Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721-0207
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ABSTRACT |
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Vascular wound healing and such pathologies as
atherosclerosis and restenosis are characterized by migration and
proliferation of the smooth muscle cells of the media after denudation
of the intima. To explore possible roles that
2-adrenergic receptors (
2-ARs) might have in these
cellular responses, we characterized the
2-ARs present in
explant-derived cultures of rat aortic smooth muscle (RASM) cells. The
results of immunofluorescence microscopy and reverse transcription
followed by the polymerase chain reaction indicated that all three
2-AR subtypes
(
2A,
2B, and
2C) were initially present.
Mitogen-activated protein kinase activity in the RASM cells was
stimulated fivefold over basal by the
2-selective agonist
dexmedetomidine (Dex) and was blocked by coincubation with the
2-selective antagonist
rauwolscine (RW) or by preincubation of the cells with the
Gi/Go-protein
inhibitor pertussis toxin.
2-AR
activation by Dex did not promote cell proliferation, as measured by
the incorporation of
[3H]thymidine.
However, Dex significantly increased RASM cell migration, and
antagonist blocked this effect. Incubation of RASM cells with Dex also
produced a marked decrease in F-actin labeling, which again was
prevented by coincubation with RW. The evidence clearly reveals the
presence of functional
2-ARs in
RASM cells. The involvement of
2-AR activation with
cytoskeletal changes and cell migration is novel and indicates a
potential role of these receptors in vascular wound healing and
pathogenesis.
atherosclerosis; G protein-coupled receptor; vascular wound healing; chemokinesis
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INTRODUCTION |
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DAMAGE OF THE INTIMAL LINING of arteries, as occurs during atherosclerosis, leads to changes in the phenotype of medial smooth muscle cells and to intimal thickening (6, 27). The phenotypic changes that may be observed include the proliferation and migration of cells, a decrease in myosin filaments, an increase in organelle number, increased deposition of extracellular matrix material, and increased protein synthesis. These phenotypic changes, which are characteristic of the transition from a primarily contractile state to a proliferative one, can also be observed in primary cultures of cells that are derived enzymatically or by tissue explant (6, 9, 11). Cultured vascular smooth muscle cells have therefore been used as a model for a variety of pharmacological and physiological studies directed toward understanding the processes involved in vascular wound healing and atherogenesis.
Three 2-adrenergic receptors
(ARs) have been identified to date based on the pharmacological profile
of ligand preference as well as on a molecular basis (5, 16, 19, 25).
In the rat, these receptors have been named
2A/D,
2B, and
2C-AR. The
2D is a homologue of the human
2A-AR. Traditionally,
2-ARs are known for their role
in presynaptic inhibition, in which their stimulation results in the
activation of K+ channels,
hyperpolarization, and the inhibition of neurotransmitter release (8).
In addition,
2-ARs are located
postsynaptically and are involved with a variety of physiological
effects, particularly in the cardiovascular system. For example, recent
studies with transgenic mice in which genes encoding the individual
2-AR subtypes have either been
deleted (18) or mutated (20), show that the
2A subtype is involved in the
central control of blood pressure, whereas the
2B subtype is involved with
peripheral effects on blood pressure. These results compliment other
studies that have shown the involvement of
2-AR activation in the
contraction of vascular smooth muscle (18, 23, 24) as well as studies
indicating that
2-ARs mediate
endothelial cell release of nitric oxide and, consequently, vascular
smooth muscle cell relaxation (2, 24). Finally, catecholamines mediate
a variety of other effects on the vasculature that are poorly
understood but certainly involve AR activation. For example, exposure
of explant-derived, cultured smooth muscle cells from rat aorta to
catecholamines leads to a dose- and time-dependent increase in cell
number (1). Furthermore, primary risk factors for atherosclerosis
include hypertension, smoking, and stress, which are all associated
with elevated plasma catecholamines and increased activation of ARs. To
further investigate the possible role(s) of
2-ARs in the cellular biology
of vascular tissue, we characterized the
2-AR subtypes and their
functional activity in primary cultures of rat aortic smooth muscle
(RASM) cells. Here we report the presence of all three subtypes and
their involvement with cell migration and cytoskeletal changes.
