From Institut für Pharmakologie (D.B., S.M., A.-A.W., K.S.) and
Institut für Klinische Anaesthesiologie, Heinrich-Heine-Universität
Düsseldorf (Germany) (T.-Ph.Z.); and Forschungszentrum für
Vaskuläre Biologie und Medizin der Friedrich-Schiller-Universität
Jena, Erfurt, Germany (E.B., E.G.).
Correspondence to Karsten Schrör, MD, Institut für Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Moorenstr 5, D-40225 Düsseldorf, Germany. E-mail schroer{at}pharma.uni-duesseldorf.de
Methods and Results—Thrombin (1 U/mL) caused a threefold to
fourfold increase of DNA synthesis in cultured bovine coronary
artery SMC as assessed from [3H]thymidine incorporation.
U 46619, a stable thromboxane A2 mimetic, had
only a minor stimulating effect on its own but potentiated the thrombin
effect sixfold to sevenfold above control (P<.05).
These findings were paralleled by a 52±5% (P<.05)
increase in cell number at 48 hours after addition of both mitogens as
compared with 24±5% with thrombin alone and no change with U 46619
alone. Thromboxane A2 receptor mRNA was found to be
upregulated sixfold 20 minutes after thrombin stimulation. Pretreatment
of SMC with thrombin for 4 hours markedly increased U 46619–induced
mitogen-activated protein kinase activity, indicating
thrombin-induced upregulation of functional thromboxane
receptors in SMC.
Conclusions—Thrombin-induced proliferation of SMC is markedly
enhanced by thromboxane A2. This might result
in an enhancement of SMC proliferation by platelet-derived
thromboxane A2 in vivo.
One issue of particular relevance in the pathophysiology of
thrombin actions is the relationship between thrombin-induced clot
formation and mitogenic activity. It has been shown that
SMC proliferation occurring in an area of thrombotic
arterial vessel occlusion is related to the size and place
of the preexisting thrombus.5 6 The thrombus
contains complex materials such as peptidergic growth factors,
serotonin, and active thrombin that can be released into
the vessel lumen and may facilitate vasoconstriction and tissue
proliferation in an area with endothelial injury.
Importantly, enzymatically active
One such thrombus-related factor is TXA2. Blood
platelets are the natural source of TXA2 in
the circulation. Previous studies have addressed the question of
whether or not TXA2 or appropriate agonists can
induce SMC proliferation. In some studies, TXA2
mimetics, such as U 46619, CTA2,
STA2 or I-BOP, can stimulate proliferation of
vascular SMC,16 17 18 19 20 21 partially doing so by
upregulating the synthesis and release of endogenous growth
factors such as platelet-derived growth factor (PDGF) or basic
fibroblast growth factor (bFGF)20 and by a
synergistic action with peptidergic growth factors in intracellular
signal transduction via the MAP kinase
pathway.19 21 In contrast, other investigators
did not detect direct mitogenic effects of
TXA2 on SMC.22 23 Part of
the explanation may be different culture conditions. This includes the
presence of fetal calf serum,18 19 species
differences, as well as the source of SMC, which in many cases are
derived from the aorta.
The number of TP receptors in platelets is regulated by hormones
(testosterone), receptor agonists (thromboxane mimetics),
peptidergic growth factors (PDGF), and disease states such as recurrent
myocardial ischemia.24 25 26 Indirect
evidence suggests that enhanced thrombin generation in blood might also
increase the number of TP receptors at the platelet
surface.27 An upregulation of TP receptor mRNA by
thrombin has also been demonstrated in a megakaryoblastic cell
line.28 There is also evidence for functional
thromboxane receptors in vascular
tissue,29 although their regulation is much less
understood. Thus the present data collectively suggest a
synergistic interaction between thrombin and
TXA2, possibly occurring at the level of
thromboxane formation, intracellular signal transduction,
and receptor regulation, respectively.
