Introduction

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Protein-tyrosine phosphorylation is an important process involved in cellular growth and regulation. Indeed, many growth factor receptors and oncogene products possess tyrosine kinase activity (Yarden and Ullrich, 1988). MAP kinase, a serine/threonine protein kinase, is activated in many proliferating cells in response to mitogenic stimulation (Ray and Sturgill, 1988). A unique property of MAP kinase is that both threonine and tyrosine residues on the enzyme must be phosphorylated for the enzyme to be fully active (Anderson et al., 1990; Ballou et al., 1991). It is believed that MAP kinase is activated by a dual-specificity threonine/tyrosine kinase, MAP kinase kinase (Ahn, 1993; Cobb et al., 1991). MAP kinase is activated during mitosis, meiosis and G₀-G₁ transition (Sturgill and Wu, 1991) and has been implicated in regulation of the cell cycle (Thomas, 1992). MAP kinases are thus implicated in the regulation of mitogen-induced cell growth and proliferation (Pages et al., 1993; Pelech and Sanghera, 1992; Seth et al., 1992) and appear to play an important role in signal transduction from growth factor receptors to the nucleus.

There is considerable interest in understanding control mechanisms of vascular smooth muscle cell growth and regulation. Abnormal proliferation of vascular smooth muscle within the arterial intima plays a key role in advancing lesions of atherosclerosis (Ross, 1986; Schwartz and Reidy, 1987) and restenosis after balloon angioplasty (Ross, 1986; Schwartz et al., 1986). Growth and proliferation of smooth muscle cells are also a characteristic feature in arteries from hypertensive patients and experimental animals (Schwartz and Reidy, 1987). PDGF released from platelets after adhesion to the injured vessel wall may be a key stimuli for smooth muscle cell migration and proliferation (Ross, 1986). The signaling pathways used by PDGF receptor stimulation in vascular smooth muscle involve stimulation of the MAP kinase pathway (Graves et al., 1993). Other growth factors, cytokines and vasoactive agents that also stimulate MAP kinase activity in vascular smooth muscle cells include epidermal growth factor (Granot et al., 1993), interleukin-8 (Yue et al., 1994), vasopressin (Granot et al., 1993; Okada et al., 1994), angiotensin II (Duff et al., 1992; Sung et al., 1994; Weber et al., 1994), endothelin-1 (Koide et al., 1992; Sung et al., 1994; Weber et al., 1994) and thromboxane A₂ (Morinelli et al., 1994). Whether the signaling pathways used by different mitogenic stimuli in vascular smooth muscle cells are different from other cell types is not clear at present. There is, however, evidence showing that vasopressin stimulates vascular smooth muscle cell MAP kinases and not NIH-3T3 cell MAP kinase (Granot et al., 1993).

Carvedilol is a neurohumoral antagonist with multiple actions (Feuerstein et al., 1993; Feuerstein and Ruffolo, 1995; Ruffolo et al., 1992). Carvedilol was originally discovered to be a beta adrenoceptor antagonist (Ruffolo et al., 1992). However, subsequent research revealed that this agent possessed potent antioxidant and free radical scavenger properties (Yue et al., 1992). In addition, carvedilol was an inhibitor of vascular smooth muscle cell proliferation induced by a broad group of mitogens, such as PDGF, fibroblast growth factor, endothelin-1, serum and thrombin (Ohlstein et al., 1993; Sung et al., 1993). These multiple actions may account for the unique in vivo effect of inhibition neointimal formation after balloon angioplasty (Ohlstein et al., 1993). The data that carvedilol inhibits the mitogenic effects of a broad group of mitogens indicate that carvedilol is not acting at the level of a single mitogen receptor but rather at a common pathway of signal transduction leading to cell growth and proliferation. The present study was therefore undertaken to examine the effects of carvedilol on MAP kinase activity and cell cycle progression in vascular smooth muscle cells.