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MATERIALS AND METHODS |
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Materials. Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin (Pen/Strep), fetal bovine serum (FBS), and 1× trypsin with EDTA (0.05 and 0.02% wt/vol) were purchased from GIBCO BRL (Grand Island, NY). Fluorescein isothiocyanate (FITC)-labeled rabbit anti-chicken and Cy5-labeled rabbit anti-chicken immunoglobin were from Jackson ImmunoResearch Laboratories, (West Grove, PA). Cell culture dishes were from Falcon (Lincoln Park, NJ).
RASM cell isolation and culture. RASM cells were prepared as adapted from Ross (26). Male rats (175-185 g, Harlan Sprague Dawley) were killed by CO2 asphyxiation, and the thoracic aorta was removed along with the aortic arch. Vessels were stripped of adventitia and cut longitudinally, and the endothelium was removed by scraping with scissors. Tissue was cut into pieces (3 mm2) and placed intima side down in six-well culture plates (3-5 pieces/well). DMEM containing Pen/Strep (50 U/ml and 50 µg/ml, respectively) and 10% FBS was added, and the plates were placed in a humidified incubator at 37°C with 5% CO2-95% air. After 7-10 days, the cells began to migrate out from the tissue sections, reaching confluence in ~20 days. Upon confluence, the cells were detached with 0.05% trypsin-0.02% EDTA and seeded on 100-mm plates at a density of 3 × 106 cells/plate (passage 1). Cells were then split 1:3 approximately every 3 days and were identified by the passage number and animal of origin (e.g., passage 2 from animal F would be labeled P2.F).
Antibody production and characterization.
Antibodies were raised in chickens to
glutathione-S-transferase (GST) fusion
proteins containing unique extracellular domains of each of the
2-AR subtypes. Methods for the
preparation of the fusion proteins and antibody purification were as
previously described (13, 30). The primers used for the initial
polymerase chain reactions (PCR) and the regions amplified for each of
the
2-AR subtypes were as
follows:
2A amino terminus
(amino acids 4-26),
2A
sense primer 5'-CCCATGGGATCCCTGCAGCCGGACGCGGGC-3',
2A antisense primer
5'-CAGGGAGAATTCGGTGGCCCGGGCGCCGCC-3';
2B second extracellular loop
(amino acids 150-177),
2B
sense primer 5'-CCGCCCGGATCCTACAAGGGCGACCAGGGC-3',
2B antisense primer
5'-GAAAGAGAATTCGCTGGAGGCCAGGATGTACCA-3';
2C amino terminus (amino acids
3-45),
2C sense
primer 5'-ACCATGGGATCCCCGGCGCTGGCGGCGGCG-3',
2C antisense primer
5'-GCCCGCGAATTCCTGGCCGCGCGGCGGC-3'. PCR products were
ligated in frame behind the flatworm GST gene of pGEX-1N (Pharmacia,
Piscataway, NJ) and were cloned in
Escherichia
coli DH5a. Fusion proteins were
soluble and were purified over glutathione-coupled agarose. Chickens
were inoculated with the purified fusion proteins by Covance (Denver,
PA), and antibodies were purified from eggs as previously described by
immunoaffinity chromatography, using the corresponding fusion proteins
covalently coupled to agarose (AminoLink, Pierce). Anti-GST antibodies
were removed using a GST-coupled subtraction column. Antibodies were
characterized as before, using COS-7 cells transiently transfected with
each of the
2-AR subtypes (13).
It was determined that each antibody was specific for its respective
2-AR subtype and did not cross react with the other subtypes (Fig. 1).
Furthermore, specific immunolabeling could be blocked by preincubation
of each antibody with its corresponding fusion protein but not with GST
or the other fusion proteins.
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Immunofluorescence microscopy. Cells were passaged onto glass coverslips, and after 24-48 h, they were rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS. After an incubation with NaBH4 (1 mg/ml), the cells were permeabilized in 0.05% saponin in PBS containing 10% goat serum and were incubated with the primary antibody for 2-4 h at room temperature. Cells were then washed (3 times, 15 min each) in PBS under gentle agitation. Incubation with the secondary antibody was for 40 min at room temperature in the same buffer in the dark. Cells were washed in PBS (3 times, 10 min each) with gentle agitation and mounted onto glass slides with 10 µl p-phenylenediamine, using Cytoseal (Stephens Scientific, Riverdale, NJ). For F-actin labeling, cells were incubated with Texas red isothiocyanate (TRITC)-labeled phalloidin (Pierce) for 2 h at room temperature, washed (3 times, 5 min each) with PBS, and mounted as before.
