© 1997 American Heart Association, Inc.
Articles |
Dopamine as a Novel Antimigration and Antiproliferative Factor of Vascular Smooth Muscle Cells Through Dopamine D1-Like Receptors
From the The First Department of Internal Medicine (K.Y., M.K., T.Ho., H.K., K.Y., M.M., J.Y.) and The Second Department of Biochemistry (T.Ha.), Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka, Japan.
Correspondence to Kenichi Yasunari, MD, The First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan.
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Abstract |
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Abstract Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine D1-like receptors in atherosclerosis, the effects of dopamine and specific D1-like agonists SKF 38393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC migration and proliferation were studied. We observed that cells stimulated by PDGF-BB (5 ng/mL), showed increased migration and proliferation. These effects were prevented by coincubation with dopamine, SKF 38393, or YM 435 (1 to 10 µmol/L), and this prevention was reversed by Sch 23390 (1 to 10 µmol/l), a specific D1-like antagonist. These actions are mimicked by forskolin (1 to 10 µmol/L), a direct activator of adenylate cyclase and 8-bromo-cAMP at 0.1 to 1 mmol/L and are blocked by a specific protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H 89), but not blocked by its negative control, N-[2-(N-formyl)-p-chlorociannamylamino)ethyl]-5-isoquinoline sulfonamide (H 85). PDGF-BB (5 ng/mL)-mediated activation of phospholipase D, protein kinase C, and mitogen activated protein kinase activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular D1-like receptor agonists inhibit migration and proliferation of VSMC, possibly through protein kinase A activation and suppression of activated phospholipase D, protein kinase C, and mitogen-activated protein kinase activity.
Key Words: dopamine • vascular smooth muscle • migration • proliferation
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Introduction |
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Migration and proliferation of VSMCs are thought to play key roles in the development of atherosclerotic lesions.1 Understanding the regulatory mechanism of VSMC migration and proliferation are, therefore, important in elucidating the pathogenesis of atherosclerosis and also in the design of therapeutic drugs or devices for treating atherogenic disorders. PDGF is one of the major mitogens in serum and is reported to stimulate VSMC proliferation.2 It has been reported that the PDGF-BB isoform induces directed migration of VSMCs3 and is a mitogen for VSMCs.4
Two distinct classes of dopamine receptor were originally thought to exist in peripheral tissue and are designated D1- and D2-like receptors.5 There are at least five dopamine receptor subtypes cloned from the brain. Types D1A and D1B are D1-like, whereas types D2, D3, and D4 are D2-like.6–8 Further, D1A receptors in coronary arterial smooth muscle cells have been reported,9 and D1-like receptors have been evaluated biochemically.10,11 Stimulation of D1-like receptors causes vasodilation.12 Vasodilator hormones such as natriuretic peptides and ß-adrenergic receptor agonists have been shown to act as the antigrowth factors.13,14
Therefore, the present study is designed to investigate the possible role of the D1-like receptors on PDGF-BB-mediated VSMC migration and proliferation and examine the potential therapeutic effect of D1-like receptor agonists on atherosclerosis.
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Methods |
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Materials
PDGF (recombinant BB), 8-bromo-cAMP, BSA, and 3-isobutyl-1-methylxanthine, were purchased from Sigma Chemical Co. DMEM, penicillin-streptomycin, trypsin EDTA (Versine), and FCS were purchased from Gibco BRL. PKC and cAMP radioimmunoassay kits, [3H]thymidine, [3H]ethanolamine, and [
-32P]ATP were purchased from Amersham Japan
Co. A Western blot system for analysis of the
phosphorylation status of p44 and p42 MAPK was
purchased from New England Biolabs Inc. Multiwell dishes, pipettes, and
flasks were purchased from Becton Dickinson. H-89,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide,
and H-85,
N-[2-(N-formyl-p-chlorocinnamylamino)ethyl]-5-isoquinoline
sulfonamide, were purchased from Seikagaku Co. YM 435 was a gift from
Yamanouchi Co.15 SKF 38393 was a gift from Smith
Kline Beecham Co Ltd.5
Cell Culture
VSMCs were grown from explants of 14-week-old normotensive
Wistar rat renal arteries, with animals handled as described
previously.16,17 Cells were identified as VSMCs
according to their morphologic and immunohistochemical characteristics
as previously reported.18,19 Briefly, these cells
showed a typical "hill-and-valley" growth pattern, were positive by
fluorescence using antibodies against 
-smooth muscle actin,
and did not express von Willebrand factor. VSMCs were grown in
DMEM supplemented with 10% FCS. Cells from passages 3 to 5 were used
and were subcultured after trypsinization on a weekly basis since cells
became confluent in 1 week. Each plate was replenished twice weekly
with fresh medium. To avoid the ß-adrenergic effect of dopamine,
propranolol (1 µmol/L) was added in each
dopamine stimulation. Propranolol (1 µmol/L)
alone did not affect cell migration and proliferation.