	Materials and Methods

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Chemicals. Human platelet derived growth factor A/B (PDGF) was purchased from Boehringer-Mannheim (Indianapolis, IN). Carvedilol 1-(carbazol-4-yloxy)-3-{(2-(o-methoxyphenoxy)ethyl)amino}-2-propanol was synthesized at SmithKline Beecham Pharmaceuticals (King of Prussia, PA). (-³²P)-ATP (specific activity, 3000 Ci/mmol) was purchased from Dupont-New England Nuclear (Wilmington, DE). All other chemicals were reagent grade from commercial sources and were used without further purification.

Culture of rat aortic smooth muscle cells. Thoracic aortae of male Sprague-Dawley rats were excised rapidly and immersed in DMEM containing gentamycin (50 µg/ml) and cleaned of connective tissue and adherent fat. Isolated arteries were cut open, and endothelium was removed by gently rubbing of the intimal surface with sharp scissors. Denuded aortae were cut into ~3-mm cubes and placed intimal face down into six-well plates. DMEM containing 10% fetal calf serum and gentamycin was gently added to the well to cover the tissues without disturbance to the orientation of the explants. Vascular smooth muscle cells were allowed to grow out from the tissue (7-10 days), and the tissues were removed by washings with culture medium. After reaching confluence in six-well plates, cells were harvested by brief trypsination and grown in T-150 flasks (passage 1). Early subcultured cells (passage 3) were used in all experiments. Purity of the vascular smooth muscle cells was estimated to be >90% by cell morphology and by the immunoexpression of myosin described previously (Ohlstein et al., 1993). Cell viability was >98% as determined by exclusion of 0.2% Trypan blue.

Cell-mediated MAP kinase activity assay. Rat vascular smooth muscle cells were seeded (1-2 × 10⁴ cells/cm²) and grown to subconfluence in 60 × 15-mm culture dishes (Corning) in DMEM-10% serum. Cells were made quiescent for 48 hr in DMEM medium and preincubated with or without carvedilol for various times (30 min-24 hr) followed by brief exposure (5-15 min) to mitogen (e.g., PDGF A/B, serum, PMA). Incubation medium was decanted, and cell monolayers were washed once with cold DPBS containing sodium orthovanadate (1 mM). Cells were lysed (30-60 min at 4°C) with lysing solution consisting of 20 mM Tris·HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 1 mM sodium orthovanadate and 1% Nonidet P-40. Lysed cells were transferred to microcentrifuge tubes and centrifuged at 16,000 × g for 10 min in 4°C. MAP kinase activity was measured according to the method of Koide et al. (1992) with modification. Briefly, assay (30 µl total volume) contained 10 µl of sample to be assayed plus 10 µl assay cocktail and 10 µl substrate, myelin basic protein peptide (APRTPGGRR) (Upstate Biotechnology, Lake Placid, NY). The peptide, which comprises the MAP kinase phosphorylation site in myelin basic protein (Clarke-Lewis et al., 1991), has been used as a substrate to detect MAP kinase activity by many investigators (Daeipour et al., 1993; Gomez-Cambronero et al., 1993) and produces a lower background in comparison to myelin basic protein when used in measuring of MAP kinase activity. The final concentrations of the assay cocktail consisted of 50 mM -glycerophosphate, pH 7.5, 10 mM magnesium acetate, 1 mM DTT, 1.5 mM EGTA, 60 µM ATP, and 1 µCi of (-³²P)-ATP. The assay was initiated by addition of substrate (final concentration, 1 mM) and terminated after 20 min by spotting 25 µl onto P-81 phosphocellulose filter paper inside the scintillation vial. The filter was washed five times with 4 ml each of 180 mM phosphoric acid, and radioactivity was counted. The background radioactivity (enzyme without substrate) was subtracted in the calculation. Thus, the nonspecific phosphorylation in the assay was minimized.