This study investigates the interactions between thrombin and a
chemically stable thromboxane mimetic, U 46619, on
proliferation of bovine coronary artery SMC. Evidence is
provided that U 46619 potentiates thrombin-induced mitogenesis and that
this response involves thrombin-induced expression of TP receptors.
DNA and Protein Synthesis
After 20 hours, [3H]thymidine (0.5 µCi/well)
or [14C]leucine (0.1 µCi/well) (both from
DuPont) were added. At the end of the total incubation period of 24
hours, the media were removed and the cells were washed twice with 1 mL
ice-cold phosphate-buffered saline (PBS), 0.3 mL ice-cold perchloric
acid (0.3 mol/L), and again with cold PBS. The cells were solubilized
with 0.3 mL NaOH (0.1 mol/L) for 30 minutes at 37°C.
[3H]Thymidine and
[14C]leucine incorporation were quantified by
liquid scintillation spectrometry. Protein concentration was measured
in 20-µL aliquots from each well with the use of the Bio-Rad
colorimetric protein assay31
(Bio-Rad Laboratories).
Determination of Number of Cells Entering S-Phase
Cell Proliferation Assay
Myelin Basic Protein Phosphorylation Assay
RNA Analysis
Statistics
Similar results as for DNA synthesis were obtained when protein
synthesis was measured by determination of
[14C]leucine incorporation. U 46619 (0.3 to
30 µmol/L) showed only small, insignificant effects on
[14C]leucine incorporation. Thrombin (1 U/mL)
1.5-fold increased [14C]leucine incorporation.
However, there was a further increase in protein synthesis when U 46619
and thrombin were combined. As seen with
[3H]thymidine, this action of U 46619 was
concentration dependent. Maximal effects were observed at 3
µmol/L U 46619. These data are summarized in Fig 2
Antagonism of U 46619–Induced Potentiation of Thrombin-Induced
Mitogenesis by Blockade of TP Receptors
Cell Proliferation
Inhibition of Thrombin-Induced Mitogenesis by Hirudin
When SMC were stimulated with thrombin and U 46619, immediate
neutralization of thrombin with hirudin completely blocked the
stimulation of DNA synthesis. Interestingly, when hirudin was added at
later time points, a remarkable stimulation of DNA synthesis by U 46619
was observed that was dependent on the duration of thrombin exposure
before neutralization with hirudin (Fig 4
Expression of TP Receptor mRNA
MAP Kinase Activity
Therefore, the level of TP receptor expression in SMC was assessed
functionally by determination of MAP kinase activity (Fig 6
Enzymatically active thrombin is a well-known mitogen in cultured
vascular SMC.10 11 12 15 35 However, growth
stimulation by thrombin is a slow-onset reaction, showing markedly
delayed kinetics in the absence of serum or peptidergic growth
factors.4 For example, several days are necessary
for thrombin to induce proliferation of bovine aortic
SMC.12 On the other hand, activation of
transcription factors, such as c-fos or nuclear factor-
This study provides evidence for TXA2 as an
amplification signal for thrombin-induced SMC proliferation. As noted
above, there are conflicting reports as to whether or not
TXA2 can stimulate
mitogenesis.16 17 18 19 20 21 22 23 We have recently shown that
thromboxane mimetics, such as U 46619, induce expression of
immediate early genes, such as c-fos or egr-1, and
potentiate PDGF-dependent responses in rat aortic
SMC.21 Similar data were obtained in SMC prepared
from bovine coronary arteries.38 The
present study is the first to demonstrate that thrombin-induced
mitogenic responses are potentiated by the
thromboxane mimetic U 46619 in a receptor-dependent manner.
First, U 46619, although a weak mitogen itself, markedly potentiated
the mitogenic actions of thrombin. Second, the
thromboxane component of this response was blocked by the
specific thromboxane receptor antagonist SQ
29,548.