Measurement of cAMP
After incubation with D1-like agonists, cells were washed three
times with 2 mL of DMEM and then stimulated for 30 minutes with
different concentrations of dopamine, SKF 38393, or YM 435 dissolved in
DMEM with 0.5 mmol/L 3-isobutyl-1-methylxanthine. Rapid
aspiration and the addition of 0.5 mL of ice-cold 65% ethanol stopped
the reaction. After evaporation in a centrifugal evaporator, the dry
residue was dissolved in an assay buffer. cAMP levels were determined
by radioimmunoassay using the Amersham cAMP radioimmunoassay kit as
previously described.20,21
Migration Assay
Migration of VSMCs was assayed by a modification of Boyden's
chamber method using microchemotaxis chambers (Neuro Probe Inc) and
polycarbonate filters (Nucleopore Corp) with pores of 5.0 µm in
diameter, as previously reported.22 In every
experiment, collagen-coated filters were used. Briefly, the membranes
were treated with 0.5 N acetic acid and then incubated for 48 to 72
hours at 25°C in a collagen solution (100 µg/mL type I
collagen in 0.5 N acetic aid). They were then air-dried. Cultured VSMCs
were trypsinized and suspended at a concentration of approximately
1.5x105 cells/mL in DMEM supplemented with 0.4%
BSA. Cell number was counted with an electronic cell counter (Model
ZB1; Coulter Electronics).23 A volume of 200 µL
of VSMC suspension (3.0x104 cells) was placed in
the upper chamber, and 40 µL of DMEM-0.4% BSA containing a migration
factor, such as PDGF, was placed in the lower chamber. The chamber was
incubated at 37°C under 5% Colo2 in air for 4 hours. After
incubation, the filter was removed, and the VSMCs on the upper side of
the filter were scraped off. The VSMCs that had migrated to the lower
side of the filter were fixed in methanol, stained with Diff-Quick
staining solution, and counted under a microscope to quantitate VSMC
migration. Migration activity was expressed as the number of cells that
had migrated per high-power field (x400). In experiments to determine
the effects of dopamine, SKF 38393, YM 435, 8-bromo cAMP, and forskolin
on VSMC migration, these agents were added to the lower chamber before
the incubation.
Growth Curves
To determine cell numbers, VSMCs were placed into six-well
culture dishes at 2x104/mL and grown in DMEM
containing 10% FCS changed for every 72 hours, then switched to the
same medium with 0.1% FCS for 48 hours to induce quiescence. Cultures
were washed with a calcium- and magnesium-free phosphate-buffered
saline and detached with trypsin EDTA solution. Counts were performed
by an electronic cell counter.
Determination of DNA Synthesis
Relative rates of DNA synthesis were assessed by determination
of [3H]thymidine incorporation, into
trichloroacetic acid-precipitable material.24
Quiescent VSMCs grown in 24-well culture dishes were pulsed 4 hours
with [3H]thymidine (10 µCi/mL), washed with
cold calcium- and magnesium-free phosphate-buffered saline and
incubated with 5% trichloroacetic acid at 4°C for 10 minutes. Cells
were dissolved in 1 N NaOH at 37°C for 30 minutes, neutralized. The
radioactivity was determined by liquid scintillation counting.