For visualization of fluorescent labeling, epifluorescent microscopy was performed using an Olympus BH-2 microscope with a ×60 oil objective (1.4 numerical aperture), with a 100-W mercury bulb, through a dichroic FITC filter cube. Alternatively, samples were visualized using a Leica-TCS confocal microscope (Deerfield, IL). All conditions were performed in duplicate or triplicate and replicated at least three times. A minimum of five fields was randomly sampled per coverslip. All images chosen for figures were taken under identical conditions (exposure time, voltage intensity, pinhole size, etc.) and were representative of each condition.Reverse transcription-PCR.
Total RNA was isolated from confluent plates (10 cm) of cultured RASM
cells using TriZol (GIBCO BRL) according to the manufacturer's instructions. The final pellets were resuspended in 100 µl of water,
and the concentration and relative purity of the RNA was determined by
spectroscopy (260/280 nm OD). Reverse transcription was done using
SuperScript II (GIBCO BRL) according to the manufacturer's specifications, using 200 ng of random primer mix (hexamers), 500 ng
oligo(dT) (12-18-mer), and 3 µg of total RNA per reaction. To
rule out amplification from genomic DNA, reverse transcription (RT)
reactions were done in parallel with a preincubation with either
ribonuclease (RNase) or RNase inhibitor for 1 h at 37°C. PCR
reactions were performed using 2 µl of the RT reaction as template.
The reactions consisted of an initial hot start at 96°C and 40 cycles of 96°C for 1 min, 55°C for 2 min, and 72°C for 3 min followed by a final incubation at 72°C for 10 min. Products were separated on 1.4% agarose gels and stained with ethidium bromide.
2-AR-specific primers were
chosen based on their uniqueness (determined using the Basic Local
Alignment Search Tool at the National Center for Biotechnology
Information) and analysis via the computer program Oligo (National
Biosciences, Plymouth, MN). Primers were synthesized by NBI (Plymouth,
MN) as follows:
2A sense primer
5'-CTCCCTGCAGCCGGATGCC-3',
2A antisense primer
5'-CCAGCGCCCTTCTTCTCTATG-3',
2A expected product size 528 base pairs (bp);
2B sense
primer 5'-CGCCATCGCGTCGGCCATC-3',
2B antisense primer
5'-GAGACCTCTGCAGTGGCTG-3',
2B expected product size 583 bp;
2C sense primer
5'-CTGGCGGCGGCGGCGGCTGA-3',
2C antisense primer
5'-TCGGGCCGGCGGTAGAAAG-3',
2C expected product size 582 bp. PCR products of the expected size were also isolated (Genclean;
Bio101) and digested with subtype-selective restriction enzymes to
further verify their identity
(BglII or
HincII, data not
shown).
MAP kinase assay.
Mitogen-activated protein (MAP) kinase assays were performed as
described by Burkey and Regan (4) with the exception that immunoprecipitation was performed using a monoclonal anti-ERK 1 (p44)
MAP kinase antibody or a combination of both polyclonal anti-ERK 1 and
ERK 2 MAP kinase antibodies (GIBCO BRL and Santa Cruz Biotechnology,
respectively). Cells were grown to confluence and then cultured
overnight in serum-free DMEM containing insulin, transferrin, and
selenium (ITS, GIBCO BRL). Cells were then incubated in the same media
containing drugs for 5-10 min at 37°C. Alternatively, some
cells were incubated with drug after a 4-h preincubation with 150 ng/ml
pertussis toxin (PTx). Cells were washed with PBS (4°C) and scraped
into lysis buffer (50 mM -glycerophosphate, 1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 2 mM MgCl2, 100 µM
NaVO3, 0.5% Triton X-100, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µM pepstatin,
and 20 µM leupeptin at pH 7.2 and 4°C). Cell debris was
sedimented by centrifugation at 12,000 g for 15 min at 4°C, and 100-µg
aliquots of the lysate were used for the MAP kinase assay. Anti-MAP
kinase antibodies (2 µg ERK 1/ERK 2, Santa Cruz Biotechnology) were
added to the lysate and incubated overnight with rotation at 4°C.
