or agonist-induced ERK1/2 activity in the presence of SB203580. Relative to the _1B-AR, _1D-AR-expressing cells displayed elevated ERK1/2 activity in the absence of agonist that was inhibited by PD98059.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   PD98059, but not SB203580, blocks PE-induced ERK1/2 activation in 1B-AR cells and decreases basal ERK1/2 activity in 1D-AR cells. Cells were treated with DMSO (0.1% v/v), 10 µM PD98059 (PD), or 10 µM SB203580 (SB) for 20 min before the addition of 100 µM PE (5 min). Cell lysates were prepared, and proteins were separated by SDS-PAGE and examined for ERK1/2 activity by the in-gel assay. +, 20 ng of activated recombinant ERK2; FBS, cells treated with 10% FBS for 5 min. A representative autoradiograph of a single experiment performed in duplicate shows bands at 42 and 44 kDa that represent activated ERK2 and ERK1, respectively.

SB203580 Blocks PE-Induced p38 Kinase Activation in _1B-AR Cells. To demonstrate the inhibitory effect of p38 kinase inhibitor SB203850 on ₁-AR-mediated responses, cells were preincubated for 20 min with DMSO (0.1% v/v) or 10 µM SB203580 before the addition of PE (100 µM; 20 min). Cells were harvested and equal volumes of cell lysates were separated by SDS-PAGE and immunoblotted for phospho- or total p38 kinase (Fig. 7). In _1B-AR cells, PE-mediated increases in phospho-38 kinase were blocked by SB203580. In contrast, PE did not activate p38 kinase in the _1D-AR cell line.

View larger version (72K): [in this window] [in a new window]   Fig. 7.   SB203580 inhibits 1B-mediated p38 kinase activation. Cells were preincubated with SB203580 (SB; 10 µM) or DMSO (C) for 20 to 30 min before PE (100 µM, 15 min). Cells were harvested and cell lysates were prepared. Equal volumes of lysate were separated by SDS-PAGE and immunoblotted for phospho- or total p38 kinase.

Role of ERK1/2 in ₁-AR-Mediated DNA and Protein Biosynthesis. Both _1B-AR and _1D-ARs inhibited DNA synthesis in Rat 1 fibroblasts (Fig. 1). Cell cycle arrest in HepG2 cells overexpressing _1B-AR occurs through an ERK1/2-dependent pathway (Auer et al., 1998). To investigate the role of ERK1/2 in ₁-AR-mediated alterations in DNA synthesis, we examined [³H]thymidine incorporation in the absence and presence of 10 µM PD98059. Although PD98059 blocked ERK1/2 activation in both cell lines (Fig. 6), PD98059 did not reverse PE-mediated decreases in [³H]thymidine incorporation in either cell line (Fig. 8, A and C), indicating that these ₁-ARs regulate DNA synthesis through an ERK1/2-independent pathway.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Role of ERK1/2 in [3H]thymidine incorporation and [35S]methionine incorporation. Rat 1 fibroblasts were treated with (, 1 µM) or without PE () for 24 h. DMSO (0.1% v/v; no inhibitor) or PD98059 (10 µM) was added 30 min before PE treatment. A, effect of PD98059 on [3H]thymidine incorporation in 1B-AR-expressing cells. PE caused an overall decrease in [3H]thymidine incorporation (p < 0.01). PD treatment also caused an overall reduction in DNA synthesis (p < 0.01). B, effect of PD98059 on [35S]methionine incorporation in 1B-AR-expressing cells. PE increases [35S]methionine incorporation (***p < 0.001). PD98059 partially blocked PE's effect on protein biosynthesis (++p < 0.01). C, effect of PD98059 on [3H]thymidine incorporation in 1D-AR-expressing cells. PE caused an overall reduction in [3H]thymidine incorporation (p < 0.01). As in A, PD treatment caused an overall reduction in protein synthesis (p < 0.01). D, effect of PD98059 on [35S]methionine incorporation 1D-AR-expressing cells. Basal protein synthesis is significantly higher in 1D-AR versus 1B-AR fibroblasts (p < 0.001). Main treatment effects of PD98059 and PE were observed (p < 0.01); however, no treatment interaction was present. Columns represent means ± S.E.M. of four to six separate experiments performed in triplicate.