Flow Cytometric Analysis of Cell Cycle
Quiescent VSMCs grown in flasks were detached with 0.25%
trypsin at 37°C for 5 minutes and then pelleted by
centrifugation (1000 rpm for 5 minutes). The cells were
resuspended in 200 µL of solution A (trypsin, 30 mg/L; citric
acid, 3.4 mmol/L; spermin, 1.5 mmol/L;
Tris-HCl, 0.5 mmol/L; Nonidet P-40. 2 mL/L). Ten minutes
later, 150 µL of solution B (trypsin inhibitor, 500
mg/L; RNase, 100 mg/L; citric acid, 3.4
mmol/L; spermin, 1.5 mmol/L; Tris HCl, 0.5
mmol/L; Nonidet P-40, 2 mL/L) was added and left for 10 minutes
at room temperature. Then 150 µL of solution C (propidium iodide,
622 µmmol/L; spermin, 3.0 mmol/L; citric
acid, 3.4 mmol/L; Tris-HCl, 0.5 mmol/L; Nonidet
P-40, 2 mL/L) was added and left for more than 10 minutes. Cell
cycle24 was analyzed within 3 hours on a
flow cytometer (EPICS PROFILE). Red blood cells were used as internal
standard of DNA analysis.
PLD Activity Measured by Ethanolamine Release
The cells in 35-mm dishes were cultured in medium containing
[3H]ethanolamine (5 µCi/mL/dish) for 24 hours
to label cellular phosphatidylethanolamine. After the labeling medium
was removed, the cells were washed twice with buffer A (20
mmol/L HEPES [pH 7.4], 120 mmol/L NaCl, 5
mmol/L KCl, 1 mmol/L MgCl2,
1.5 mmol/L CaCl2, 0.1% (wt/vol) BSA,
22.2 mmol/L glucose). After 10 minutes incubation with
buffer A containing agonists with or without antagonists,
the reaction was terminated by removing buffer A and adding 0.75 mL of
methanol. The cells were harvested by gentle scraping. The dishes were
then washed again with 0.75 mL of methanol. Distilled water (0.6 mL)
and 0.75 mL of chloroform were added to the cells. The aqueous and
lipid phases were obtained from the cell extracts by adding 0.75 mL of
chloroform and 0.75 mL of water, followed by
centrifugation (3000g, 10 minutes).
Ethanolamine metabolites from the aqueous phases were fractionated on
Dowex 50w (H+)–packed columns (Bio-Rad Econo
columns, 1-mL bed volume) as already described.25
The initial flow-through (1 mL) along with the following 3 mL of water
contained glycerophosphoethanolamine. Ethanolamine phosphate was eluted
with 15 mL of water. Finally, ethanolamine was eluted with 8 mL of 1
mol/L HCl.
Cell Fractionation and Assay of PKC
VSMCs were washed twice with an ice-cold assay buffer
(50 mmol/L Tris-HCl [pH 7.5] buffer containing 2
mmol/L EDTA, 2 mmol/L EGTA, 0.25 mol/L sucrose, 10
mmol/L 2-mercaptoethanol, 0.21 mmol/L leupeptin, and
0.23 mmol/L phenylmethylsulfonyl fluoride). Then
cells were scraped and were sonicated with three 10-second bursts. The
homogenates were centrifuged at
100 000g for 60 minutes at 4°C to separate the cytosolic
and particulate fractions. The cytosolic fraction was kept on ice with
Nonidet P-40 at a final concentration of 1%. The pellet resuspended in
assay buffer containing 1% Nonidet P-40 was stirred on ice for 1 hour,
and then was centrifuged at 100 000g for 30
minutes. PKC activity was measured by a modification of the method
previously reported using the Amersham PKC assay
system.19 In brief, a sample of the reaction
mixture (50 mmol/L Tris-HCl [pH 7.5], 3
mmol/L calcium acetate, 100
µmol/L--phosphatidyl-L-serine, 1
µmol/L phorbol myristate acetate, 225
µmol/L substrate peptide, 7.5 mmol/L
dithiothreitol, and 0.05% wt/vol sodium azide) were mixed with
magnesium [32P]ATP and incubated at 25°C for
15 minutes. The substrate peptide interacts at 8%,13%, and 38% of
PKC activity with cAMP-dependent protein kinase,
phosphorylase kinase, and proteolytic PKC fragment,
respectively. However, it does not interact with hexokinase and myosin
light-chain kinase. An acidic reaction-quenching reagent was added to
stop the reaction. Phosphorylated peptide was separated
on binding paper. After the paper was washed, the extent of
phosphorylation was detected by scintillation counting.
