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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1274-1282

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1274-1282.)
© 1997 American Heart Association, Inc.

Articles

Low-Affinity Thromboxane Receptor Mediates Proliferation in Cultured Vascular Smooth Muscle Cells of Rats

Feng N. Ko

From the Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.

Correspondence to F.N. Ko, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd, Section 1, Taipei, Taiwan.


*    Abstract
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 *Abstract
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Abstract We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by mea-suring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5±2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3±0.6 nmol/L and 1.4±0.5 µmol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2{alpha} showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.


Key Words: thromboxane receptor • mitogen-activated protein kinase • vascular smooth muscle cell • proliferation • protein kinase C


*    Introduction
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 *Introduction
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 down arrowResults
 down arrowDiscussion
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Recent studies suggest that the growth and migration of medial smooth muscle cells play important roles in the remodeling of the vascular structure in a hypertensive state or in arterial restenosis after angioplasty.1 2 It is also well known that SHR show hypertrophy and/or hyperplasia in their cardiovascular system even in the prehypertensive stage.3 4 A number of studies have thus been conducted to investigate the regulatory mechanism for the proliferation of VSMCs and have provided evidence for the involvement of various vasoactive substances in the modulation of VSMC growth.5 6 7 8

TxA2 is a potent inducer of platelet aggregation and constrictor of VSMCs. It is produced in activated platelets and vascular walls.9 Particularly in some genetic rat models (eg, SHR and Dahl salt-sensitive strains) for human hypertension, TxA2 generation in the vascular wall is enhanced and therefore could contribute in part to the hypertrophy of the media observed in hypertension.10 11 In addition, some studies in vitro indicate that Tx receptor agonists induce VSMC proliferation.5 11 Antagonism of the Tx receptor with specific receptor antagonists not only reduces the deposition of cholesterol in the aortic wall but also retards plaque formation in coronary arteries of hypercholesterolemic rabbits.12 Thus, TxA2 released from the vascular wall as well as aggregated platelets may play a role in the remodeling of the vascular structure in a hypertensive state or in cardiovascular diseases including atherosclerosis and arterial restenosis after angioplasty.1 2

These observations suggest that a Tx receptor is involved in TxA2-stimulated events. Recent studies reveal that Tx receptor subtypes may exist in various tissues and/or species.13 14 However, it is not known which subtype of the Tx receptor is involved in the growth of VSMCs. In previous work, we have reported that there are two TxA2 binding sites existing in A10 VSMCs. The high-affinity site is responsible for TxA2-induced vasoconstriction, while the low-affinity site is responsible for TxA2-mediated VSMC proliferation.15 Although the mechanism for TxA2-induced blood vessel contraction is well known, the cellular mechanism for TxA2-mediated VSMC proliferation has not been fully addressed. Herein, we report the results of a series of experiments in an attempt to elucidate the pathway TxA2 activates for proliferation of VSMCs, by using cultured aortic VSMCs from WKY and SHR.


*    Methods
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 *Methods
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Reagents
PDGF-BB homodimers and Py20 were obtained from Upstate Biotechnology Inc. Monoclonal antibodies against ERK1 (p44 mapk), ERK 2 (p42 mapk), Ras (Y13-259), and Raf-1 were from Santa Cruz Biotechnology Inc. PMA, MBP, staurosporine, U46619 (9,11-dideoxymethanoepoxy-9{alpha},11{alpha}-prostaglandin F2{alpha}) and PTX were purchased from Sigma Chemical Company. Protein G–Sepharose was from Pharmacia LKB Biotechnology Inc. [3H]U46619 (>10 Ci/mmol) was obtained from DuPont NEN, and [3H]thymidine (25 Ci/mmol), [{gamma}-32P]ATP (>5000 Ci/mmol), and myo-[2-3H]inositol (17.1 Ci/mmol) were purchased from Amersham. All cell culture reagents were purchased from GIBCO. All other chemicals were reagent grade. Unless otherwise noted, the concentrations of the drugs used were U46619 10 µmol/L, SQ29548 10 µmol/L, staurosporine 30 nmol/L, and PMA 500 nmol/L.

Cell Culture
Medial explants were dissected from freshly harvested rat aortic strips and plated in 100-mm Petri dishes. VSMCs (aortic) from explants were cultured as described by Ross.16 After the cells reached confluence, they were harvested by brief exposure to trypsin/EDTA (0.1%/4 mmol/L) and transferred onto a new plate. At this stage, the cells were designated as the first passage. Harvesting was repeated when the cell growth reached confluence. VSMCs from passages 3 to 17 were used in the studies. Cell viability was determined by trypan-blue exclusion and generally exceeded 95%. Cells were characterized as smooth muscle cells by morphology and immunostaining with an antibody to smooth muscle {alpha}-actin.

Binding of [3H]U46619
For ligand-binding assays, confluent cell monolayers were washed with Krebs-Henseleit solution (KHS; composition, in mmol/L: 117.5 NaCl, 5.4 KCl, 1.2 NaH2PO4, 25.0 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 5.5 glucose, and 25.0 HEPES), pH 7.4, and then 250 µL of various concentrations of [3H]U46619 was added for 60 minutes at 37°C. The incubation was terminated and the bound radioactivity counted in a liquid scintillation counter (Beckman 5000 TC) as previously described.15 Nonspecific binding was defined as binding in the presence of 200 µmol/L U46619. The protein content of the cells was measured by the method of Lowry et al,17 using bovine serum albumin as a standard.