A possible explanation for the synergistic action between thrombin and
U 46619 is that thrombin might regulate the expression of
thromboxane receptors in SMC. A marked, sixfold increase in
TP receptor mRNA levels was seen at 20 minutes. This is much earlier
than the autoinduction of thrombin receptor mRNA expression in bovine
or rat vascular SMC by thrombin.15 39 An
upregulation of TP receptor gene expression by thrombin in a human
megakaryoblastic cell line has been previously
reported.28
Although TP receptor protein could not be detected by means of Western
blotting or ligand binding studies probably because of low receptor
protein expression, two lines of evidence support the concept of
thrombin-induced upregulation of functional TP receptors in SMC. First,
a brief (1.5 to 4 hours) incubation of SMC with thrombin resulted in an
increased mitogenic responsiveness to U 46619 that was
dependent on the duration of thrombin exposure before neutralization
with hirudin. Second, SMC that have been treated with thrombin for 4
hours showed a markedly higher stimulation of MAP kinase by U 46619
compared with nontreated SMC. In concert with the demonstration of
increased TP receptor mRNA expression by thrombin, these functional
data provide strong evidence for an increased expression of
functionally active TP receptors by thrombin. Because of the common
pathways of thrombin- and TXA2-induced signaling,
including MAP kinase activation,19 21 37 an
enhanced expression of thromboxane receptors is probably
also translated into growth promoting signals within SMC. Accordingly,
we have shown that cell proliferation (cell number 48 hours after
stimulation) was potentiated by the combined use of thrombin and U
46619 compared with either agent alone.
Several in vivo studies indicate a synergistic procoagulatory effect
between thrombin and TXA2 during
formation40 and
resolution41 of thrombi within coronary
arteries. There is also evidence for early upregulation of vascular
thrombin receptors in vivo and in SMC tissue culture by peptidergic
growth factors.42 An increased number of
prostaglandin
H2/TXA2-receptors has been
observed in platelets from patients with active unstable angina and
was related to increased thrombin activity.27
Inhibition of thrombin by hirudin in vivo significantly reduced
platelet-rich thrombi and neointima proliferation in
experimentally injured vessel segments.6 These
data suggest that active TXA2 may be generated in
platelet-rich thrombi long enough to enhance the
mitogenic responses to thrombin. However,
thromboxane formation may be limited by autoinactivation of
platelet
cyclooxygenase.43
To address this possibility, we have measured platelet-derived
thromboxane formation in vitro under conditions similar to
those associated with clot-related thromboxane formation in
vivo (data not shown). In these studies, a marked, aspirin-sensitive
thromboxane generation was found, persisting for at least 2
hours after addition of thrombin. This suggests continued
cyclooxygenase and thromboxane synthase
activity in the platelets over at least this period of time.
Clearly, an increased thromboxane receptor number in SMC
and associated thromboxane formation will act in concert
and might synergize in thrombin-induced mitogenesis not only in vitro
but also in vivo.
In summary, we have shown that thrombin-dependent upregulation of
vascular TP receptors occurs in thrombin-stimulated SMC and that active
thromboxane potentiates proliferative thrombin responses.
If these findings are translated into the in vivo situation, they would
support the combination of selective thromboxane receptor
antagonists in combination with thrombin
inhibitors for prevention of SMC proliferation after
coronary angioplasty.44 45 46
Received July 14, 1997;
revision received September 8, 1997;
accepted September 30, 1997.
© 1998 American Heart Association, Inc.
Basic Science Reports
Thrombin-Induced Mitogenesis in Coronary Artery Smooth Muscle Cells Is Potentiated by Thromboxane A2 and Involves Upregulation of Thromboxane Receptor mRNA
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Previous studies have
shown that thrombin is a potent though slow-acting mitogen for vascular
smooth muscle cells (SMC). Because thrombin generation in vivo is
accompanied by platelet activation, it has been suggested that
platelet-derived factors might enhance thrombin-induced SMC
proliferation. No information is available so far on the possible role
of thromboxane A2.