The PKC assay was linear for 15 minutes. PKC activity was determined by
subtracting the initial rate of protein kinase activity (in the absence
of activators) from the initial rate of protein kinase
activity in the presence of phosphatidylserine,
calcium acetate, and phorbol myristate acetate.
MAPK Assay
MAPK activity was measured by the Amersham MAPK assay
system.26 Briefly, cell aliquots
(1x106 cells) were challenged with ligand at
37°C, and the reaction was terminated by directly adding lysis buffer
containing 20 mmol/L Tris (pH 8), 20 mmol/L
ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 2
mmol/L EGTA, 2 mmol/L dithiothreitol, 0.1
mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL
aprotinin, and 0.1% Triton X-100 (final concentration) in a total
volume of 200 µL. Extracts (15 µL) were then assayed by adding 10
µL of the substrate buffer (containing 6 mmol/L substrate
peptide, 75 mmol/L HEPES, 300 µmol/L sodium
orthovanadate, and 0.05% sodium azide, pH 7.4) and 5 µL of ATP
buffer (containing 0.3 mmol/L
[-32P]ATP (300 µCi/mL) and 90
mmol/L MgCl2). The substrate peptide
interacts with p34cdc2 at 3.4% and PKC at 0.1% of MAPK activity.
After a 30-minute incubation at 37°C, 10 µL of 300 nmol/L
orthophosphoric acid was added to terminate the reaction. Thirty
microliters of each sample was spotted onto phosphocellulose discs,
washed three times for 30 minutes in 0.5% phosphoric acid, and washed
once for 5 minutes in distilled water. The radioactivity on each disk
was then determined by scintillation counting.
Immunoblotting
VSMCs grown on a six-well plate were stimulated with
agonists at 37°C in serum-free DMEM for specified durations. The
reaction was terminated by the replacement of medium with 100 µL of
SDS–polyacrylamide gel electrophoresis buffer, pH 6.8,
containing 62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol,
50 mmol/L dithiothreitol, and 0.1% bromphenol blue. After
brief sonication (5 seconds), samples were boiled for 5 minutes at
95°C and centrifuged (14 000g, 5 minutes) at
4°C, and the supernatant (25 µL) was subjected to
SDS–polyacrylamide gel electrophoresis. Proteins in the gel
were transferred to a polyvinylidene difluoride membrane
(Schleicher & Schuell) by electroblotting. The membrane was treated
with rabbit polyclonal phospho-specific MAPK antibodies that detect
p42MAPK and p44MAPK only
when catalytically activated by phosphorylation
at Tyr-204. After incubation with secondary antirabbit antibodies,
immunoreactive proteins were detected by the CDP-Star chemiluminescence
system (New England Biolabs Inc).
Statistical Methods
Statistical analysis was performed by ANOVA and
Scheffé's modified t test.27
Values of P<.05 were considered to be significant.
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Results |
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Effect of D1-Like Receptor Agonists on cAMP Formation in VSMCs
Fig 1
shows the effect of the
D1-like agonists, dopamine, SKF 38393 and YM 435, on cAMP formation in
VSMCs. As shown in Fig 1, D1-like agonists dose dependently increased
the intracellular cAMP formation in VSMCs with the rank-order potency
YM 435
dopamine>SKF 38393. This cAMP formation was blocked
completely by the specific D1-like antagonist Sch
23390.
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) or without (
) 10 µmol/L Sch
23390, a D1-like specific antagonist, on cAMP formation in
VSMCs. cAMP responses of VSMCs to increasing concentrations of D1-like
agonists were assessed as described in "Methods." Each point
represents the mean±SD of quadruplicate determinations in a
single representative experiment.
Effect of D1-Like Receptor Agonists on VSMC Migration Stimulated
With PDGF
The effect of the D1-like receptor agonists, dopamine, SKF 38393,
and YM 435, on the migration of VSMCs treated with PDGF-BB for 4 hours
is shown in Fig 2. D1-like receptor
agonists inhibited clearly in a concentration-dependent manner
PDGF-BB-induced VSMC migration. Although nonstimulated VSMCs exhibited
little migration activity, D1-like receptor agonists did not inhibit
this basal activity (data not shown). There was a significant
correlation between percent increase in D1-like agonists-mediated cAMP
formation and the percent decrease in migration activity
(r=.89, P<.01).