Determination of DNA and Protein Synthesis
DNA and protein synthesis were measured by using [3H]thymidine and [3H]leucine incorporation, respectively.15 Quiescent cells were incubated with U46619 or U46619 plus antagonists in 1 mL DMEM/FCS-free medium for 20 hours, when 1 µCi/mL [3H]thymidine or [3H]leucine was added for pulse labeling. The cells were further incubated for 4 hours and then washed twice with 1 mL of PBS. The cells were treated with 10% trichloroacetic acid to precipitate the acid-insoluble material, from which the DNA or protein was collected on a Whatman GF/B filter. The filter was then shaken in 3.5 mL scintillation fluid for 24 hours before counting.

[3H]IP Accumulation
Confluent cell monolayers were loaded with [3H]myo-inositol (5 µCi/mL) for 24 hours in inositol-free DMEM. Prelabeled cells were then washed twice with KHS solution and incubated for 15 minutes in the presence of 10 mmol/L LiCl. U46619 was added and incubation continued for 1 hour. The incubation was terminated and the total [3H]IP isolated and the radioactivity counted as described previously.18

Measurement of [Ca2+]i Level
VSMCs were cultured on glass coverslips and incubated in a medium containing 5 µmol/L fura 2-AM, a Ca2+ indicator, for 45 minutes at 37°C. The fluorescent measurements were made at 37°C with a spectrophotometer (CAF-110, Jasco) as described previously.15 The [Ca2+]i was then calculated as described by Grynkiewicz et al.19

MAPKK and MAPK Assays
Quiescent VSMCs were washed twice with PBS and then stimulated with U46619 for the indicated times. After washing once with ice-cold PBS, the cells were lysed in ice-cold lysis buffer that consisted of (mmol/L): 20 Tris-HCl (pH 7.5), 50 ß-glycerophosphate, 1 PMSF, 2 EGTA, 2 DTT, 1 Na3VO4, and 20 mg/mL aprotinin. The cells were then sonicated for 5 seconds and centrifuged at 14 000g for 10 minutes. The supernatant was used as the source of MAPK and MAPKK. Aliquots containing an amount of protein (2 mg) were used for immunoprecipitation as described by Tobe et al.20

The kinase assay was performed using 32P phosphorylation of MBP as a measurement of MAPK activity. The reaction mixture (containing, in mmol/L, 20 Tris-HCl, pH 7.5; 10 MgCl2; 1 MnCl2; and 40 ATP, as well as 1 µCi [{gamma}-32P]ATP and 1 mg/mL MBP) was incubated with 20 mL of protein sample for 30 minutes at 25°C. The reactions were terminated by spotting 12 µL of the reaction mixture onto Whatman p81 paper. The paper was washed five times with phosphoric acid (0.5%) and dried. The amount of 32P incorporated into MBP was determined in a scintillation counter.21 In some experiments, MAPK activity "in gel" was measured by the phosphorylation of MBP. The phosphorylated MBP was separated by 10% SDS-PAGE and the extent of MBP phosphorylation determined by autoradiography.

MAPKK activity was assayed by using a recombinant rat MAPK fused to glutathione-S-transferase as a substrate. Cell lysates were immunoprecipitated with 5 µg of the mouse monoclonal antibody against the N-terminal 16–amino acid peptide (PKKKPTPIQLNPNPEG) of MAPKK and protein G–Sepharose. After washing, the immunoprecipitates were incubated with kinase buffer containing 100 µg of recombinant MAPK and 5 µCi [{gamma}-32P]ATP at 25°C for 5 minutes. The reaction was then stopped and the mixture was assayed for MAPKK activity on p81 paper or autoradiographically after separation in 10% SDS-PAGE as described above for the measurement of MAPK activity.

Immunoblot Analysis of Tyrosine Phosphorylation of MAPK
As above, quiescent cells were incubated with U46619 for the indicated times. Protein from the cell lysates was immunoprecipitated with anti-MAPK antibody at 4°C for 2 hours. All immune complexes were incubated with protein G–Sepharose at 4°C for 1 hour, and the immune complex bound to protein G–Sepharose was precipitated by centrifugation. After the immunoprecipitates were washed with 1 mL of the immunoprecipitation buffer, they were treated with 20 µL of Laemmli's sample buffer. For immunoblot analysis, the immunoprecipitates were electrophoresed in 7.5% SDS-PAGE, transferred onto nitrocellulose membrane, and then incubated with Py20 at 25°C for 4 hours. After washing, the membranes were incubated with goat anti-mouse IgG conjugated to alkaline phosphatase. The blots were developed by adding the alkaline phosphatase substrate BCIP/NBT in 0.1 mol/L Tris (pH 8.9) for 30 minutes.