Role of p38 Kinase in ₁-AR-Induced DNA and Protein Biosynthesis. In contrast to PD98059, 10 µM SB203580 reversed _1B-AR-mediated inhibition of DNA synthesis by ~50% (Fig. 9A; ⁺⁺p < 0.01 compared with PE alone), suggesting that inhibition of DNA replication occurs, in part, through a p38 kinase-dependent pathway. Agonist-induced protein biosynthesis in _1B-AR cells was also inhibited ~50% by SB203580 (Fig. 9B; ⁺⁺p < 0.01 versus PE alone). In contrast, SB203580 had no effect on either [³H]thymidine incorporation (Fig. 9C) or [³⁵S]methionine incorporation (Fig. 9D) in _1D-AR-expressing fibroblasts. The effect of SB203580 on unstimulated DNA synthesis was not significantly different between the two cell lines. Combined PD and SB treatment in _1B-AR cells completely blocked the effect of PE on protein synthesis [control, 7.8 ± 0.9 cpm (× 10³); PE, 20.5 ± 2.6 cpm (× 10³), p < 0.001; PD + SB, 6.7 ± 0.6 cpm (× 10³), N.S.; PE + PD/SB, 8.0 ± 1.0 cpm (× 10³), N.S.], indicating that the _1B-AR uses both ERK1/2 and p38 kinase cascades to promote protein biosynthesis.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Role of p38 kinase in [3H]thymidine incorporation and [35S]methionine incorporation. Rat 1 fibroblasts were treated with (, 1 µM) or without PE () for 24 h. DMSO (0.1% v/v; no inhibitor) or SB203580 (10 µM) was added 30 min before PE treatment. A, effect of SB203580 on [3H]thymidine incorporation in 1B-AR-expressing cells. PE significantly decreased basal [3H]thymidine incorporation (*p < 0.05). SB203580 significantly reversed the inhibitory effect of PE (++p < 0.01). B, effect of SB203580 on [35S]methionine incorporation in 1B-AR-expressing cells. As in Fig. 8B, PE significantly increases [35S]methionine incorporation (***p < 0.001) and this effect is reversed by SB203580 (++p < 0.01). C, effect of SB20350 on [3H]thymidine incorporation in 1D-AR-expressing cells. PE caused an overall reduction in [3H]thymidine incorporation (p < 0.01). No significant effect of SB203580 on PE-mediated inhibition of [3H]thymidine incorporation was observed. D, effect of SB20350 on [35S]methionine incorporation in 1D-AR-expressing cells. PE causes an overall increase in [35S]methionine incorporation. No significant effect of SB203590 was observed. Columns represent means ± S.E.M. of four to six separate experiments performed in triplicate.

	Discussion

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₁-ARs are important mediators of arterial blood pressure, vascular smooth muscle contraction, and growth. Multiple ₁-AR subtypes are expressed in peripheral arteries; however, whether the same ₁-AR subtype regulates both contractile and growth responses in vivo and whether this growth represents hypertrophy, hyperplasia, or both have not been adequately defined. Definitive assessment of ₁-AR-mediated growth responses in vivo is hindered by the lack of ₁-AR subtype-selective compounds and differential regulation of receptor expression by prolonged agonist exposure (Chen et al., 1995; Rokosh et al., 1996). However, several laboratories have shown differential coupling of ₁-AR subtypes to MAPK activation (Zhong and Minneman, 1999; Keffel et al., 2000; McCune et al., 2000) and [³H]thymidine incorporation (Keffel et al., 2000) in heterologous cell expression systems, suggesting that ₁-AR subtypes might differentially activate cellular growth responses.

To further examine the regulatory functions of the _1B-AR and _1D-AR, we compared cell proliferation, protein biosynthesis, and the MAPK isoforms mediating these growth responses in Rat 1 fibroblasts stably expressing these receptor subtypes. Despite similar levels of receptor expression (between 5.5 and 10 pmol/mg of protein; McCune et al., 2000) and the overall similarity of effect on cell proliferation and protein biosynthesis, our results reveal differential requirements for agonist and MAPK activation between these receptor subtypes. Agonist treatment of _1B-AR-expressing fibroblasts induces protein biosynthesis (Fig. 2), ERK1/2 activity (Fig. 3; McCune et al., 2000), and p38 kinase activity (Fig. 5), but has no effect on JNK activity (Fig. 4) and inhibits DNA synthesis (Fig. 1). In contrast, protein biosynthesis and ERK1/2 activity are elevated in the absence of agonist in _1D-AR cells. Although ERK1/2 activity and protein biosynthesis are agonist-independent for the _1D-AR, JNK activity and inhibition of [³H]thymidine incorporation are agonist-dependent, suggesting that the _1D-AR is not constitutively linked to these responses in Rat 1 fibroblasts.

The overall pattern of MAPK activation and cellular growth responses observed in Rat 1 fibroblasts is distinct from ₁-AR subtypes expressed in either CHO or PC12 cells. Similar to results obtained in PC12 cells, activation of _1B-AR in Rat 1 fibroblasts cells increases ERK1/2 and p38 kinase activity, but does not activate JNK. However, although _1B-AR mediates increased protein biosynthesis and inhibition of DNA replication in Rat 1 fibroblasts, this receptor subtype does not affect DNA replication when expressed in CHO cells (Keffel et al., 2000). Expression and activation of the _1D-AR stimulate DNA replication, p38 kinase, and JNK in CHO cells (Keffel et al., 2000) but activate only ERK1/2 in PC12 cells (Zhong and Minneman, 1999). In contrast, agonist activation of the _1D-AR inhibits DNA replication (Fig. 1), activates JNK (Fig. 4), and has no effect on p38 kinase activity (Fig. 5) in Rat 1 fibroblasts. Furthermore, studies in PC12 and CHO cells failed to report constitutive regulation of MAPK isoforms or growth responses for _1D-AR, although this receptor constitutively activates calcium transients in Rat 1 fibroblasts (Garcia-Sainz and Torres-Padilla, 1999). Several reports have shown that the _1D-AR is weakly coupled to intracellular signals (Theroux et al., 1996; Ruan et al., 1998; Taguchi et al., 1998). An apparent lack of agonist-mediated, growth-related responses is consistent with constitutive or agonist-independent signaling by the _1D-AR. The overall conclusion from heterologous expression studies is that growth-related responses are dependent on ₁-AR subtype and host cell. Whether ₁-AR subtypes differentially regulate MAPK activity and growth of various peripheral arteries is the focus of our ongoing investigations.