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Inhibition of PDGF-BB-Induced Cell Proliferation by D1-Like
Agonists
As shown in Fig 3, the number of
cells stimulated by PDGF-BB for 48 hours was higher than those
stimulated by control (vehicle). The D1-like agonists, dopamine, SKF
38393, and YM 435, exhibited dose-dependent inhibition of this
PDGF-BB-induced VSMC proliferation. There was a significant correlation
between the percent increase in D1-like agonist-mediated cAMP formation
and the percent decrease in cell number (r=.87,
P<.01).
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Antiproliferative Action of D1-Like Agonists on Postconfluent
VSMCs
Fig 4 shows the effect of D1-like
receptor agonists on [3H]thymidine
incorporation by postconfluent VSMCs in 0.1% FCS media or when
stimulated by PDGF-BB for 48 hours. The D1-like receptor agonists,
dopamine, SKF 38393 and YM 435, inhibited DNA syntheses of VSMCs in a
dose-dependent manner. The D1-like agonists did not cause the loss of
cells at the confluent stage. After addition of D1-like receptor
agonists, <1% of cells were found to be present in the
supernatant media. Cell viability was also assessed by trypan blue
staining, confirming that >98% of cells were alive. There was a
significant correlation between the percent increase in D1-like
agonist-mediated cAMP formation and the percent decrease in
[3H]thymidine incorporation (r=.94,
P<.01).
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Inhibition of Dopamine Action by a Specific D1-Like
Antagonist Sch 23390
To confirm further that dopamine acts through D1-like receptors,
the specific D1-like antagonist, Sch 23390, was used. Sch
23390 alone had no effect on migration, number, or
[3H]thymidine incorporations by VSMCs. Sch
23390 reversed significantly the dopamine-induced decrease in
migration, number, and [3H]thymidine
incorporation by VSMCs (Table 1).
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Flow Cytometric Analysis of Cell Cycle
VSMCs cultured with 0.1% FCS for 48 hours are in the
G0-G1 stage (100%). A
summary of the cell cycle data is shown in Table 2. PDGF-BB treatment for 24 hours induced
the change of the cell cycle from
G0-G1 (64.8%) to the S
(27.5%) or G2 M (7.6%) stage. D1-like agonists
suppressed significantly this PDGF-BB-induced change.
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Effect of Forskolin and 8-Bromo cAMP on VSMC Migration and
Proliferation Stimulated With PDGF-BB
To elucidate whether the inhibitory effect of dopamine
on the migration and proliferation of VSMCs after stimulation with PDGF
is causally linked to the increase in cellular cAMP, we examined the
effect of an activator of adenylate cyclase,
forskolin, on VSMC migration and proliferation treated with PDGF-BB.
The addition of forskolin reduced PDGF-BB-induced VSMC migration and
proliferation in a dose-dependent manner (Fig 5). Further, a cAMP analogue,
8-bromo-cAMP, also reduced VSMC migration and proliferation treated
with PDGF-BB. Inhibition of PDGF-BB-induced VSMC migration and
proliferation by dopamine could be reproduced by this analogue at
concentrations of 100 µmol/L and 1 mmol/L
(Fig 5).
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Effect of PKA Inhibitor on D1-Like Agonist-Mediated
Suppression of [3H]Thymidine Incorporation
Incubation of VSMCs with the PKA inhibitor, H 89,
reversed significantly D1-like agonist-mediated suppression of
[3H]thymidine incorporation activated
by PDGF-BB (5 ng/mL) for 48 hours (Fig 6). This action was not exerted by H 85,
a negative control of H 89.28
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Effect of Dopamine on PLD Activities and PKC Activities in Normal
and PDGF-BB Stimulated Cells
As shown in Table 3, PLD activities
in cells treated by PDGF-BB for 10 minutes were greater than those in
control cells. This increase was blocked significantly by the
coincubation of the cells with dopamine (10 µmol/L), forskolin
(1 µmol/L), or 8-bromo cAMP (100 µmol/L). The
action of dopamine was also blocked by coincubation of the cells with
Sch 23390 (10 µmol/L), a specific D1-like
antagonist or H 89 (10 µmol/L), a PKA
inhibitor.