Immunoblot Analysis of PKC Isozymes
Confluent VSMCs were washed with PBS and then incubated in DMEM with or without PMA for the indicated times. After the incubation, the cells were washed with ice-cold PBS, scraped, and collected by centrifugation at 1000g for 10 minutes. The collected cells were lysed in ice-cold homogenization buffer, which contained (mmol/L): 20 Tris-HCl (pH 7.5), 1 EDTA, 1 EGTA, 2 DTT, 20 leupeptin, 0.1 PMSF, and 10 benzamidine. The homogenates were centrifuged at 45 000g for 1 hour at 4°C to yield the supernatant (cytosolic extracts) and pellets (membrane fractions). Samples from these two fractions (100 mg of protein) were electrophoresed in 10% SDS-PAGE and the separated proteins transferred onto nitrocellulose paper. The nitrocellulose membrane was incubated with specific PKC isozyme monoclonal antibodies (against PKC-a, PKC-b, PKC-t, PKC-e, PKC-q, PKC-m, PKC-d, and PKC-z, respectively) in PBST buffer for 4 hours, followed by goat anti-mouse IgG conjugated to alkaline phosphatase for 1 hour. Then the blots were developed as described above.

Analysis of Ras-Bound GTP and GDP
VSMCs were assayed for levels of Ras-GTP complexes after U46619 stimulation as described.22 23 Briefly, cells were labeled with 0.2 mCi/mL [32P]phosphate in phosphate-free DMEM for 4 hours. After addition of agonists for 2 minutes, cell lysates were subjected to immunoprecipitation with Y13-259 antibody with the aid of protein G–Sepharose. The immune complex was washed extensively and the guanine nucleotides bound to Ras were eluted and analyzed by thin-layer chromatography on a polyethyleneimine-cellulose plate. The results were expressed as GTP/(GTP+GDP)x100%, reflecting the amount of GTP-bound Ras before and after exposure to agonists.

p74raf-1 Activity Assay
As above, quiescent VSMCs were incubated with U46619 or PDGF for the indicated times. Protein from the cell lysates was immunoprecipitated with anti-Raf-1 antibody. The MAPKK (recombinant, baculovirus-expressed MAPKK) activity, activated by Raf-1, in the immune complex was measured in 10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, 150 mmol/L NaCl, 2 mmol/L DTT, 0.8 µg recombinant MAPKK, 0.2 µCi [{gamma}-32P]ATP, and 1 mmol/L ATP at 37°C for 30 minutes. The reaction mixture was subjected to 10% SDS-PAGE and autoradiography. The phosphorylation bands were visualized and quantitated by using a computing densitometer with ImageQuant software (Molecular Dynamics).

Cell-Cycle Analysis
To estimate the proportions of cells at various stages in different phases of the cell cycle, cellular DNA content was measured by flow cytometry as described by March et al.25 Briefly, cells (2x106/mL) were fixed with 70% ethanol (in PBS) in ice for 30 minutes and then resuspended in PBS containing 40 µg/mL propidium iodide and 0.1 mg/mL RNase. After 30 minutes at 37°C, cells were analyzed with a FACstar cytofluorometer (Becton Dickinson) with excitation at 488 nm and emission at 585 nm.

Cell Counts
To determine the effect of U46619 on cell growth, quiescent cells were cultured for 1 or 2 days in medium supplemented with various agents. The culture medium was changed daily and cell numbers were determined by dissociation of adherent cells with trypsin and counting with a hemocytometer.15

Data Analysis
Saturation and displacement binding data were analyzed by the weighted least-squares iterative curve-fitting program LIGAND.26 The data were fitted to a one- and then a two-site model; if the residual sums of squares were statistically less for a two-site fit of the data than for a one-site as determined by F-test comparison, then the two-site model was accepted.

The experimental results are expressed as the mean±SEM and accompanied by the number of observations. A one-way ANOVA was used for multiple group comparisons. If there was a significant variation between treatment groups, then the mean values for inhibitors were compared with those for the control group with the Student's t test, and values of P<.05 were considered to be statistically significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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[3H]U46619 Binding in VSMCs
[3H]U46619 at concentrations ranging from 1 to 3000 nmol/L was used to label Tx receptors on cultured VSMCs. The specific binding of [3H]U46619 was 62.7±3.1% of the total binding at 3000 nmol/L [3H]U46619 in VSMCs from SHR. Scatchard plots of the binding data were curvilinear, suggesting more than a single class of binding site. LIGAND analysis fitted the data to a two-site model. The high-affinity site showed a Bmax of 15.0±0.2 fmol/mg protein, with a Kd value of 2.3±0.6 nmol/L, while the low-affinity site showed a Bmax of 176.0±13.3 fmol/mg protein and a Kd value of 1.4±0.5 µmol/L (Fig 1ADown and Table 1Down). In contrast, [3H]U46619 showed a saturable tendency at concentrations of 30 to 100 nmol/L in WKY. The specific binding of 100 nmol/L [3H]U46619 was 58.5±4.5% of the total binding. Scatchard plots of the binding data were linear, resulting in a better fit to a one-site model by computerized analysis, with a Kd value of 15.5±2.6 nmol/L and a Bmax of 11.0±0.4 fmol/mg protein (Fig 1BDown and Table 1Down). The binding properties of [3H]U46619 were not changed between passage 3 and passage 17 (eg, Kd value 13.7±2.1 versus 14.9±2.3 nmol/L; Bmax 12.1±0.6 versus 11.6±0.5 fmol/mg protein in WKY, n=4). Thus, VSMCs from passages 3 through 17 were used for further experiments. SQ29548 displaced specific [3H]U46619 binding to VSMCs in a concentration-dependent manner with Ki values of 9.6±1.9 and 7.6±1.6 nmol/L (n=4) at the high-affinity sites of SHR and WKY, respectively, and 3.2±0.6 µmol/L at the low-affinity site of SHR (Table 2Down).



