Regulatory of vascular
effect of thromboxane A2 on proliferation smooth muscle cells from rats
TAIJI NAGATA, YOSHIO UEHARA, ATSUSHI NUMABE, TOSHIHIKO ISHIMITSU, NOBUHITO HIRAWA, TOSHIO IKEDA, HIROAKI MATSUOKA, AND TSUNEAKI
SUGIMOTO
The Second Department of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113; Division of Cardiorenal Disease and Hypertension, Department of Medicine, Dokkyou University School of Medicine, Mibu, Tochigi 321-02; The Second Department of Medicine, Yokohama City University, Yokohama, Kanagawa 232; and Department of Nephrology, Kantou-Teishin Hospital, Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan Yoshio Uehara, Atsushi Numabe, Nagata, Taiji, Toshihiko Ishimitsu, Nobuhito Hirawa, Toshio Ikeda, Hiroaki Matsuoka, and Tsuneaki Sugimoto. Regulatory effect of thromboxane A, on proliferation of vascular smooth muscle cells from rats. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): Hl331-H1338, 1992.-We investigated the regulatory effects of the vasoconstrictor thromboxane AZ on the proliferation of vascular smooth muscle cells (VSMC) from WistarKyoto rats using 9,11-epithio-11,12-methano-thromboxane A, (STA,), a stable analogue of thromboxane AZ. STAZ dose dependently increased incorporation of [“HI thymidine into DNA in randomly cycling VSMC and significantly shortened the doubling time. Cell cycle analysis revealed that the increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G,/mitotic (M) phase. Moreover, STA2 enhanced protein synthesis in VSMC during the G,/M phase, whereas the protein synthesis was unaffected in the G,/G, period. In fact, STA, prompted the cells in G,/M phase to synthesize actin, a major cytoskeleton protein. Conversely, inhibition of protein synthesis by puromycin retarded the transition from S to GJM. In addition, depolymerization of the actin molecules by cytochalasin D offset the quick progression to the G,/M phase by STA,. These data indicate that thromboxane A, stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G,/M. This enhanced progression is attributable partly to a rapid buildup of the cytoskeleton proteins during the GJM period.
thromboxane AZ. Particularly in some genetic rat models for human hypertension, thromboxane A2 generation in the vascular wall is enhanced and thereby conceivably contributes in part to the medial hypertrophy observed in hypertension (14, 40). Moreover, recent studies in vitro indicate that exogenous prostaglandins, e.g., prostaglandin (PG) E2, prostacyclin (PG12), and PGD2, retard the proliferation of VSMC, whereas vasoconstrictor thromboxane A2 enhances the VSMC proliferation (13, 15, 37). On the basis of these studies, it seems possible that thromboxane A2 released from the vascular wall as well as aggregated platelets plays some role in the remodeling of the vascular structure in a hypertensive state or in various forms of arterial injuries. Although this hypothesis is intriguing, the possible roles of thromboxane AZ in the proliferation of VSMC have not been fully addressed. We therefore have investigated the mechanism by which the vasoconstrictor thromboxane AZ influences the proliferation of smooth muscle cells derived from rat thoracic aorta, using 9,11-epithio-11,12-methano-thromboxane AZ (STA2), a stable analogue of thromboxane AZ, and cell culture techniques.