Key Words: growth substances • cells • muscle, smooth
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Generation of the
serine protease thrombin is the key event in hemostasis and of vital
importance for clot formation and thrombotic vessel occlusion after
vascular injury.1 2 This involves a combined
action of the enzyme on circulating zymogens of clotting factors as
well as a receptor-mediated stimulation of platelet aggregation and
contraction of vascular SMC. Activation of protease-dependent receptors
resulting in the generation of a tethered ligand after binding of
thrombin to its receptor allows one molecule of thrombin to
activate many thrombin receptors.3 4
These findings have stimulated the search for control mechanisms that
determine the specificity of this response and focus the biological
action of thrombin to specific cellular targets. 
-thrombin7
will escape inactivation by circulating antithrombins after binding
within the clot8 or
subendothelial matrix.5 9 This
thrombin might further stimulate platelets or the clotting cascade
and maintains its activity over hours.10 In
addition, enzymatically active thrombin is a potent direct mitogen for
SMC.10 11 12 13 14 15 This effect is antagonized by direct
thrombin inhibitors such as
hirudin.10 Thus the mitogenic action
of thrombin in vivo might result from both a direct action on SMC and
thrombin-induced generation of platelet-derived
mitogenic factors in the thrombus.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cell Culture
Bovine SMC were isolated enzymatically from the left anterior
descending coronary artery of adult female animals, following
the method of Fallier-Becker et al.30 The cells
were cultivated in an 80% Ham's F-12/20% Dulbecco's modified eagle
medium (DMEM), supplemented with 15% fetal calf serum (FCS), 100 U/mL
penicillin, and 0.1 mg/mL streptomycin (all cell culture material was
from Gibco or Becton & Dickinson). Cells were grown in a humidified
atmosphere of 5% CO2 and 95% air at 37°C. SMC
were characterized microscopically by the typical "hill-and-valley"
growth pattern and by indirect immunostaining with a
specific monoclonal -actin antibody (Boehringer Mannheim).
The media were exchanged twice a week. Monolayers of the cells were
passaged on 150-mm tissue culture dishes once a week with trypsin
(0.05%)/EDTA (0.5 mmol/L). Passages 4 to 10 were used for the
experiments. Cell viability was assessed by means of trypan blue
exclusion test and was found to be >95%.
SMC were seeded into 24-well plates
(5x104 cells/well) and cultivated for 72 hours
under standard conditions until subconfluency was reached. For the
following 24 hours, cells were exposed to serum-free medium to allow
defined stimulation with mitogens. All further incubations were also
carried out in serum-free medium, supplemented with 3 µmol/L
indomethacin (Luitpold Pharma). -Thrombin (1 U/mL), the TXA2 mimetic U 46619
(Upjohn Diagnostics) (0.3 to 30 µmol/L) and the
thromboxane receptor antagonist SQ 29,548
(Bristol-Myers-Squibb Pharmaceuticals) (1 µmol/L) were added to
duplicate or triplicate wells. In additional experiments, hirudin (3
ATU/mL) was added 0 to 4 hours subsequent to stimulation with either
thrombin alone or thrombin combined with U 46619, respectively.
DNA synthesis was assayed by
immunofluorescence labeling of incorporated
5-bromo-2'-deoxyuridine (BrdU) with a cell proliferation kit (BrdU
labeling and detection kit I, Boehringer Mannheim). Cells were
grown on round glass microscope slides (diameter, 12 mm),
serum-deprived, and stimulated with mitogens as described above. Twenty
hours after addition of the agonists, BrdU incorporation was monitored
according to the manufacturer's instructions.
Representative areas of the slides were screened in a
standardized way for nuclei stained positive for BrdU. At the time of
the BrdU labeling, no differences in cell count (see below) were
seen.
Cells were grown to 
50% confluence, then made quiescent and
stimulated with mitogens as described above. After 24 and 48 hours,
respectively, cells were trypsinized and total cell number was assessed
by counting the cells per well, using a hemocytometer.
MAP kinase activity was assessed by the MBP
phosphorylation assay as described by Huwiler et
al.32 Subconfluent, serum-deprived SMC were
treated with indomethacin (3 µmol/L) as
described above. Thrombin was added to every second dish for 4 hours.