Immunoblotting of smooth muscle cell lysates with MAP kinase antibodies and anti-phosphotyrosine. Proteins in whole-cell lysates from control, PDGF-stimulated and drug-pretreated cells were separated by 12% sodium dodecyl sulfate-PAGE and transferred to nitrocellulose membranes (Immobilon-P). For determination of MAP kinase protein and tyrosine phosphorylation, the following procedures were used. Membranes were treated (60 min) with 3% bovine serum albumin in TPBS solution (20 mM Tris, 160 mM NaCl in PBS, pH 7.4) followed by 3 washings (5 min each) in TPBS solution containing 0.05% Tween 20 (Tween-TPBS). The membranes were then blotted with a rabbit anti-human MAP kinase (UBI) or a mouse monoclonal anti-phosphotyrosine (IgG2b_k clone 4G10; UBI) at a concentration of 1 µg/ml for 2 hr. After 3 washings (5 min each) in Tween-TPBS, membranes were blotted with horseradish peroxidase conjugated donkey anti-rabbit antibodies or rabbit anti-mouse antibodies at 1 µM for 45 min. After three washings with Tween-TPBS (10 min each), the membranes were treated with a chemiluminescent detection reagent, ECL reagents (reagent 1 and 2, 1:1 mixture) (Amersham, RPN 2106) for 30 sec. Blots were then subjected to autoradiography with Kodak X-Omat film.

Purification of MAP kinases. MAP kinases were purified from PDGF- or serum-stimulated cell extract by FPLC using a Mono Q HR 5/5 column (Bogoyevitch et al., 1993; Koide et al., 1992). Briefly, quiescent vascular smooth muscle cells were exposed to PDGF (3 nM) or serum (10%) for 5 min. Cells were washed three times and harvested by scraping into ice-cold Dulbecco's PBS containing sodium orthovanadate (1 mM). Cell suspensions were homogenized using a Polytron PTA7K1 probe. The homogenates were centrifuged at 1000 × g for 10 min followed by 10,000 × g for 10 min in an Eppendorf centrifuge. The supernatant was filtered through 0.2-µm filter unit (Acrodisk), and the filtrate was applied to a Mono Q HR 5/5 column equilibrated with 15 mM -glycerophosphate, 1.5 mM EDTA, 0.1 mM Na₃VO₄ and 1 mM DTT, pH 7.3. After washing with the same buffer, MAP kinase activity was eluted with a linear gradient of NaCl in the same buffer at a flow rate of 1 ml/min. Fractions (0.5 ml each) were collected and assayed (10 µl) for MAP kinase activity as described above except 1 mM myelin basic protein (Sigma Chemical, St. Louis, MO) was used as a substrate.

Cell cycle analysis by propidium iodide staining. Rat vascular smooth muscle cells were grown to near-confluence in T-150 flasks in culture medium as described above. The cell monolayer was washed once with DMEM without serum, harvested by brief trypsinization and suspended into the same medium. Cells were then seeded (5 × 10⁵ cells/cm²) into dishes (10-cm diameter) and incubated for 24 hr at 37°C in DMEM without serum. After replenishment with fresh DMEM, cells were preincubated with carvedilol for 15 min followed by the addition of serum (10% final) and incubated for 1, 2 or 3 days. At the end of each time point, cells were trypsinized and collected into 12 × 75-mm polypropylene tubes and pelleted. Cells were washed once with cold Ca⁺⁺/Mg⁺⁺-free PBS containing 1% bovine serum albumin and once with cold Ca⁺⁺/Mg⁺⁺-free PBS and suspended in 1.5 ml of cold PBS. Cells were gently vortexed and resuspended into a single-cell suspension after each centrifugation/washing cycle. To the cell suspension undergoing gentle vortexing, 3 ml of cold ethanol (100%) was quickly added. The ethanol-fixed cells were stored at 4°C for 1 to 2 days before proceeding with the staining procedure. Excess ethanol was removed by centrifugatio, and cells were washed once with PBS. Cells were resuspended in 4.5 ml of PBS, and 0.5 ml of RNase (100 µg/ml) was then added for 30-min incubation at 37°C. After centrifugation, cells were resuspended in 5 ml of 70 µM propidium iodide in PBS. The staining process was performed in the dark for 30 min at room temperature. The excess staining solution was removed and cells resuspended in PBS to 2 to 5 × 10⁶ cells/ml. The DNA content of the cells was analyzed by a FACScan flow cytometer (Becton Dickinson Immunocytometry SystemS, San Jose, CA) coupled with a HP CONSORT 32 computer (Hewlett Packard, Palo Alto, CA).