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  		Figure 1. Saturation experimental data and Scatchard plots (insets) of [3H]U46619 binding to VSMCs from SHR (A) and WKY (B). Nonspecific binding was defined in the presence of 200 µmol/L unlabeled U46619 and was subtracted from the total binding to afford specific binding. The data shown were those from a single experiment of six experiments determined in duplicate samples.


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  		Table 1. Characteristics of Thromboxane Receptors in Cultured VSMCs from SHR and WKY


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  		Table 2. Potencies of SQ29548 Against Responses to U46619 in VSMCs from SHR and WKY

Effects of U46619 on DNA Synthesis
As shown in Fig 2Down, U46619 produced a concentration-dependent increase of [3H]thymidine incorporation into serum-free quiescent VSMCs from SHR and WKY. The concentrations of U46619 required to evoke half-maximal (EC50) DNA synthesis were 1.6±0.3 and 3.0±0.3 µmol/L in SHR and WKY, respectively. These concentrations were close to the Kd value for [3H]U46619 binding to the low-affinity site in SHR. However, the maximal efficacy of U46619-induced [3H]thymidine incorporation into DNA in VSMCs from WKY was only about 30% of that obtained from SHR. In contrast, PGE2 (1 to 10 µmol/L) and PGF2{alpha} (1 to 10 µmol/L) did not increase the [3H]thymidine incorporation into the DNA of VSMCs from WKY (-5±4% and 8±6% increase over basal level for 10 µmol/L PGE2 and PGF2{alpha}, respectively; n=4 for quadruplicate samples) and SHR (-9±6% and 7±4% increase over basal level for 10 µmol/L PGE2 and PGF2{alpha}, respectively; n=4 for quadruplicate samples). We have examined the synergistic stimulation of [3H]thymidine incorporation between U46619 and other mitogens in VSMCs from SHR. We found that low concentrations of FCS (1%), PDGF (0.2 ng/mL), or ADP (1 µmol/L), which displayed little mitogenic activity alone (11±6%, 10±3%, and 20±7% increase over the basal level, respectively; n=5), markedly potentiated U46619-stimulated DNA synthesis (from 15±5% to 60±5%, 54±5%, and 87±9% increase over basal level, respectively; n=5). SQ29548 (10 µmol/L) caused rightward shifts of the concentration-response curves for U46619 in VSMCs from SHR and WKY, with pKB values of 6.0±0.3 and 5.7±0.7, respectively (Fig 3Down and Table 2Up).



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  		Figure 2. Increase in [3H]thymidine incorporation into VSMCs from SHR ({circ}) or WKY ({bullet}) stimulated with U46619. [3H]Thymidine was added 20 hours after the quiescent cells had been exposed to U46619 and then incubations were continued for another 4 hours. Basal [3H]thymidine incorporation was 278±35 and 325±48 dpm in VSMCs from SHR and WKY, respectively. Data are the mean±SEM (n=5, each assayed in quadruplicate).



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  		Figure 3. Antagonism of U46619-induced [3H]thymidine incorporation by SQ29548 in VSMCs from SHR (A) and WKY (B). Quiescent cells were stimulated with U46619 in the presence of DMSO (0.1% for solvent control, {circ}) or SQ29548 (10 µmol/L, {bullet}) for 24 hours. [3H]Thymidine was added 20 hours after the quiescent cells had been exposed to U46619, and then incubations were continued for another 4 hours. Basal [3H]thymidine incorporation was 310±19 and 295±20 dpm in VSMCs from SHR and WKY, respectively. SQ29548 alone had no effect on basal incorporation. Data are the mean±SEM (n=5, each assayed in quadruplicate).

Effect of U46619 on the Formation of IP and [Ca2+]i
The hydrolysis of phosphoinositides is a major signal-transduction pathway in the control of cell proliferation.25 U46619 increased [3H]IP formation in cultured VSMCs from SHR and WKY in a concentration-dependent manner (Fig 4Down for SHR), with EC50 values of 2.5±0.9 and 3.7±1.3 µmol/L, respectively (Table 2Up). U46619 also caused a rapid rise in [Ca2+]i in cultured VSMCs from SHR and WKY, with EC50 values of 1.5±0.6 and 3.0±1.2 µmol/L, respectively (Fig 5Down for SHR and Table 2Up). Again, SQ29548 inhibited U46619-induced [3H]IP formation and increase in [Ca2+]i in VSMCs from SHR, with pKB values of 6.2±0.4 and 6.4±0.3, respectively (Figs 4Down and 5Down; Table 2Up).