Subsequently, cells were stimulated with U 46619 (3 µmol/L) for
10 minutes. In some experiments, SMC were preincubated with SQ 29,548
(1 µmol/L) for 2 minutes before stimulation with U 46619.
After stimulation, SMC were washed with PBS and harvested into kinase
buffer (20 mmol/L Tris/HCl, pH 7.5, 1 mmol/L EGTA, 2
mmol/L MgCl2, 0.1 mmol/L sodium
orthovanadate, 1 mmol/L phenylmethylsulfonyl-fluoride, 25
µg/mL leupeptin, and 1 µg/mL pepstatin). Harvested cells were
briefly sonicated and centrifuged for 5 minutes at
14 000g and 4°C. Cell extracts (60 µg protein) were
incubated for 15 minutes at 30°C in the presence of 20 µg MBP
(Sigma), 10 µmol/L ATP, and 4 µCi
[
-32P]-ATP (Hartmann Analytik). The reaction
was terminated by addition of 4x Laemmli buffer and heating of the
samples for 5 minutes at 95°C. Proteins were separated by SDS/PAGE
(13.5%), gels were fixed in 25% propan-2-ol/10% acetic
acid, and bands were visualized by autoradiography.
TP receptor mRNA expression was analyzed by RT-PCR with
total RNA from SMC prepared with trizol reagent (Gibco BRL), following
standard methods.33 First-strand cDNA was
synthesized from total RNA using oligo d(T) and murine leukemia virus
reverse transcriptase (Perkin Elmer). The nucleotide
sequence for the bovine prostaglandin
H2/TXA2 (TP) receptor has
been deposited in the EMBL genebank database with the accession numbers
U 53484 and U 53485, respectively. PCR primers were designed according
to the cDNA sequence corresponding to the putative fourth and seventh
transmembrane domain of the receptor (sense:
5'-TGGGGCGCTACACCGTGCAG-3'; antisense: 5'-ATAGACCCAGGGATCCAAGA-3'). A
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)
fragment of 238 bp length was amplified in addition to the 410 bp
TP-receptor fragment (sense: 5'-TGATGACATCAAGAAGGTGGTGAAA-3';
antisense: 5'-TCCTTGGAGGCCATGTAGGCCAT-3'). PCRs were carried out using
AmpliTaq DNA Polymerase (Perkin Elmer). After a 2-minute
predenaturation step at 95°C, the reactions were cycled 30 times in a
cycle profile of 1 minute at 95°C, 1.5 minutes at 57°C, and 2
minutes at 72°C, followed by a final extension step for 5 minutes at
72°C. The PCR products were analyzed by automated laser
fluorescence with an ALF express sequencing system (Pharmacia
Biotech).
All data are mean±SEM of n independent experiments, performed
in duplicates or triplicates as indicated. Three-way ANOVA followed by
linear contrasts and a Bonferroni-Holm procedure for multiple testings
or one-way ANOVA followed by Bonferroni multiple comparison tests were
used for statistical analysis. Values of P<.05 were
considered significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Potentiation of Thrombin-Induced Stimulation of DNA and Protein
Synthesis by U 46619
Incubation of SMC with the stable TXA2
mimetic U 46619 (0.3 to 30 µmol/L) for 24 hours resulted only in
a minor increase of DNA synthesis. Some stimulation (1.5-fold above
control) was seen at 3 µmol/L U 46619. Thrombin (1 U/mL)
stimulated [3H]thymidine incorporation 3- to
4-fold above control. Coincubation of thrombin (1 U/mL) with U 46619
resulted in a concentration-dependent stimulation of DNA synthesis at
increasing concentrations of U 46619. For example, in the presence of
3 µmol/L U 46619, thrombin-induced DNA synthesis was about twice
as high as with thrombin alone, indicating a potentiation of
thrombin-induced mitogenic reactions by the
thromboxane mimetic. Further increase in U 46619
concentrations resulted in a disappearance of this effect. These data
are summarized in Fig 1
. A similar
potentiation of thrombin-induced DNA synthesis by U 46619 was observed
when the incorporation of BrdU was measured (Table).