Thymidine kinase assay. Rat vascular smooth muscle cells were seeded (1-2 × 10⁴ cells/cm²) and grown to sub-confluence in 100 × 15-mm culture dishes in DMEM-10% serum. Cells were made quiescent for 48 hr in DMEM medium and preincubated with or without carvedilol for 15 min followed by the addition of 5% to 10% serum for an additional 24-hr incubation. The incubation medium was decanted, and cell monolayers were washed once with cold DMEM. Cells were scraped into 1.5 ml of 50 mM Tris buffer (pH 8.0) containing 0.5 mM mercaptoethanol and sonicated. Cell homogenates were assayed for thymidine kinase activity according to the method described by Desgranges et al. (1991).

Analysis of data. Results are expressed as the mean ± S.E.M. from n number of experiments done in triplicate or quadruplicate. Statistical significance was determined using a one-way analysis of variance with a value of P < .05 accepted as significant.

	Results

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Effect of carvedilol on PDGF-mediated MAP kinase activity in rat vascular smooth muscle cells. PDGF was a potent stimulator of MAP kinase in rat vascular smooth muscle cells. PDGF (1 nM) stimulated MAP kinase activity ranging from 10- to 15-fold over basal activity (basal activity: 150 ± 16 pmol/mg/20 min, n = 6). The stimulation was transient, reached maximal stimulation between 5 and 10 min and returned to the basal activity after 20 to 30 min (fig. 1A). When vascular smooth muscle cells were preincubated with carvedilol (10 and 30 µM) for 24 hr, the basal activity of MAP kinase was not significantly altered by 10 µM carvedilol but was lowered (39%) by 30 µM carvedilol (fig. 1B). Carvedilol inhibited PDGF-induced MAP kinase activity in rat aortic smooth muscle cells in a concentration-dependent manner. The inhibition achieved by 10 and 30 µM carvedilol on PDGF-induced activity were 22% and 54%, respectively (fig. 1B).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Carvedilol inhibits PDGF-induced MAP kinase activity in vascular smooth muscle cell extracts. Quiescent cells were exposed to PDGF (1 nM) for various times (A) or preincubated with carvedilol for 24 hr followed by 10-min exposure to vehicle (control) or 1 nM PDGF (B). Results are expressed as mean ± S.E.M. from three independent experiments with triplicate determinations (A) or from a representative triplicate experiment from 3 experiments with similar results (B).

Effect of carvedilol on PDGF-induced MAP kinases and their tyrosine phosphorylation in immunoblotting assay. Immunoblotting is shown of control, PDGF-stimulated and carvedilol-pretreated whole cell lysates for MAP kinase and phosphotyrosine (fig. 2). Brief exposure (5-10 min) of rat aortic smooth muscle cells with PDGF (1 nM) did not significantly alter the 44- and 42-kDa protein bands recognized by MAP kinase antibody (left). However, PDGF significantly increased tyrosine phosphorylation of protein bands near 130, 120, 75, 44, 42 and 38 kDa (right). A 24-hr preincubation of cells with carvedilol (10 µM), a concentration that inhibited MAP kinase and cell proliferation, did not significantly alter the intensity of 44- and 42-kDa protein bands recognized by MAP kinase antibody (fig. 2, left). However, carvedilol treatment significantly decreased the intensity of phosphotyrosine bands recognized by phosphotyrosine antibodies (fig. 2, right). Four other protein bands were also recognized by phosphotyrosine antibodies. Among the protein bands that showed tyrosine phosphorylation, only protein bands of 44 and 42 kDa were recognized by anti-MAP kinase antibodies (fig. 2, left).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Carvedilol does not inhibit expression of p42 and p44 MAP kinase isozymes but inhibits the tyrosine phosphorylation in vascular smooth muscle cells. Quiescent cells were preincubated with carvedilol for 24 hr and exposed to PDGF (1 nM) for 5 min. Western blots were examined for MAP kinase (left) and phosphotyrosine (right) as described in the Materials and Methods.