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Membrane-bound (particulate) PKC activities in PDGF-BB treated cells were greater than those in control cells with a corresponding decrease in cytosolic PKC activities. When cells were coincubated with dopamine (10 µmol/L), forskolin (1 µmol/L), or 8-bromo-cAMP (100 µmol/L), PDGF-BB-induced PKC activation was significantly inhibited. The action of dopamine was also blocked by the coincubation of the cells with Sch 23390 or H 89.
Inhibition of MAPK Activity by D1-Like Agonists
Incubation of VSMCs with the D1-like agonists significantly
inhibited MAPK activity (Fig 7A).
Treatment of VSMC with D1-like agonists alone, at the concentration
used in these experiments, did not alter the basal MAPK activity (data
not shown). The PKA inhibitor H 89 (10
µmol/L) reversed significantly the effect of D1-like agonists.
Inhibition of PDGF-BB-induced MAPK activation by dopamine, PKA agonists
(forskolin [1 µmol/L] or 8-bromo-cAMP [100
µmol/L]) and a PKC inhibitor (calphostin C
[1 µmol/L]) was also observed by
immunoblotting (Fig 7B).
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Discussion |
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This study has demonstrated for the first time that D1-like agonists inhibit the migration and proliferation of VSMCs stimulated with PDGF in a concentration-dependent manner. Although it has been reported that immunoreactive dopamine is present in human and rat plasma,29,30 the plasma free dopamine concentrations (approximately 0.1 to 1 nmol/L) are much lower than the concentration of dopamine that inhibited VSMC migration significantly in our in vitro study. However, local levels of dopamine in vascular tissues may be much higher than their plasma concentration, because it has been shown that a considerable amount of dopamine is present in the conjugated form (0.1 to 1 µmol/L).31 Further, it has been reported that free dopamine may be formed through a deconjugation reaction when necessary.32 Our results suggest, therefore, that dopamine acting locally in a paracrine manner inhibits the migration of VSMCs after stimulation with factors such as PDGF. The migration of arterial medial VSMCs into the intima is important in intimal thickening not only in atherosclerotic lesions but also in restenosis after angioplasty.1,33 Consequently, it is possible that dopamine antagonizes the development of these vascular lesions as a local antimigratory factor for VSMCs, although we have no direct evidence in vivo at this time.
In the present study, dopamine did not inhibit the basal migration activity of nonstimulated VSMCs. According to the trypan blue exclusion test, dead cells stained with trypan blue were not found 24 hours after treatment with 10 µmol/L dopamine. Based on these observations and the finding that cultured VSMCs actively produce cAMP induced by dopamine, it is unlikely that the inhibitory effect of dopamine on VSMC migration observed in this study was due to its cytotoxicity.
We have obtained some evidence for a causal link between cAMP
production and the inhibition of VSMC migration and
proliferation on treatment with PDGF-BB. For example, cAMP formation by
1 µmol/L forskolin, which is greater than cAMP formation
by 10 µmol/L dopamine, induced greater antimigration
activity. Further, cAMP formation by 0.1 µmol/L
forskolin, which is greater than cAMP formation by 1
µmol/L dopamine, induced a greater decrease in
[3H]thymidine incorporation (Figs 1
, 2, and 4).
Moreover, as shown in Fig 6, the PKA inhibitor H 89
reversed this D1-like agonist-mediated inhibition of DNA synthesis.
These results suggest that dopamine inhibits VSMC migration and
proliferation stimulated with PDGF-BB, probably through a
cAMP-dependent process and PKA activation.