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  		Figure 4. Inhibition of U46619-stimulated [3H]inositol monophosphate formation by SQ29548 in VSMCs from SHR. [3H]myo-Inositol–labeled cells were incubated with DMSO (0.1% for solvent control, {circ}) or SQ29548 (10 µmol/L, {bullet}) at 37°C for 15 minutes, and then various concentrations of U46619 were added for another 60 minutes. Data are the mean±SEM (n=4, each assayed in triplicate).



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  		Figure 5. Effects of SQ29548 on the intracellular Ca2+ concentration in VSMCs from SHR. Fura 2–loaded quiescent cells incubated with DMSO (0.1% for solvent control) or SQ29548 for 15 minutes were stimulated with U46619.

Activation of MAPK and MAPKK by U46619
MAPK, another group of components in the signal-transduction pathway, has been shown to be activated during DNA synthesis and cell-cycle progression.20 21 Therefore, whether U46619 activated MAPK was checked. U46619 at 10 µmol/L rapidly activated MAPK and its upstream MAPKK in VSMCs from SHR and WKY. MAPK and MAPKK activities were measurable 1 minute after stimulation, peaked at 2 minutes, and decreased gradually thereafter (data not shown). U46619 activated MAPK in a concentration-dependent manner with EC50 values of 1.9±0.7 and 2.5±0.9 µmol/L in VSMCs from SHR and WKY, respectively (Fig 6Down and Table 2Up).



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  		Figure 6. Concentration-response curves of U46619-stimulated MAPK activity in VSMCs of SHR ({circ}) and WKY ({bullet}). Quiescent cells were stimulated with various concentrations of U46619 for 2 minutes and MAPK activity in the cell lysate was determined. Basal MAPK activities were 450±50 and 538±47 pmol/min per milligram protein, respectively, in VSMCs from SHR and WKY. Data are the mean±SEM (n=5 to 6).

Stimulation of MAPK Tyrosine Phosphorylation by U46619
Two isoforms of MAPK have been identified: one with a molecular weight of 42 kD and the other 44 kD. The applied anti-MAPK antibodies are able to recognize both 42- and 44-kD MAPK isoforms. The activity of the p42 mapk and p44 mapk can also be monitored by the electrophoretic mobility of the phosphorylated MAPK, which possesses a higher apparent molecular weight compared with the inactivated form, resulting in a decrease in the electrophoretic mobility. Fig 7ADown shows the time course for the shift in mobility of the 42- and 44-kD MAPK bands after stimulation with U46619. When cells were stimulated with U46619, the mobility of p42 mapk and p44 mapk were maximally shifted within 2 to 5 minutes. Also, the activation of MAPK requires phosphorylation at both tyrosine and threonine residues.21 Therefore, we next asked whether the U46619-induced activation of MAPK was accompanied by ty-rosine phosphorylation. Cell lysates were immunoprecipitated with anti-MAPK (ERK 1 or ERK 2) antibodies and followed by Western blotting with Py20. As shown in Fig 7BDown, tyrosine phosphorylation in both 42- and 44-kD MAPK peaked 2 minutes after stimulation with U46619 and then decreased gradually thereafter, consistent with the above result. Since U46619 has been shown to stimulate the hydrolysis of phosphoinositide, resulting in intracellular Ca2+ mobilization and PKC activation, the role of PKC in U46619-induced MAPK activation was examined. To determine which PKC isoform is involved in the U46619-induced MAPK activation, the expression of PKC isoforms in VSMCs was characterized by Western blot analysis. Immunoblot analysis revealed that PKC-{alpha}, -{delta}, and -{zeta} were present in VSMCs of SHR. PKC-{alpha} and -{delta} were in the cytosol, while PKC-{zeta} was in both cytosol and membrane fractions of VSMCs (Fig 8Down). After treatment of VSMCs with PMA for 1 hour, PKC-{alpha} (94%) and -{delta} (95%) were translocated from cytosol to the membrane. However, after a 24-hour treatment with PMA, a dramatic decline in the total amount of PKC-{alpha} and -{delta} immunoreactivity in the membrane and cytosol was seen. In contrast, the expression of PKC-{zeta} in both cytosol and membrane was not altered by PMA treatment (Fig 8Down). When cells were pretreated with PMA for 24 hours and subsequently challenged with U46619, 42- and 44-kD MAPK tyrosine phosphorylation failed to be induced (Fig 7CDown). Furthermore, the activation of MAPK by U46619 was almost completely inhibited by SQ29548, staurosporine, or pretreatment of cells with PMA for 24 hours (Fig 9Down).



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  		Figure 7. Kinetics of the activation of MAPK (A) or of tyrosine phosphorylation of 42- and 44-kD MAPK (B and C) in VSMCs stimulated by U46619. Quiescent VSMCs, untreated (A and B) or pretreated with PMA (500 nmol/L, C) for 24 hours, were stimulated with U46619 (10 µmol/L) for the indicated time periods. Arrows denote the two isoforms of MAPK in VSMCs, 42- and 44-kD.