cell growth; thymidine uptake; cell cycle; G, resting mitotic period; cytoskeleton protein; actin
MATERIALS
period;
Cell Culture
have emphasized the role of the growth and migration of medial smooth muscle cells in the remodeling of the vascular structure in a hypertensive state (6, 22) or in the remodeling processes involved in arterial restenosis after angioplasty (12, 30). It is therefore of great interest to investigate the regulatory mechanism of the proliferation of vascular smooth muscle cells (VSMC). A number of these studies have thus far been conducted, thereby providing evidence for the involvement of various vasoactive substances derived from vascular vessels, e.g., prostaglandins (37), serotonin (23), various growth factors like platelet-derived growth factor (PDGF) (31) and transforming growth factor (20), angiotensin II (5), endothelin (lo), and endothelium-derived relaxing factor(s) (7), in the modulation of VSMC growth. The vascular wall is not only the container for the blood supply but also functions as an autocrine or paracrine gland to secrete various vasoconstrictor and relaxing substances (8,19) and a number of growth factors (1, 25). In this context, it is well postulated that the vascular wall produces significant amounts of vasoconstrictor
RECENT
STUDIES
0363-6135/92
$2.00
Copyright
AND METHODS
VSMC were isolated from the thoracic aortas of 8-wk-old Wistar-Kyoto rats, according to the method of Ross and coworkers (31, 32). Briefly, thoracic descending aortas were obtained, and the adventitia and intima were carefully removed. The explants were then placed on a polystyrene dish with the intimal side attached to the dish, and Dulbecco’s modified Eagle’s medium (DMEM; GIBCO Laboratories, Grand Island, NY), containing 20% (vol/vol) fetal bovine serum (FBS; GIBCO Laboratories), penicillin (100 U/ml), and streptomycin (100 pg/ml), was gently added. The dishes were incubated at 37°C in a humidified atmosphere of 95% air-5% COa. The cells reached confluency 10 days after inoculation. They were harvested by brief exposure to Hanks’ balanced salt solution supplemented with 0.05% (wt/vol) trypsin and 0.02% (wt/vol) EDTA and transferred into a new dish. At this stage, the cells were designated as the first passage. The percentage of FBS was changed to 10% (vol/vol) in subsequent media. The harvest was repeated when the cells became confluent. VSMC from passages 3 to 6 were used in this study. The cells in culture exhibited the characteristic “hills and valleys” growth pattern, and the presence of cu-actin molecules was demonstrated by using a monoclonal anti-cY-actin antibody and immunoenzyme assay method (32, 37).
0 1992 the American
Physiological
Society
H1331
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
H1332
CELL
GROWTH
AND
Cell Synchrony Cell synchronization was performed according to the standard methods as described by Ashihara and Baserga (2). To synchronize VSMC in the G,/G, boundary (quiescent VSMC), randomly cycling cells were cultured in serum-free media for 48 h. Replacement of serum-free media by 10% fresh sera initiated cell proliferation. Quiescent VSMC were cultured for 24 h in media containing 10% sera and 1 mM hydroxyurea. Hydroxyurea stopped the proliferative process at the G,/S boundary. DNA replication was started again by washout of the hydroxyurea with 10% fresh sera. To synchronize the cells in the premitotic period, we utilized 15 rig/ml colchicine. G,/S-synchronized VSMC were cultured for 12 h in DMEM containing 10% sera and colchicine. The mitotic process could be resumed by washout of the colchicine with 10% fresh sera. Assessment
’
of
Cell Proliferation
Doubling time. To assess the proliferating activity, we measured the doubling time of VSMC and [:3H]thymidine uptake into DNA fragments. The doubling time was determined as follows: 5 x lo4 cells were seeded onto g-cm2 dishes with 2 ml DMEM containing 10% sera and cultured for 24 h. Then, the media were changed to culture media containing a given concentration of STA, (Ono Pharmaceuticals, Osaka, Japan), a stable thromboxane A, analogue (16). Cell number was determined by light microscopy before the addition of STA2 and after 24 h. The doubling time was calculated according to the following equation: doubling time = (tl - t2) x log 2/(log N, log N2), where t, and t2 are the times when cell numbers are counted and N, and N2 are the cell numbers at tl and t2, respectively. Measurement of r3H]thymidine uptake. The rate of DNA replication was assessed by measuring incorporation of [:3H]thymidine into