View larger version (14K):
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Figure 1. Potentiation of thrombin (1 U/mL)-induced
[3H]thymidine incorporation by the
thromboxane mimetic U 46619 (0.3 to 30 µmol/L). Circles
indicate the absence, squares the presence of thrombin. Data are
mean±SEM of n=5 independent experiments (performed in
triplicate).
View this table:
[in a new window]
Table 1. Potentiation of Thrombin-Induced Mitogenesis (BrdU
Incorporation Into Cellular DNA) and Cell Proliferation (Cell Count) by
the Thromboxane Mimetic U 46619 .
View larger version (14K):
[in a new window]
Figure 2. Synergistic effects of U 46619 (0.3 to 30
µmol/L) on thrombin (1 U/mL)-induced [14C]leucine
incorporation. Circles indicate the absence, squares the presence of
thrombin (1 U/mL). Data are mean±SEM of n=5 independent experiments
(performed in duplicate).
To answer the question of whether the potentiation of
thrombin-induced mitogenesis by U 46619 is receptor mediated, thrombin
(1 U/mL) and U 46619 (3 µmol/L) were coincubated with the
selective thromboxane receptor antagonist SQ
29,548.34 SQ 29,548 (1 µmol/L) alone did
not influence [3H]thymidine incorporation in
unstimulated cells (data not shown). However, coincubation with SQ
29,548 (1 µmol/L) resulted in a complete prevention of U
46619-mediated amplification of thrombin-induced DNA synthesis. This
indicates that the potentiation of thrombin-induced DNA synthesis by U
46619 was mediated by TP receptors. These data are shown in Fig 3.
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Figure 3. Potentiation of thrombin (Thr; 1 U/mL)-induced
mitogenesis by U 46619 (U46; 3 µmol/L) and its prevention by the
selective thromboxane receptor antagonist SQ
29,548 (SQ29; 1 µmol/L). Data are mean±SEM of n=4 independent
experiments (performed in triplicate). *P<.05 vs Thr,
#P<.05 vs Thr+U46.
The effects of thrombin and/or U 46619 on SMC mitogenesis were
further studied in a cell proliferation assay. At the time relevant for
BrdU labeling (24 hours after stimulation), no differences in the cell
count were observed between the groups. In contrast, 48 hours after
addition of thrombin, a significant progression of the cell cycle into
the M-phase was observed that was reflected by an increase in cell
number. Again, thrombin-induced SMC proliferation was significantly
amplified by U 46619. The data are summarized in the Table
.
SMC mitogenesis requires prolonged exposure to thrombin, and
stimulation of DNA synthesis can be inhibited by addition of hirudin
from 0 to 8 hours after stimulation with
thrombin.15 Therefore, we have studied the
effects of a brief exposure to thrombin on U 46619–induced SMC
mitogenesis by inhibiting thrombin activity with hirudin from 0 to 4
hours after stimulation. When added up to 4 hours after stimulation,
hirudin (3 ATU/mL) completely inhibited thrombin-induced DNA synthesis
(Fig 4).
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Figure 4. Inhibition of thrombin-induced mitogenesis by
hirudin. Thrombin (Thr, 1 U/mL)-induced mitogenesis was completely
blocked by hirudin (Hir, 3 ATU/mL) added from 0 to 4 hours after
stimulation. U 46619 (U46, 3 µmol/L) did not stimulate SMC
mitogenesis but potentiated the effects of thrombin. When SMC were
stimulated with thrombin and U 46619, immediate neutralization (0 min)
of thrombin with hirudin also completely blocked the stimulation of DNA
synthesis. When hirudin was added at later time points (90 to 240
minutes), a marked stimulation of mitogenesis by U 46619 was observed
that was dependent of the duration of thrombin exposure before
neutralization with hirudin. Data are mean±SEM of a
representative experiment performed in triplicate.