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  		Figure 8. Translocation and downregulation of PKC isoforms in VSMCs in response to PMA. Cells were treated with 0.1% DMSO or 500 nmol/L PMA for 1 or 24 hours, and then cytosolic and membrane fractions were prepared. The proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose paper, and immunoblotted with PKC-{alpha}, -ß, -{tau}, -{epsilon}, -{theta}, -µ, -{delta}, and -{zeta}–specific antibodies.



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  		Figure 9. Effects of SQ29548, staurosporine, and PMA on U46619-induced MAPK activity in VSMCs from SHR. Quiescent cells were treated with DMSO (0.1%), SQ29548 (10 µmol/L, ), or staurosporine (30 nmol/L, ) for 15 minutes or pretreated with PMA (500 nmol/L) for 24 hours (), then DMSO (0.1%, {square}) or U46619 (10 µmol/L, {blacksquare}) was added for another 2 minutes. MAPK activity in the cell lysate was determined. Basal MAPK (resting) activity was 450±50 pmol/min per milligram protein. Data are the mean±SEM (n=5). *P<.001 compared with the U46619-treated cells.

Effects of U46619 on Ras-GTP Accumulation and p74raf-1 Activation
In quiescent VSMCs from SHR, endogenous p21ras was almost entirely (90%) in the inactive form. However, the ratio of GTP/GTP+GDP bound to p21ras was increased to 93.2±3.9% by PDGF-BB (10 ng/mL), while U46619 (10 µmol/L) did not have any effect (data not shown). Raf-1 activity of VSMCs was also rapidly increased by PDGF-BB (10 ng/mL) in a time-dependent manner. The Raf-1 activity was measurable 1 minute after stimulation, peaked at 2 minutes (1.7±0.1-fold increase over the basal level), and decreased gradually thereafter. U46619 (10 µmol/L) again did not increase the p74raf-1 activity in VSMCs from SHR (data not shown).

Effects of U46619 on Cell-Cycle Progression
The mechanism of the enhanced cell-cycle progression by U46619 was examined using flow cytometry. As shown in Fig 10ADown, in quiescent VSMCs from SHR, 93.2% of the cells were in the growth-arrested (G0/G1) phase of the cell cycle, whereas after 20 or 24 hours' stimulation with U46619, 50.4% and 33.3%, respectively, of the VSMCs entered the S phase of the cell cycle. The G2/M phase began at 20 hours and reached its maximum at 24 hours (Fig 10ADown). These results clearly indicated that U46619 promoted cell-cycle progression. To further clarify whether U46619 directly caused the transition from the S to the G2/M phase, we examined alterations in the cell-cycle progression, using VSMCs synchronized in the G1/S boundary. The cycling process was stopped at this boundary with hydroxyurea (10 µmol/L for 24 hours) and started again by washing out this reagent with fresh medium containing U46619. DNA content of the G2/M phase was increased after the washout and reached a maximum 2 hours after the addition of U46619 (Fig 10BDown). In contrast, FCS (10%) promoted the transition of S into the G2/M phase with a rate slower than U46619. FCS did not significantly increase the DNA content of the G2/M phase at 2 hours but reached a maximum at 4 hours after addition to the culture medium (Fig 10BDown).



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  		Figure 10. Effect of U46619 on cell-cycle progression in VSMCs from SHR. Quiescent cells or cells synchronized in G1/S boundary were stimulated with U46619 (10 mmol/L) or FCS (10%) for the indicated times. 0 hour indicates the DNA content for the untreated resting cells.

Effects of U46619 on Cell Growth and Protein Content
The 48-hour quiescent VSMCs from SHR were grown in serum-free medium in the absence or presence of 10% FCS or U46619 without or with various inhibitors, and the number of cells was counted. As shown in Table 3Down, the number of viable cells increased 2.4±0.2- and 3.2±0.3-fold after 24 and 48 hours of reexposure of quiescent cells to 10% FCS. The numbers of viable cells were also increased by 10 and 30 µmol/L U46619 to 1.4±0.1- and 1.9±0.2-fold, respectively, after a 24-hour reexposure, and 1.7±0.1- and 2.2±0.2-fold, respectively, after a 48-hour reexposure (Table 3Down). Incubation of serum-starved VSMCs with U46619 or 10% FCS for 24 hours also significantly increased the protein content of VSMCs (Table 3Down). U46619 at concentrations of 10 and 30 µmol/L increased the amount of protein by 158±19% and 190±10%, respectively. The effect of U46619 was again markedly inhibited by SQ29548 or staurosporine (n=5).


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  		Table 3. Stimulation of Cell Growth by Reexposure to U46619 or FCS


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, U46619 stimulated [3H]thymidine incorporation into DNA and growth of rat VSMCs, consistent with previous reports.5 12 27 However, others have found that TxA2 caused only protein synthesis and hypertrophy but not mitogenesis of VSMCs.28 29 30 An explanation for the discrepancy may lie in one or more of the following methodological differences: the methodology employed to measure the DNA synthesis (4-hour pulse-labeling versus 24-hour labeling); the age of the animals chosen for the source of VSMCs (young versus adult); or the different species (SHR versus WKY) or tissues (aorta versus coronary VSMCs) used. Another reason for the discrepancy is that the low-affinity TxA2 binding sites may be differentially expressed in VSMCs from various species and tissues.