It has been reported that stimulation of phosphatidylinositol turnover,
diacylglycerol formation, and intracellular Ca2+
flux is likely to be required for chemotaxis of human
VSMCs.13 Since PKA activation decreases PLD
(Table 3) and cAMP decreases the intracellular
Ca2+ level in VSMCs,34 it
is possible that decreased diacylglycerol formation due to the
suppression of PLD by PKA activation or the decreased cytosolic
Ca2+ induced by cAMP suppresses the migratory
activities of VSMCs stimulated with PDGF-BB. However, we have not fully
elucidated the exact cellular mechanism by which cAMP inhibits
PDGF-BB-induced migration of VSMCs. Very recently, it was reported that
MAPK activation is involved in PDGF-directed migration in
VSMC.35 Therefore, inhibition of PDGF-directed
migration by D1-like agonists may be explained by PKA-mediated
inhibition of MAPK activity.
cAMP inhibits serum- or PDGF-BB-induced DNA synthesis in VSMCs
affecting, therefore, their proliferation.36,37
Moreover, we have demonstrated recently that adrenomedullin, which
increases cAMP in VSMCs, inhibits the FCS-induced proliferation of
VSMCs.38 It is possible that cAMP elevation by
D1-like agonists inhibits PDGF-induced proliferation of VSMCs. We have
demonstrated for the first time that dopamine-induced PKA activation
inhibits PLD, PKC, and MAPK activation by PDGF (Fig 7). It has been
reported that PKA mediates inhibition of PDGF-BB-induced by MAPK
signaling in VSMCs,39 which is consistent
with the present study. The antagonism by PKA does not appear to be
at the level of the PDGF receptor ß subunit, MAPK kinase, or MAPK but
is likely to occur between the receptor and MAPK kinase. A number of
upstream activators of MAPK kinase have been reported,
including Raf-1.40 It has been reported that
activation of PKC is required to activate
MAPK,41–43 as shown in the present study
(Fig 7B), and hyperphosphorylation of Raf-1 in
VSMCs43 and that activation of Raf-1 is inhibited
by PKA in Rat-1 fibroblast cells.45,46 PKC may
therefore contribute to the suppression of the MAPK activity by PKA.
Because PLD activates PKC through the formation of
diacylglycerol in VSMCs,47 PLD also may
contribute to this suppression. However, since PLD is also reported to
be activated by PKC,48 the possibility
that PLD suppression is the result of PKC suppression cannot be
excluded. Possible sites of action remain to be elucidated. In rat
glioma C6 Bul cells, PLC-, which plays an important role in
mediating the PDGF mitogenic
signal,49 is reported to be
phosphorylated by PKA.50 Further,
the D1-like receptor agonist femoldopam is reported to decrease
PLC-1 activity in renal tissue,51 and
inhibition by cAMP of basal and induced inositol phosphate
production in VSMCs has been also
reported.52 It is possible that D1-like
agonist-mediated PKA activation phosphorylates PLC-1 and
affects PLD and PKC signal transduction, which, in turn, suppress the
MAPK pathway. Moreover, as shown in Table 2, PDGF-BB significantly
changed the cell cycle from
G0-G1 to the S phase.
Activation of MAPK isoforms is required for cell cycle progression and
S phase entry of fibroblast in response to mitogenic
factors.53 The D1-like agonists inhibited this
progression. These results indicate that D1-like agonists
activate PKA, which modifies PLD, PKC and MAPK activity. These
changes, in turn, suppress the cell cycle and cell proliferation.
In the present study, we have used VSMCs from the renal artery. Because approximately two-thirds of all lesions of renovascular hypertension in adults are caused by atherosclerosis,54 this protective effect of D1-like agonists may have a potential therapeutic significance for preventing this disease. However, whether D1-like agonists will prevent the development of atherosclerosis is another critical question raised by these studies. VSMC proliferation is an intermediate event in the atherogenesis, which follows endothelial cell damage and the deposition of fatty streaks in the vessel wall and results in the formation of the organized atherosclerotic plaque.55
In conclusion, activation of D1-like receptors suppresses PDGF-BB mediated VSMC migration and proliferation by activating PKA and suppressing PLD, PKC, and MAPK activity.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Atsumi Ohnishi and Yuka Inoshita for excellent technical assistance. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, Osaka City University Medical Research Foundation, Kimura Memorial Foundation, and Uehara Memorial Foundation. A part of the study was presented at the 50th Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research. Kenichi Yasunari was given a 1997 Young Investigator Award by the Japan Atherosclerosis Society for this study.
Received June 24, 1996; accepted July 22, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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