DNA fragments (37). First, VSMC in a logarithmic growth state were cultured for 24 h in media containing 10% FBS, 0.25 &i [3H]thymidine (20-30 Ci/mmol), and a given concentration of STA,. After the culture period, the cells were harvested, washed with Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ and then homogenized by a model UR-20 ultrasonicator (Tomy-Seiko, Tokyo, Japan). The homogenate was treated with 5% perchloric acid at 4°C for 30 min and spun at 3,000 g for 30 min. The precipitate was repeatedly washed with 5% perchloric acid solution, and radioactivity in this acid-insoluble fraction was measured by an automatic liquid scintillation counter. Similarly, [ ZH] thymidine uptake by G,/G 1-synchronized VSMC or G,/S-synchronized VSMC was measured. In these experiments, the cells were cultured in fresh media containing [:3H]thymidine and lo-’ M STA, and harvested at various culture periods. Radioactivity in the acid-insoluble DNA fragments was determined by the same procedures as described in the method for randomly cycling VSMC. Mitotic index. The percent population of mitotic cells (mitotic index) was determined by light microscopy. Briefly, after the proliferating process was resumed in the VSMC synchronized in various phases, the cells were fixed with 5% acetate ethanol solution after culture for various time periods. The fixed cells were stained by Giemsa solution. The cells in the mitotic process were detected by light microscopy. Five hundred nuclei were examined in each sample. Cytoskeleton Protein Synthesis To examine STA, effect on cell body formation, mined incorporation of [“HI leucine into cellular VSMC synchronized in the G,/G, or G,/S boundary
we deterproteins. were cul-
THROMBOXANE
A2
tured for 12 h in 10% FBS containing a given concentration of STA, and 0.05 &i of [“Hlleucine (120-190 Ci/mmol). The cells were harvested and homogenized by an ultrasonicator in 25 mM phosphate-buffered saline containing 1 mM EDTA. The homogenate was treated with ice-chilled 5% (vol/vol) trichloroacetate. The precipitate was washed with the trichloroacetate solution and dissolved in 1 ml of 0.5 N NaOH solution. The radioactivity of this solution was measured with an automatic liquid scintillation counter. To reveal whether STA2 directly affected the synthesis of cytoskeleton proteins during the G,/M period, we then examined the alterations in the formation of actin, a major cytoskeleton protein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21). G,/S-synchronized cells were cultured for 12 h in media containing 0.05 &i [“Hlleucine and a given concentration of STA,. Afterward the cells were harvested and homogenized by an ultrasonicator. The homogenate was spun at lo4 g for 30 min, and the pellet was washed three times with 5 mM phosphate-buffered solution at pH 7.2 including 1 mM EDTA and 1 mM dithiothreitol. The combined supernatant was used for analysis of cytoskeleton proteins. Proteins in the supernatant were denatured at 95°C for 10 min in 50 mM tris(hydroxymethyl)aminomethane hydrochloride at pH 6.8 containing 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 1 mM dithiothreitol, and 0.0025% (wt/vol) bromphenol blue. Samples were applied to a 7.5% polyacrylamide slab gel, using a Protean II Slab Cell (Bio-Rad, Richmond, CA) and MultiDrive XL power supply unit (Pharmacia Biotechnology, Bromma, Sweden). Proteins were stained with 0.1% Coomassie Blue in fixative (water-methanol-acetate, 5:4: 1, vol/vol). After destaining was completed, the density of the separated proteins was quantitatively scanned, using a model EPA-3000 densitypattern analyzer (Maruzen Petrochemicals, Tokyo, Japan). The amount of actin was determined by reference to the relative mobility of an cu-actin standard. Moreover, alterations in actin formation were determined by measuring incorporation of [“HI leucine into actin molecules. For this analysis, the actin band was cut out of the gel and the radioactivity in it was eluted with a tissue solubilizer (NCS, Amersham International, Buckinghamshire, UK). The radioactivity was determined using an automatic liquid scintillation counter. To confirm the association of cytoskeleton protein formation with cell growth, we investigated the effects of protein synthesis inhibition and functional disturbance of actin on the mitotic process in the M phase. G,/S-synchronized VSMC were cultured in fresh media containing a given concentration of puromycin, an inhibitor of protein synthesis, or cytochalasin D, a depolymerizing agent of actin. The mitotic index was measured at various culture periods. Reagents Reagents were all of analytic grade. Eicosanoids and related compounds were offered by Ono Pharmaceuticals. Radioactive materials were purchased from Amersham International. Statistical