The presence of receptor subtypes for TxA2 is controversial. It has been reported by Nusing et al31 that only one Tx receptor gene may exist in human placenta, megakaryocytic cells, and mesangial cells. In a more recent study, Abe et al32 also have cloned only one rat Tx receptor, which is expressed specifically in renal glomerulus, arterial VSMCs, and transitional cell epithelium of renal pelvis. In contrast, many pharmacological and binding studies indicate that there are two Tx receptors existing in VSMCs and platelets. Furthermore, Yamamoto et al33 reported that Tx receptor stimulation results in at least two signaling pathways in rabbit aortic VSMCs: one is the activation of PI hydrolysis via a PTX-insensitive G protein, and the other is the inhibition of PI hydrolysis via a PTX-sensitive G protein. These results imply that there may be two Tx receptor subtypes existing in rabbit aortic VSMCs, although the possibility that a single receptor couples to two G proteins cannot be ruled out. Indeed, Borg et al14 have purified two proteins (52 and 55 kD) from rat brain and rabbit aorta by immunoaffinity chromatography employing anti-peptide and anti-receptor antibodies. The 55-kD protein is clearly a Tx receptor that specifically binds [3H]SQ29548. Since the 52-kD protein does not appear to be a digestion product of the 55-kD receptor protein, it may represent a Tx receptor subtype present in rat brain and rabbit aorta.

In the present study, [3H]U46619 bound to cultured VSMCs from SHR at two classes of sites, whereas only the high-affinity site was detected in VSMCs from WKY. The EC50 values of U46619 for the stimulation of [3H]thymidine incorporation into DNA of VSMCs were closely comparable to the Kd value of the low-affinity binding site for [3H]U46619 in cultured VSMCs from SHR. Moreover, SQ29548, a TxA2 receptor antagonist, antagonized U46619-induced [3H]thymidine incorporation of VSMCs from SHR and WKY, with pKB values similar to its Ki value for inhibiting [3H]U46619 binding to the low-affinity site in SHR (Table 2Up). Thus, the growth-promoting effect of U46619 may be mediated by the low-affinity binding site in VSMCs. However, no low-affinity [3H]U46619 binding site was detected in VSMCs from WKY, although U46619 induced [3H]thymidine incorporation into DNA in VSMCs from WKY. This may be due to the small number of low-affinity U46619 binding sites existing in VSMCs from WKY, which could be undetectable due to the limitations of Scatchard or LIGAND analysis. This notion is partially supported by the fact that the maximal efficacy of U46619-induced [3H]thymidine incorporation into DNA in VSMCs from WKY was only about 30% of that obtained from SHR.

TxA2, PGF2{alpha}, and PGE2 are powerful vasoconstrictors in a variety of species and vascular beds. It has been reported that U46619 cross-reacted with PGF2{alpha} and PGE2 receptors at concentrations in the micromolar range.29 34 It is unlikely that U46619-stimulated proliferation of VSMCs is due to its action on PGF2{alpha} and PGE2 receptors, since PGE2 and PGF2{alpha} at concentrations of 1 to 10 µmol/L did not stimulate DNA synthesis in VSMCs. Furthermore, Dorn et al29 have reported that PGE2 and PGF2{alpha} induced protein synthesis but not [3H]thymidine incorporation into DNA in VSMCs. The protein synthesis induced by PGE2 and PGF2{alpha} was not affected by SQ29548, suggesting that the growth-promoting effects of the prostaglandins are due to agonism at a prostaglandin receptor. Moreover, in this study and that of Dorn et al, U46619-stimulated proliferation of VSMCs was inhibited by SQ29548. Thus, the growth-promoting effects of PGE2, PGF2{alpha}, and TxA2 are apparently mediated by interactions with different receptors. Indeed, many previous works indicate that PGE1, PGE2, and PGF2{alpha} inhibit proliferation of aortic smooth muscle cells from human, guinea-pig, rabbit, and rat.35 36 37 38

VSMCs alter their phenotypes in response to altered functional demands. Two types of VSMCs have been proposed: one is a "contractile type" and the other is a "synthetic type."39 The synthetic type of VSMC is stimulated to proliferate by growth factors, whereas the contractile type is not. The migration of VSMCs from media to intima after endothelial denudation may cause the cells to change functionally from the contractile to the synthetic type and cause abnormal proliferation of VSMCs. It would appear, therefore, that in most cases phenotypic modulation of VSMCs from the contractile toward the synthetic state is a prerequisite for proliferation. Thus, it is tempting to speculate that, through binding to its receptors, TxA2 mainly provokes contractile effects on VSMCs from WKY but induces contraction and proliferation in VSMCs from SHR. Previous results indicate that TxA2 generation is higher in SHR than in age-matched WKY.10 11 Our results also indicate that the low-affinity Tx receptor is expressed at much higher levels in VSMCs from SHR than from WKY. In addition, TxA2 can synergize with other factors such as PDGF, epidermal growth factor, and ADP in stimulating VSMC proliferation. Thus, TxA2 is at least partly responsible for the rapid proliferation of VSMCs of SHR and thereby would contribute to the media hypertrophy observed in hypertension.