Protein G agarose (20 µl; Calbiochem, La Jolla, CA) was then added,
incubated for 2 h at 4°C and spun at 12,000 g. The pellet was washed two times in
lysis buffer and one time in kinase buffer [lysis buffer containing 12.5 µg/ml MgCl2 and
25 µg/ml protein kinase A inhibitor (Sigma)]. The washed pellet
was resuspended in kinase buffer with 1 mg/ml myelin basic protein
(GIBCO BRL) and 5 µM
[
-32P]ATP (NEN,
Boston, MA) in a final volume of 40 µl. Reactions proceeded for 15 min at 37°C and were terminated with the addition of 10 µl of
25% (wt/vol) trichloroacetic acid. Aliquots (20 µl) were filtered
onto Whatman GF/B filters through a Brandel cell harvester
(Gaithersburg, MD) and washed with 75 mM phosphoric acid (10 ml each).
Radioactivity was determined by liquid scintillation counting.
Multiple induction was calculated by dividing the sample values by the unstimulated control values (after subtracting
background, e.g., absence of lysate).
Proliferation assay. [3H]thymidine incorporation assays were performed as a marker of cell proliferation. Initially, primary culture aortic smooth muscle cells were plated at a density of 150 cells/mm2 in a 96-well plate (Corning, Corning, NY) and grown >14 h in DMEM(+), 95% air-5% CO2. The next day, cells were washed with PBS (37°C) and incubated for 24-48 h in DMEM without FBS (length of time was a function of the assay, and a number of times were examined; generally, a 24-h serum starvation period was applied). Cells were then exposed to DMEM alone or containing 2% FBS (positive control), agonist [1 µM dexmedetomidine (Dex)], or agonist plus antagonist [1 µM Dex + 1 µM rauwolscine (RW)] and 0.1 µCi [3H]thymidine (Amersham, Arlington Heights, IL). Next, cells were incubated overnight, lysed in H2O, and harvested onto Whatman GF/B filters, using a Brandel cell harvester. The filters were then washed repeatedly (~10 ml/well) with ice-cold H2O, dried in an oven (55°C), and counted in Safety-Solve (Research Products International). A number of parameters were examined, including incubating with agonist in the presence of ITS, 0.1% FBS, 2% FBS, and lower cell densities (1 × 104 and 0.5 × 104 cells/well).
In addition, the assays were performed on cells seeded at 300% confluence (a density of 2,000 cells/mm2) in 12-well plates containing inserts designed to yield a 3 × 20-mm rectangular cell reservoir to mimic the conditions of cell migration assays. Cells were allowed to adhere for 2.5 h in DMEM containing 10% FBS and Pen/Strep, after which time the inserts were removed and the cell strips were washed and incubated overnight in DMEM containing ITS and Pen/Strep. Cells were next incubated in DMEM alone, containing 2% FBS (positive control) or with agonist (1 µM Dex), and 0.1 µCi [3H]thymidine was added to each for 48 h. Cells were then lysed in H2O and harvested onto Whatman GF/B filters, using a Brandel cell harvester. The filters were washed repeatedly (~10 ml/well) with ice-cold H2O, dried in an oven (55°C), and counted in Safety-Solve (Research Products International).Cell migration assay. The effects of drugs on the migration of RASM cells was determined using the linear migration assay described by Hoying and Williams (12; see also Ref. 28). Cells were seeded at 300% confluence (~2,000 cells/mm2) in 12-well plates containing inserts designed to yield a 3 × 20-mm rectangular cell reservoir from which cell migration could occur. In the present study, inserts were made of Teflon. We found that inserts made out of Delrin, as used previously, were toxic to RASM cells. Cells were allowed to adhere for 2.5 h in DMEM containing 10% FBS and Pen/Strep, after which time the inserts were removed and the cell strips were washed and incubated overnight in DMEM containing ITS and Pen/Strep. The cells were then incubated with or without drugs for 48 h. The media was changed every 24 h, and drug was readministered to the appropriate wells. The cells were then fixed for 30 min in PBS containing 4% paraformaldehyde, permeabilized for 1 min in PBS containing 0.2% Triton X-100 at room temperature, and rinsed in PBS. Nuclei were stained by incubating the cells for 5 min in PBS containing 5 µg/ml bisbenzimide (Molecular Probes, Eugene, OR). The migration profile of each well was determined by counting fluorescent nuclei using Image 1 analysis software (Universal Imaging, West Chester, PA) interfaced to a microscope with an automated stage (Nikon SCU-1). Random motility coefficients were calculated by computer for both sides of the rectangular cell strip, according to Hoying and Williams (12; see also Ref. 28).