Analysis
The values are expressed as means * SE. Statistical differences were analyzed by the Student’s t test, the paired t test, or one- or two-way analysis of variance. RESULTS
STA2 Effect on Randomly Cycling VSMC
First, we investigated the effect of STA2, a stable thromboxane A2 analogue, on the cell proliferation of randomly cycling VSMC. STAz increased incorporation of [:‘H]thymidine into acid-insoluble DNA fragments in
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
CELL
GROWTH
AND
THROMBOXANE
H1333
A2
VSMC in a dose-dependent manner ranging from lOAs to lOA M STAz (Table 1). To confirm this property of STA2, we examined the alteration in the doubling time when the cells were maximally stimulated with 10es M STA2. As shown in Fig. 1, this dose of STA2 significantly shortened the doubling time of VSMC in a logarithmic growth state (19.24 t 0.32 vs. 20.25 t 0.40 h). These data clearly indicate that STA2, a stable analogue of thromboxane AZ, enhances the rate of cell cycle progression in cycling VSMC populations. Analysis
P
effect of thromboxane A2 on proliferation smooth muscle cells from rats
TAIJI NAGATA, YOSHIO UEHARA, ATSUSHI NUMABE, TOSHIHIKO ISHIMITSU, NOBUHITO HIRAWA, TOSHIO IKEDA, HIROAKI MATSUOKA, AND TSUNEAKI
SUGIMOTO
The Second Department of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113; Division of Cardiorenal Disease and Hypertension, Department of Medicine, Dokkyou University School of Medicine, Mibu, Tochigi 321-02; The Second Department of Medicine, Yokohama City University, Yokohama, Kanagawa 232; and Department of Nephrology, Kantou-Teishin Hospital, Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan Yoshio Uehara, Atsushi Numabe, Nagata, Taiji, Toshihiko Ishimitsu, Nobuhito Hirawa, Toshio Ikeda, Hiroaki Matsuoka, and Tsuneaki Sugimoto. Regulatory effect of thromboxane A, on proliferation of vascular smooth muscle cells from rats. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): Hl331-H1338, 1992.-We investigated the regulatory effects of the vasoconstrictor thromboxane AZ on the proliferation of vascular smooth muscle cells (VSMC) from WistarKyoto rats using 9,11-epithio-11,12-methano-thromboxane A, (STA,), a stable analogue of thromboxane AZ. STAZ dose dependently increased incorporation of [“HI thymidine into DNA in randomly cycling VSMC and significantly shortened the doubling time. Cell cycle analysis revealed that the increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G,/mitotic (M) phase. Moreover, STA2 enhanced protein synthesis in VSMC during the G,/M phase, whereas the protein synthesis was unaffected in the G,/G, period. In fact, STA, prompted the cells in G,/M phase to synthesize actin, a major cytoskeleton protein. Conversely, inhibition of protein synthesis by puromycin retarded the transition from S to GJM. In addition, depolymerization of the actin molecules by cytochalasin D offset the quick progression to the G,/M phase by STA,. These data indicate that thromboxane A, stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G,/M. This enhanced progression is attributable partly to a rapid buildup of the cytoskeleton proteins during the GJM period.
thromboxane AZ. Particularly in some genetic rat models for human hypertension, thromboxane A2 generation in the vascular wall is enhanced and thereby conceivably contributes in part to the medial hypertrophy observed in hypertension (14, 40). Moreover, recent studies in vitro indicate that exogenous prostaglandins, e.g., prostaglandin (PG) E2, prostacyclin (PG12), and PGD2, retard the proliferation of VSMC, whereas vasoconstrictor thromboxane A2 enhances the VSMC proliferation (13, 15, 37). On the basis of these studies, it seems possible that thromboxane A2 released from the vascular wall as well as aggregated platelets plays some role in the remodeling of the vascular structure in a hypertensive state or in various forms of arterial injuries. Although this hypothesis is intriguing, the possible roles of thromboxane AZ in the proliferation of VSMC have not been fully addressed. We therefore have investigated the mechanism by which the vasoconstrictor thromboxane AZ influences the proliferation of smooth muscle cells derived from rat thoracic aorta, using 9,11-epithio-11,12-methano-thromboxane AZ (STA2), a stable analogue of thromboxane AZ, and cell culture techniques.