Effect of carvedilol on PMA-induced MAP kinase activity in vascular smooth muscle cells. To investigate whether other mitogen-stimulated (i.e., protein kinase C) signal transduction pathways leading to MAP kinase were also affected by carvedilol, the effect of carvedilol on PMA-stimulated MAP kinase activity in vascular smooth muscle cells was studied. Carvedilol inhibited PMA-induced MAP kinase activity in a concentration- and time-dependent manner (fig. 3). At 30-min pretreatment of vascular smooth muscle cells with 10 to 100 µM carvedilol, the highest concentration tested (100 µM) produced complete inhibition of PMA-induced MAP kinase activity (fig. 3A). At a concentration of 30 µM, carvedilol inhibited PMA-induced MAP kinase activity by 93%. Carvedilol (10 µM) did not significantly inhibit PMA-induced MAP kinase activity in the 30-min preincubation study. However, when smooth muscle cells were preincubated with carvedilol for 24 hr, carvedilol became more potent for inhibiting PMA-induced MAP kinase activity. The inhibition by 10, 30 and 100 µM carvedilol was 36%, 96% and 100%, respectively (fig. 3B).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Carvedilol inhibits PMA-induced MAP kinase activity in vascular smooth muscle cells. Quiescent cells were pretreated with carvedilol for 30 min (A) and 24 hr (B) followed by exposure to PMA (500 nM) for 15 min. MAP kinase activity was then determined. Results are from a typical triplicate experiment. Similar results were obtained in two other experiments.

Purification of MAP kinases from PDGF- or serum-stimulated cell free extract. MAP kinases from vascular smooth muscle cells stimulated (5 min) with PDGF or serum were purified by FPLC using Mono Q HR 5/5 column. When cells were exposed to PDGF (3 nM) (fig. 4A) or 10% serum (fig. 4B), two major peaks with MAP kinase activity were found. The specific activity (pmol/mg/20 min) of the enzyme were considerably higher in peak 2 than in peak 1. Figure 4A also shows that unstimulated cells (basal) had minimal MAP kinase activity under either peak 1 or peak 2. The MAP kinases in peak 1 and peak 2 had molecular masses of 42 and 44 kDa, respectively, and were recognized by MAP kinase- and phosphotyrosine-specific antibodies (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   FPLC column (Mono Q HR5/5) chromatography of unstimulated or mitogen-stimulated (5 min) MAP kinase activity. Cell extracts from quiescent cells or PDGF (3 nM)- (A) or serum (10%)-stimulated (B) cells were subjected to FPLC column chromatography as described in Materials and Methods. Peaks 1 and 2 showed MAP kinase activity determined by the phosphorylation of myelin basic protein.