Similar results were obtained in three independent experiments. ). Since direct thrombin
effects on DNA synthesis were completely inhibited by addition of
hirudin within the time window studied, these results suggest a priming
effect of thrombin on SMC resulting in an increased
mitogenic responsiveness to U 46619.
As a possible mechanism to explain the potentiation of
thrombin-induced mitogenic effects by U 46619, we studied
the hypothesis that TP receptors are upregulated by thrombin.
Stimulation of quiescent SMC with thrombin (1 U/mL) for up to 3 hours
resulted in a marked though transient increase in TP receptor mRNA
expression. A maximum effect was seen at 20 minutes. At this time,
thrombin stimulated TP receptor mRNA expression about sixfold above
control. After continuous stimulation with thrombin for 3 hours, TP
receptor mRNA expression returned to levels of unstimulated cells. Fig 5 shows representative
original tracings of one experiment out of three with similar
results.
View larger version (17K):
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Figure 5. Time-dependent stimulation of TP receptor–mRNA
expression by thrombin (original registration). SMC were stimulated
with thrombin (1 U/mL) for 20, 60, and 180 minutes, and a 410 bp
receptor fragment was amplified by RT-PCR. A 238 bp–GAPDH fragment was
used as an internal standard. Similar data were obtained in three
independent experiments.
Experiments were performed to determine the effects of thrombin on
the expression of TP receptor protein. However, TP receptor could not
be detected in membrane preparations from control cells or from
thrombin-treated cells by Western blot analysis with polyclonal
antibodies raised against an epitope deduced from the cloned bovine TP
receptor. Similarly, no receptor binding was detected in ligand-binding
studies with [3H]SQ 29,548,
[3H]I-BOP, and
[125I]-BOP, respectively. Since TP receptors
could be detected by both, Western blotting and ligand-binding studies
in COS-7 cells transiently transfected with TP receptor cDNA (not
shown), the lack of detection in SMC is most likely a result of a low
level TP receptor expression in SMC. ). In control cells, addition of U 46619
did not significantly stimulate MAP kinase activity. These data are
consistent with a low basal TP receptor expression and are in
line with our findings that U 46619 alone was not capable to stimulate
DNA synthesis in SMC. However, when SMC were pretreated with thrombin
for 4 hours, a marked stimulation of MAP kinase activity by U 46619 was
seen (Fig 6). This effect was inhibited by the TP receptor
antagonist SQ 29,548. In concert with our findings on TP
receptor mRNA, these data provide functional evidence for a
thrombin-induced upregulation of TP receptors.
View larger version (16K):
[in a new window]
Figure 6. U 46619 (U 46, 3 µmol/L)-induced MAP kinase
stimulation. In control cells (Con), U 46619 did not stimulate MAP
kinase activity. When SMC were pretreated with thrombin (1 U/mL) for 4
hours, a marked stimulation of MAP kinase activity by U 46619 was
observed that was prevented by the TP receptor antagonist
SQ 29,548 (SQ, 1 µmol/L). A representative
experiment is shown. Similar data were obtained in three independent
experiments.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present data indicate that thrombin-induced mitogenesis of
coronary artery SMC is markedly enhanced in the presence of a
TXA2 mimetic, which, alone, has only minor
mitogenic activity. It is also shown that this potentiation
of the thrombin-induced response is TP receptor mediated because it is
prevented by a selective antagonist. We have demonstrated a
marked increase in thromboxane receptor mRNA in SMC and
provided functional evidence for TP receptor upregulation by thrombin.
These findings may provide a mechanistic basis for the proliferative
responses to the two agonists. 
B,
occurs within a few hours after thrombin
stimulation.36 Similarly,
phosphorylation of several signal transduction
molecules, including MAP kinases, has been observed within minutes
after thrombin stimulation.37 Thus despite rapid
thrombin receptor activation, the maximal mitogenic effect
of thrombin in cultured SMC requires a prolonged exposure to active
thrombin.15 This was explained by a progressive
thrombin-dependent recruitment of additional cells into the growth
fraction. Alternatively, thrombin-induced mitogenesis in vivo might
involve the generation or amplification of additional growth promoting
signals, present in the thrombus or in the injured vessel wall in
vivo but not in cultured SMC.