cell growth; thymidine uptake; cell cycle; G, resting mitotic period; cytoskeleton protein; actin
MATERIALS
period;
Cell Culture
have emphasized the role of the growth and migration of medial smooth muscle cells in the remodeling of the vascular structure in a hypertensive state (6, 22) or in the remodeling processes involved in arterial restenosis after angioplasty (12, 30). It is therefore of great interest to investigate the regulatory mechanism of the proliferation of vascular smooth muscle cells (VSMC). A number of these studies have thus far been conducted, thereby providing evidence for the involvement of various vasoactive substances derived from vascular vessels, e.g., prostaglandins (37), serotonin (23), various growth factors like platelet-derived growth factor (PDGF) (31) and transforming growth factor (20), angiotensin II (5), endothelin (lo), and endothelium-derived relaxing factor(s) (7), in the modulation of VSMC growth. The vascular wall is not only the container for the blood supply but also functions as an autocrine or paracrine gland to secrete various vasoconstrictor and relaxing substances (8,19) and a number of growth factors (1, 25). In this context, it is well postulated that the vascular wall produces significant amounts of vasoconstrictor
RECENT
STUDIES
0363-6135/92
$2.00
Copyright
AND METHODS
VSMC were isolated from the thoracic aortas of 8-wk-old Wistar-Kyoto rats, according to the method of Ross and coworkers (31, 32). Briefly, thoracic descending aortas were obtained, and the adventitia and intima were carefully removed. The explants were then placed on a polystyrene dish with the intimal side attached to the dish, and Dulbecco’s modified Eagle’s medium (DMEM; GIBCO Laboratories, Grand Island, NY), containing 20% (vol/vol) fetal bovine serum (FBS; GIBCO Laboratories), penicillin (100 U/ml), and streptomycin (100 pg/ml), was gently added. The dishes were incubated at 37°C in a humidified atmosphere of 95% air-5% COa. The cells reached confluency 10 days after inoculation. They were harvested by brief exposure to Hanks’ balanced salt solution supplemented with 0.05% (wt/vol) trypsin and 0.02% (wt/vol) EDTA and transferred into a new dish. At this stage, the cells were designated as the first passage. The percentage of FBS was changed to 10% (vol/vol) in subsequent media. The harvest was repeated when the cells became confluent. VSMC from passages 3 to 6 were used in this study. The cells in culture exhibited the characteristic “hills and valleys” growth pattern, and the presence of cu-actin molecules was demonstrated by using a monoclonal anti-cY-actin antibody and immunoenzyme assay method (32, 37).
0 1992 the American
Physiological
Society
H1331
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
H1332
CELL
GROWTH
AND
Cell Synchrony Cell synchronization was performed according to the standard methods as described by Ashihara and Baserga (2). To synchronize VSMC in the G,/G, boundary (quiescent VSMC), randomly cycling cells were cultured in serum-free media for 48 h. Replacement of serum-free media by 10% fresh sera initiated cell proliferation. Quiescent VSMC were cultured for 24 h in media containing 10% sera and 1 mM hydroxyurea. Hydroxyurea stopped the proliferative process at the G,/S boundary. DNA replication was started again by washout of the hydroxyurea with 10% fresh sera. To synchronize the cells in the premitotic period, we utilized 15 rig/ml colchicine. G,/S-synchronized VSMC were cultured for 12 h in DMEM containing 10% sera and colchicine. The mitotic process could be resumed by washout of the colchicine with 10% fresh sera. Assessment
’
of
Cell Proliferation
Doubling time. To assess the proliferating activity, we measured the doubling time of VSMC and [:3H]thymidine uptake into DNA fragments. The doubling time was determined as follows: 5 x lo4 cells were seeded onto g-cm2 dishes with 2 ml DMEM containing 10% sera and cultured for 24 h. Then, the media were changed to culture media containing a given concentration of STA, (Ono Pharmaceuticals, Osaka, Japan), a stable thromboxane A, analogue (16). Cell number was determined by light microscopy before the addition of STA2 and after 24 h. The doubling time was calculated according to the following equation: doubling time = (tl - t2) x log 2/(log N, log N2), where t, and t2 are the times when cell numbers are counted and N, and N2 are the cell numbers at tl and t2, respectively. Measurement of r3H]thymidine uptake. The rate of DNA replication was assessed by measuring incorporation of [:3H]thymidine into DNA fragments (37). First, VSMC in a logarithmic growth state were cultured for 24 h in media containing 10% FBS, 0.25 &i [3H]thymidine (20-30 Ci/mmol), and a given concentration of STA,. After the culture period, the cells were harvested, washed with Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ and then homogenized by a model UR-20 ultrasonicator (Tomy-Seiko, Tokyo, Japan). The homogenate was treated with 5% perchloric acid at 4°C for 30 min and spun at 3,000 g for 30 min. The precipitate was repeatedly washed with 5% perchloric acid solution, and radioactivity in this acid-insoluble fraction was measured by an automatic