Effect of carvedilol on FPLC-purified MAP kinases. The effects of carvedilol on cell-free, FPLC-purified MAP kinase activity are shown in figure 5. Preincubation of FPLC-purified enzyme with carvedilol (1 or 10 µM) for 15 min followed by enzymatic assay (20 min in room temperature) resulted in the concentration-related inhibition of enzyme activity. At the highest concentration tested, carvedilol (10 µM) inhibited peak 1 and peak 2 enzyme derived from PDGF-stimulated cells by 55 ± 3% and 43 ± 1%, respectively. Similar inhibition was observed with FPLC-purified enzymes from serum (10%)-stimulated cell extracts (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of carvedilol on FPLC-purified MAP kinase activity. Cell extracts from PDGF-stimulated cells were purified by FPLC as described in figure 4. Three fractions containing highest activity of MAP kinase activity in peak 1 or peak 2 were pooled for the study. Peak 1 or peak 2 enzymes were preincubated with carvedilol (1 or 10 µM) for 15 min (in room temperature) followed by 20-min enzyme assay (in room temperature). Peak 1 and peak 2 enzymes corresponded to 42- and 44-kDa MAP kinase isozymes. Results are expressed as mean ± S.E.M. from 3 experiments done in triplicate.

Effect of carvedilol on serum-induced cell cycle progression. The cell cycle progression of quiescent cells induced by serum and the effect of carvedilol (10 µM) are shown in figure 6. When rat vascular smooth muscle cells were grown in DMEM in the absence of serum for 24 to 48 hr (quiescent cells), the distribution of cells in G₀/G₁, S and G₂/M phases were 96%, 1% and 3%, respectively. Subsequent addition of serum (10%) significantly increased the population of cells in S or G₂/M phase (fig. 6, left). After 24 hr of incubation with serum, cells in S and G₂/M phase increased to 44% and 23% respectively. In the presence of carvedilol (10 µM) (fig. 6, right), cells in S and G₂/M phase were substantially less than that in the control. The percentage inhibition by carvedilol for S and G₂/M was 48% and 35%, respectively. After 48-hr exposure to serum, vascular smooth muscle cells still showed relatively high percentage in S and G₂/M phase (31% and 15%). Carvedilol (10 µM) inhibited cell number in S and G₂/M phase by 39% and 47%, respectively. After 72 hr, the majority of the cells in both control and carvedilol-treated cells remained in G₀/G₁ phase.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Carvedilol inhibits vascular smooth muscle cell entering S phase of cell cycle. Quiescent cells were pretreated with carvedilol (10 µM) for 15 min followed by an addition of serum (10%) for 24-, 48- and 72-hr incubation. Propidium iodide-stained cells were subjected to flow cytometry as described in the Materials and Methods. Results were expressed as cell number (ordinate) vs. fluorescence intensity (abscissa). Similar results were obtained in 3 other experiments.

Effect of carvedilol on serum-induced thymidine kinase activity. Because carvedilol inhibits vascular smooth muscle cell entry from G₀/G₁ phase to S phase in cell cycle, the effect of carvedilol on the S phase-specific enzyme marker thymidine kinase activity was investigated. Figure 7 illustrates that serum (5% or 10%, 24 hr) stimulated thymidine kinase activity of smooth muscle cells in a concentration-dependent manner. The upregulation of thymidine kinase activity by 5% and 10% serum was 4- and 8-fold, respectively. Carvedilol (10 µM) markedly inhibited (>70%) serum-induced thymidine kinase activity in rat vascular smooth muscle cells.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Carvedilol inhibits thymidine kinase in vascular smooth muscle cells. Quiescent cells were pretreated with carvedilol (10 µM) for 15 min followed by an addition of serum (5%) for 24 hr. Results are a typical experiment done in quadruplicate with duplicate assays.