The activation of the Tx receptor is linked to the stimulation of PI hydrolysis, which produces two second messengers, IP3 and DAG, in platelets and VSMCs.40 41 IP3 is known to release Ca2+ from intracellular stores, and DAG activates PKC. The activation of PKC and increase in [Ca2+]i appear to account for most of the early proliferative events.42 We showed that U46619 caused increases in IP formation and [Ca2+]i. The EC50 values for both U46619-induced IP formation and increase in [Ca2+]i are closely comparable to the Kd value for [3H]U46619 binding to the low-affinity site in VSMCs from SHR and to the EC50 values for U46619-induced [3H]thymidine incorporation of VSMCs from SHR and WKY. Moreover, SQ29548 inhibited U46619-induced [3H]thymidine incorporation into DNA, IP formation, and increase in [Ca2+]i, with identical pKB values, which were also close to its Ki value for inhibiting [3H]U46619 binding to the low-affinity site. Thus, the PI hydrolysis is presumably stimulated by U46619 via a low-affinity Tx receptor and may be essential for the mitogenic effect in VSMCs.

U46619 facilitates the transition of the cells from G0/G1 phase to the S phase of the cell cycle, as shown in the result of flow cytometry. Recently, a novel group of serine/threonine kinases, MAPKs (also known as ERKs), have been shown to be activated during the transition of entering the S phase from the G0/G1 phase of the cell cycle.43 44 Consistent with the previous report,45 U46619 was shown to trigger the activation of 42- and 44-kD MAPK in a time- and concentration-dependent manner, with EC50 values close to the Kd value of the low-affinity binding site. This EC50 value was also close to the EC50 for U46619-induced [3H]thymidine incorporation into DNA of VSMCs. Furthermore, U46619 also activated MAPKK, a direct upstream activator of MAPK.46 The Tx receptor is associated with a G protein, and on the basis of its predicted sequence, it lacks an intracellular tyrosine kinase domain.40 41 Thus, the stimulation of the MAPK activity by U46619 is probably indirect. Vasopressin, angiotensin II, thrombin, and phorbol ester stimulate PKC in VSMCs42 that also induce tyrosine phosphorylation and MAPK activity in these cells. Thus, we postulate that the activation of MAPK is downstream of PKC in the signal-transduction-pathway response to U46619 in VSMCs. This is supported by the observation that the activation of MAPK by U46619 was sensitive to staurosporine, a PKC inhibitor. Furthermore, downregulation of PKC-{alpha} and -{delta} by pretreatment of VSMCs with PMA abolished the activation of MAPK induced by U46619.

Another pathway for activating MAPKK involves the Ras-Raf activation initiated by the activation of tyrosine kinase receptors. However, activation of MAPKK by G protein–coupled receptors via p21ras and p74raf-1 has also been reported.47 48 49 To analyze further the pathway that links U46619 to MAPKK, we explored the role of Ras and Raf in U46619-induced MAPKK activation. No significant Ras or Raf activation induced by U46619 could be observed. This result indicates that U46619 does not activate the Ras-MAPKK pathway in VSMCs.

In conclusion, there are two classes of Tx binding sites in VSMCs. The high-affinity site is responsible for Tx-induced vasoconstriction, while the low-affinity site is responsible for Tx-mediated VSMC proliferation. TxA2 may be responsible for stimulating proliferation of VSMCs as a competence factor, since it stimulates cell-cycle progression and increases cell number of VSMCs. The mechanism of VSMC proliferation induced by U46619 may be as follows. U46619 binds to the low-affinity Tx receptor–Gq complex and stimulates the hydrolysis of PI, producing IP3 and DAG, leading to the rise of [Ca2+]i and activation of PKC, respectively. The activation of PKC in turn activates MAPK, leading to DNA synthesis that results in growth of the cells. Future studies will be directed toward analyzing whether other cytoplasmic kinases are activated through the stimulation of the low-affinity Tx receptor.


*    Selected Abbreviations and Acronyms
 
DAG = diacylglycerol
DMEM = Dulbecco's modified Eagle's medium
ERK = extracellular signal-regulated kinases
FCS = fetal calf serum
IP = inositol monophosphate
MAPK = mitogen-activated PK
MAPKK = MAPK kinase
MBP = myelin basic protein
PAGE = polyacrylamide gel electrophoresis
PDGF = platelet-derived growth factor
PG = prostaglandin
PK = protein kinase
PMA = phorbol 12-myristate 13-acetate
PTX = pertussis toxin
Py20 = monoclonal anti-phosphotyrosine antibody
SHR = spontaneously hypertensive rats
Tx = thromboxane
VSMC = vascular smooth muscle cell
WKY = Wistar-Kyoto rats


*    Acknowledgments
 
This work was supported by a research grant from the National Science Council of the Republic of China (NSC83-0420-B002-067).

Received March 28, 1996; accepted September 2, 1996.


*    References
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 up arrowIntroduction
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 *References
 

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