liquid scintillation counter. Similarly, [ ZH] thymidine uptake by G,/G 1-synchronized VSMC or G,/S-synchronized VSMC was measured. In these experiments, the cells were cultured in fresh media containing [:3H]thymidine and lo-’ M STA, and harvested at various culture periods. Radioactivity in the acid-insoluble DNA fragments was determined by the same procedures as described in the method for randomly cycling VSMC. Mitotic index. The percent population of mitotic cells (mitotic index) was determined by light microscopy. Briefly, after the proliferating process was resumed in the VSMC synchronized in various phases, the cells were fixed with 5% acetate ethanol solution after culture for various time periods. The fixed cells were stained by Giemsa solution. The cells in the mitotic process were detected by light microscopy. Five hundred nuclei were examined in each sample. Cytoskeleton Protein Synthesis To examine STA, effect on cell body formation, mined incorporation of [“HI leucine into cellular VSMC synchronized in the G,/G, or G,/S boundary
we deterproteins. were cul-
THROMBOXANE
A2
tured for 12 h in 10% FBS containing a given concentration of STA, and 0.05 &i of [“Hlleucine (120-190 Ci/mmol). The cells were harvested and homogenized by an ultrasonicator in 25 mM phosphate-buffered saline containing 1 mM EDTA. The homogenate was treated with ice-chilled 5% (vol/vol) trichloroacetate. The precipitate was washed with the trichloroacetate solution and dissolved in 1 ml of 0.5 N NaOH solution. The radioactivity of this solution was measured with an automatic liquid scintillation counter. To reveal whether STA2 directly affected the synthesis of cytoskeleton proteins during the G,/M period, we then examined the alterations in the formation of actin, a major cytoskeleton protein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21). G,/S-synchronized cells were cultured for 12 h in media containing 0.05 &i [“Hlleucine and a given concentration of STA,. Afterward the cells were harvested and homogenized by an ultrasonicator. The homogenate was spun at lo4 g for 30 min, and the pellet was washed three times with 5 mM phosphate-buffered solution at pH 7.2 including 1 mM EDTA and 1 mM dithiothreitol. The combined supernatant was used for analysis of cytoskeleton proteins. Proteins in the supernatant were denatured at 95°C for 10 min in 50 mM tris(hydroxymethyl)aminomethane hydrochloride at pH 6.8 containing 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 1 mM dithiothreitol, and 0.0025% (wt/vol) bromphenol blue. Samples were applied to a 7.5% polyacrylamide slab gel, using a Protean II Slab Cell (Bio-Rad, Richmond, CA) and MultiDrive XL power supply unit (Pharmacia Biotechnology, Bromma, Sweden). Proteins were stained with 0.1% Coomassie Blue in fixative (water-methanol-acetate, 5:4: 1, vol/vol). After destaining was completed, the density of the separated proteins was quantitatively scanned, using a model EPA-3000 densitypattern analyzer (Maruzen Petrochemicals, Tokyo, Japan). The amount of actin was determined by reference to the relative mobility of an cu-actin standard. Moreover, alterations in actin formation were determined by measuring incorporation of [“HI leucine into actin molecules. For this analysis, the actin band was cut out of the gel and the radioactivity in it was eluted with a tissue solubilizer (NCS, Amersham International, Buckinghamshire, UK). The radioactivity was determined using an automatic liquid scintillation counter. To confirm the association of cytoskeleton protein formation with cell growth, we investigated the effects of protein synthesis inhibition and functional disturbance of actin on the mitotic process in the M phase. G,/S-synchronized VSMC were cultured in fresh media containing a given concentration of puromycin, an inhibitor of protein synthesis, or cytochalasin D, a depolymerizing agent of actin. The mitotic index was measured at various culture periods. Reagents Reagents were all of analytic grade. Eicosanoids and related compounds were offered by Ono Pharmaceuticals. Radioactive materials were purchased from Amersham International. Statistical
Analysis
The values are expressed as means * SE. Statistical differences were analyzed by the Student’s t test, the paired t test, or one- or two-way analysis of variance. RESULTS
STA2 Effect on Randomly Cycling VSMC
First, we investigated the effect of STA2, a stable thromboxane A2 analogue, on the cell proliferation of randomly cycling VSMC. STAz increased incorporation of [:‘H]thymidine into acid-insoluble DNA fragments in
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 11, 2019.
CELL
GROWTH
AND
THROMBOXANE
H1333
A2
VSMC in a dose-dependent manner ranging from lOAs to lOA M STAz (Table 1). To confirm this property of STA2, we examined the alteration in the doubling time when the cells were maximally stimulated with 10es M STA2. As shown in Fig. 1, this dose of STA2 significantly shortened the doubling time of VSMC in a logarithmic growth state (19.24 t 0.32 vs. 20.25 t 0.40 h). These data clearly indicate that STA2, a stable analogue of thromboxane AZ, enhances the rate of cell cycle progression in cycling VSMC populations. Analysis
P