	Discussion

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MAP kinase, a serine/threonine specific protein kinase, is a unique protein kinase that is active only when both threonine and tyrosine (Thr¹⁸³ and Tyr¹⁸⁵ in mammalian; Thr¹⁸⁸ and Tyr¹⁹⁰ in Xenopus 42-kDa MAP kinase) within the protein kinase subdomain VIII are phosphorylated (Ray and Sturgill, 1988). MAP kinase thus has been considered as a "switch kinase" responsible for tyrosine to serine/threonine phosphorylation. MAP kinase is believed to be a key enzyme in the signal transduction pathway and, as such, has been widely studied in many proliferating cells in response to mitogenic stimulation (Pages et al., 1993; Ray et al., 1988). The present study using a whole-cell system demonstrated that carvedilol inhibited mitogen (e.g., PDGF)-induced activation of MAP kinase activity measured by the inhibition of phosphorylation of myelin basic protein peptide and tyrosine phosphorylation of the enzymes. In addition, carvedilol also directly inhibited FPLC-purified MAP kinase from mitogen-stimulated cell extract.

In agreement with Koide et al. (1992), rat aortic smooth muscle cells have two MAP kinase isozymes identifiable by antibodies that recognize both 42- and 44-kDa isozymes (with higher affinity toward 44-kDa isozyme; fig. 2), and these isozymes can be separated by Mono Q chromatography (fig. 5). Mono Q partially purified enzyme was indeed MAP kinases as determined by their immunoreactivity to MAP kinase antibodies, up-regulation by mitogens (fig. 4), molecular mass of 42 and 44 kDa with tyrosine phosphorylation in both sodium dodecyl sulfate-PAGE and myelin basic protein polymerized PAGE resolving gel (data not shown).

The different intensity of 42- and 44-kDa MAP kinase protein bands recognized by anti-MAP kinase antibody in the present study should not be interpreted that smooth muscle cells have more 44-kDa isozyme because the antibody used in this study may have higher affinity toward 44-kDa isozyme. However, PDGF- or serum-stimulated cell lysates consistently showed greater 44- than 42-kDa isozyme activity after Mono Q chromatography, which may indicate that vascular smooth muscle cells have more 44-kDa isozyme (fig. 4) or 44-kDa isozyme is more readily activated (fig. 2, right). These data suggest that carvedilol may regulate both the levels of MAP kinase protein, as well as the activation of MAP kinases through tyrosine phosphorylation. The inhibition was not due to cytotoxicity because not all protein bands detected by silver staining after carvedilol treatment were affected (data not shown). Furthermore, the concentrations tested in these experiments have been previously shown not to be cytotoxic and reversible in a variety of smooth muscle cell proliferation assays (Sung et al., 1993).

Although carvedilol inhibits the catalytic activity of MAP kinase partially purified from mitogen-stimulated vascular smooth muscle cells, it still is not certain whether carvedilol can have additional upstream effects on another kinase leading to the activation of MAP kinase or an inhibitor of other signal transduction pathways. The later possibility seems plausible because carvedilol also inhibited tyrosine phosphorylation of several protein bands (e.g., 75- and 38-kDa bands) in addition to MAP kinase. However, this inhibition was not a nonspecific effect as carvedilol did not inhibit MAP kinase kinase protein expression of as determined by Western analysis (data not shown). The precise molecular mechanism for the inhibition of MAP kinase activity is not yet known. However, carvedilol, as well as some of its hydroxylated metabolites, are also potent antioxidants and free radical scavengers, and this activity largely results from the unique carbazol moiety in its structure. Extensive studies in a variety of test systems, including physicochemical, biochemical, cellular and in vivo models, have established the ability of carvedilol to scavenge oxygen-derived free radicals, and these studies have been reviewed recently (Feuerstein and Ruffolo, 1995; Yue et al., 1992). As such, redox-sensitive reactions may be sensitive to the antioxidant properties of carvedilol.

Additional support for the possibility that the inhibitory effect of carvedilol on MAP kinase activity in vascular smooth muscle may be mediated by its antioxidant properties comes from the observations that reactive oxygen intermediates can stimulate MAP kinase activity in NIH-3T3 cells (Stevenson et al., 1994). Furthermore, oxidative stress can activate a number of early genes, including c-fos, c-myc (Crawford et al., 1988), c-jun (Datta et al., 1992) and NF-B (Schreck et al., 1991), and many of these early genes products are known to be phosphorylated by MAP kinases (Alvarez et al., 1991; Chen et al., 1992; Pulverer et al., 1991).