Selected Abbreviations and Acronyms
MAP kinase
=
mitogen-activated protein kinase
MBP
=
myelin basic protein
RT-PCR
=
reverse transcription–polymerase chain reaction
SMC
=
smooth muscle cell(s)
TP
=
thromboxane receptor(s)
TXA2
=
thromboxane A2
Acknowledgments
This study was supported by grants of the Deutsche
Forschungsgemeinschaft (GL 178/3–1; SCHR 194/10–1, SFB 351 D7, SFB
242 A9). The authors thank Dr J. Stürzebecher (Forschungszentrum
für Vaskuläre Biologie und Medizin der
Friedrich-Schiller-Universität Jena; Erfurt, Germany) for a
generous supply of purified -thrombin, Dr M. Ogletree
(Bristol-Myers-Squibb Pharmaceuticals, Princeton, NJ) for SQ
29,548, and Dr R. Schmedemann (Upjohn Co, Heppenheim, Germany) for U
46619. The authors wish to thank Dr Dieter Hafner (Institut für
Pharmakologie, Heinrich-Heine-Universität, Düsseldorf,
Germany) for statistical advice. The competent technical
assistance of Sabine Menzel and secretarial assistance of Erika Lohmann
are gratefully acknowledged.
Footnotes
This study was presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13 to 16, 1995, and published in abstract form in Circulation (1995; 92(suppl I):I-111).
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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  A. Haider, I. Lee, J. Grabarek, Z. Darzynkiewicz, and N. R. Ferreri Dual Functionality of Cyclooxygenase-2 as a Regulator of Tumor Necrosis Factor-Mediated G1 Shortening and Nitric Oxide-Mediated Inhibition of Vascular Smooth Muscle Cell Proliferation Circulation, August 26, 2003; 108(8): 1015 - 1021. [Abstract] [Full Text] [PDF]
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 M. Yaekashiwa and L.-H. Wang Transcriptional Control of the Human Thromboxane Synthase Gene in Vivo and in Vitro J. Biol. Chem., June 14, 2002; 277(25): 22497 - 22508. [Abstract] [Full Text] [PDF]
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 E. Connolly, D. J. Bouchier-Hayes, E. Kaye, A. Leahy, D. Fitzgerald, and O. Belton Cyclooxygenase Isozyme Expression and Intimal Hyperplasia in a Rat Model of Balloon Angioplasty J. Pharmacol. Exp. Ther., February 1, 2002; 300(2): 393 - 398. [Abstract] [Full Text] [PDF]
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 S.-i. Saitoh, F. Onogi, K. Aikawa, M. Muto, T. Saito, K. Maehara, and Y. Maruyama Multiple endothelial injury in epicardial coronary artery induces downstream microvascular spasm as well as remodeling partly via thromboxane A2 J. Am. Coll. Cardiol., January 1, 2001; 37(1): 308 - 315. [Abstract] [Full Text] [PDF]
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 W. Young, K. Mahboubi, A. Haider, I. Li, and N. R. Ferreri Cyclooxygenase-2 Is Required for Tumor Necrosis Factor-{alpha}- and Angiotensin II-Mediated Proliferation of Vascular Smooth Muscle Cells Circ. Res., April 28, 2000; 86(8): 906 - 914. [Abstract] [Full Text] [PDF]
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Y. Ikeda, A. Sugawara, Y. Taniyama, A. Uruno, K. Igarashi, S. Arima, S. Ito, and K. Takeuchi Suppression of Rat Thromboxane Synthase Gene Transcription by Peroxisome Proliferator-activated Receptor gamma in Macrophages via an Interaction with NRF2 J. Biol. Chem., October 13, 2000; 275(42): 33142 - 33150. [Abstract] [Full Text] [PDF]
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