Our results indicate that ERK1/2 activation is associated with increased protein biosynthesis rather than proliferative growth of Rat 1 fibroblasts. For example, the ERK1/2 pathway inhibitor PD98059 blocks ERK1/2 activity in _1B-AR- and _1D-AR-expressing cells (Fig. 6), but does not reverse agonist-mediated inhibition of [³H]thymidine incorporation in either cell line (Fig. 8, A and C), suggesting that ERK1/2 activation is not required for ₁-AR-mediated inhibition of DNA synthesis. In contrast to its effects on PE-mediated DNA replication, PD98059 attenuates PE-mediated increases in [³⁵S]methionine incorporation in _1B-AR-expressing cells (Fig. 8B), suggesting this receptor couples agonist binding to protein biosynthesis through ERK1/2. Basal [³⁵S]methionine incorporation is elevated in _1D-AR- relative to _1B-AR-expressing fibroblasts (Fig. 8, compare B and D). The ERK1/2 pathway inhibitor PD98059 blocks basal ERK1/2 activity (Fig. 6) and [³⁵S]methionine incorporation (Fig. 8D), suggesting that constitutive ERK1/2 activity is coupled to increases in protein biosynthesis in _1D-AR-expressing cells. The physiological significance of agonist-dependent increases in ERK1/2 and protein biosynthesis by the _1B-AR and constitutive activity of these responses by the _1D-AR are not known. We did not observe increases in cell size (hypertrophy) in unstimulated _1D-AR cells or in agonist-treated _1B-ARs (data not shown). It is possible that constitutive activation of ERK1/2 for _1D-ARs or agonist-mediated increases in ERK1/2 via _1B-ARs induces a differentiated or synthetic phenotype characterized by constitutive synthesis of extracellular matrix proteins in Rat 1 fibroblasts.

In contrast to the requirement for ERK1/2 activation in [³⁵S]methionine incorporation, inhibition of DNA synthesis occurs through an ERK1/2-independent pathway for both ₁-AR subtypes (Fig. 10). Similar to a previous study examining DNA synthesis in CHO cells expressing the _1A-AR (Keffel et al., 2000), the p38 kinase inhibitor SB203850 reverses PE-mediated inhibition of [³H]thymidine incorporation in the _1B-AR cell line (Fig. 9A). The lack of effect of SB203580 on PE-mediated inhibition of [³H]thymidine incorporation and elevated basal [³⁵S]methionine incorporation in the _1D-AR cell line is consistent with the inability of this receptor subtype to induce p38 kinase activation (Fig. 5). Therefore, inhibition of [³H]thymidine incorporation in response to _1D-AR activation may occur through JNK and/or other signaling cascades in this cell line.

View larger version (20K): [in this window] [in a new window]   Fig. 10.   Proposed model of signaling by 1B-AR and 1D-ARs in Rat 1 fibroblasts: agonist-induced activation of the 1B-AR increases p38 kinase and ERK1/2 activity. Activation of both p38 kinase and ERK1/2 are linked to 1B-AR-mediated increases in protein biosynthesis. In contrast, ERK1/2 activity and protein biosynthesis are increased in the absence of agonist in 1D-AR cells (bold arrow). However, agonist treatment of 1D-AR-expressing cells promotes JNK activation and inhibition of DNA synthesis. ERK1/2 activation is not coupled to inhibition of DNA synthesis mediated by either receptor subtype.

As illustrated in Fig. 10, we propose that agonist activation of the _1B-AR induces p38 kinase and ERK1/2 activation and increases protein biosynthesis. In addition, our results indicate that the _1B-AR uses both ERK1/2 and p38 kinase-dependent pathways to induce protein biosynthesis. Use of two parallel pathways may explain the robust effect of agonist on protein synthesis in the _1B-AR relative to the _1D-AR cell line (Fig. 2). In contrast to the regulatory properties of _1B-ARs, increases in protein biosynthesis occur primarily through constitutive ERK1/2 activity for the _1D-AR. Furthermore, inhibition of DNA replication occurs through an ERK1/2-independent pathway in both cell lines, suggesting that the profile of MAPK activity (i.e., ERK1/2 versus JNK or p38 kinase) may differentiate growth-related responses in Rat 1 fibroblasts.