Vasoconstrictors such as endothelin, vasopressin, angiotensin II and thrombin activate p42 MAP kinase, and activation of this enzyme has been shown to require the activation of protein kinase C (Kribben et al., 1993). The present study confirms previous observations (Kribben et al., 1993) that phorbol esters, esters that are thought to act exclusively through activation of protein kinase C, stimulate MAP kinase activity in rat vascular smooth muscle cells and further supports that MAP kinase may play an important role in signal transduction mediated through protein kinase C activation in this cell type. Furthermore, it has been shown in fibroblasts (Kazlauskas and Cooper, 1988) and myocytes (Bogoyevitch et al., 1993) that phorbol esters also elicit tyrosine phosphorylation on 42-kDa MAP kinase. Therefore, the present data support the existing literature that protein kinase C and MAP kinase participate in the regulation of vasoconstriction, as well as vascular smooth muscle proliferation.

Inasmuch as MAP kinase is known to be activated by a wide variety of mitogens and implicated in cell proliferation and cell cycle control (Boulton et al., 1990), the direct involvement of MAP kinase in the regulation of the cell cycle has not been demonstrated definitively. It has been suggested, however, that treatment of quiescent (G₀) vascular smooth muscle cells with a mitogen (e.g., PDGF, serum) induces rapid activation of MAP kinase that may be involved in the mitogenic response several hours after activation (i.e. at late G₁ or early S phase before DNA synthesis) (Pelech and Sanghera, 1992). Furthermore, MAP kinase is highly related to p34^cdc2, a cyclin-dependent protein kinase (Schreck et al., 1991) that is also implicated in regulation of cell cycle (Draetta, 1990; Pelech et al., 1990; Pelech and Sanghera, 1992). The present results demonstrate that carvedilol (10 µM) significantly inhibited the quiescence cells from entering S and G₂/M phases in response to 10% serum (fig. 6). The inhibitory effect of carvedilol was not due to cytostasis, or a delay in entering the cell cycle, since the inhibition is seen after 24 hr and lasted for 48 hr (fig. 6). Under these experimental conditions, serum-stimulated cells reached confluence after 72 hr and remained in G₀/G₁ phase (70-80%). As for carvedilol-treated cells, the cell count after 72 hr was significantly (40%) less than control (1.6 × 10⁶ vs. 2.7 × 10⁶ cells/dish), but 80% of the cells were in G₀/G₁ phase.

Thymidine kinase, which catalyzes the conversion of deoxythymidine to deoxythymidine monophosphate, is an important enzyme involved in the synthesis of DNA precursors that lead to DNA synthesis during the cell cycle. Thymidine kinase activity is low in G₁, increases during S phase and declines in G₂ (He et al., 1991) and has been described as a model S phase-specific enzyme (Sherley and Kelly, 1988). The present study shows that carvedilol inhibits thymidine kinase activity (fig. 7) under the similar experimental conditions for cell cycle determination. These data further support that carvedilol inhibits vascular smooth muscle cells from entering S phase on serum stimulation.

In summary, carvedilol inhibits MAP kinase activity in mitogen-stimulated smooth muscle cell extracts and directly inhibits MAP kinase partially purified from mitogen-stimulated smooth muscle cellular extracts. Carvedilol also significantly inhibits quiescent vascular smooth muscle cells entering S phase from G₀/G₁ in response to mitogenic stimulation. Therefore, the in vitro and in vivo antiproliferative effects of carvedilol reported previously (Ohlstein et al., 1993; Sung et al., 1993) may involve the inhibition of MAP kinase and entry of vascular smooth muscle cells into S phase of the cell cycle. The present study also suggests that carvedilol represents a new tool for the study of MAP kinase activity and will be a useful agent for further understanding of the regulation of vascular smooth muscle cell growth.