Statistical analysis. All experiments were done on at least three occasions. Independent experiments were defined as those performed on different days, with cells derived from different animals or from different passage numbers. Analysis of variance (ANOVA) was used to evaluate statistical differences between means. Bonferroni tests were used to identify between-group differences when the ANOVA was significant (P < 0.05).
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RESULTS |
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Characterization of the antibodies and identification of subtypes
present in RASM cells.
Figure 1 illustrates the relative specificity and lack of
cross-reactivity of the individual
2-AR-specific antibodies. COS-7 cells were transiently transfected with the
2A-ARs
(A-C),
2B-ARs (D-F),
or
2C-ARs
(G-I).
Immunofluorescent microscopy was then done using the individual
antibodies. Figure 1, A,
D, and
G show fluorescence of cells after
incubation with
anti-
2A-NH2
polyclonal antibodies. Figure 1, B,
E, and
H show fluorescence of cells after incubation with anti-
2B-2ECL
polyclonal antibodies. Figure 1, C,
F, and
I show fluorescence of cells after
incubation with
anti-
2C-NH2 polyclonal antibodies. The antibodies appear to be specific for their
individual receptor subtype and do not present any immunoreactivity to
cells transfected with a different receptor subtype.
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2-AR-stimulated MAP
kinase activity.
MAP kinase activity was measured in primary cultures of RASM cells
after stimulation with the
2-AR-selective agonist Dex. Figure 4 shows the multiple stimulation of
MAP kinase activity, as determined by the incorporation of
32P into myelin basic protein,
after immunoprecipitation of enzyme with antibodies to both ERK 1 and
ERK 2. Incubation of RASM cells with Dex resulted in a statistically
significant stimulation of MAP kinase activity at a 100 nM
concentration that was blocked by coincubation with the
2-selective antagonist RW (100 nM). The maximal stimulation, approximately fivefold at
10
6 M Dex, was comparable
to the stimulation obtained with 100 nM phorbol myristate acetate. In
addition, the Dex-stimulated increase in MAP kinase activity was
blocked after pretreatment of the cells with a
Gi/Go-selective
inhibitor PTx.
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[3H]thymidine incorporation
assay.
Because the stimulation of MAP kinase activity is frequently associated
with cellular proliferation, we examined the effects of
2-AR stimulation on the
incorporation of
[3H]thymidine.
Incubation of cultured RASM cells for 24 or 48 h with 1 µM Dex did
not stimulate
[3H]thymidine
incorporation over basal levels (Fig. 5).
This was repeated under conditions (in strip) that were identical to
the cell migration assays described below, and there was no stimulation of [3H]thymidine
incorporation by Dex. Cells incubated with 2% FBS for 24 or 48 h did,
however, show a significant stimulation of [3H]thymidine
incorporation.
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2-AR stimulation of RASM
cell migration.
Using automated video microscopy in a linear migration assay (12, 28),
we calculated random motility coefficients for either untreated RASM
cells, RASM cells that had been incubated for 48 h with 1 µM Dex or
with 1 µM Dex plus 1 µM RW. In Fig. 6,
the mean results of four independent experiments show that incubation
with Dex produced an 11-fold increase in the migration of the RASM
cells compared with the untreated controls. Coincubation of the RASM
cells with Dex and RW blocked this increase and indicated that the
effects of Dex on RASM cell migration were specific and mediated by the
activation of
2-ARs.
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Effects of 2-AR
stimulation on F-actin polymerization.
To investigate the possibility that the increase in cell motility might
be associated with cytoskeletal changes, we used fluorescence microscopy to examine the extent of F-actin polymerization in RASM
cells that had been treated with Dex. Figure
7 shows the labeling of F-actin by
TRITC-phalloidin in untreated RASM cells (A-C)
and in cells that had been incubated either with 1 µM Dex (D-F)
or with 1 µM Dex plus 1 µM RW
(G-I)
for 6 h. Treatment with agonist
(D-F)
caused a marked decrease in the intensity of F-actin labeling compared
with control
(A-C).
This effect can be blocked by coincubation with antagonist
(G-I).
Nearly identical results were obtained with RASM cells prepared from
three different animals and from cells plated at both high and low
density. As with the effects of Dex on cell migration, the effects of
Dex on F-actin polymerization were consistent with the activation of
2-ARs.
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DISCUSSION |
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2-ARs are present in cultured
RASM cells, as determined by immunofluorescence microscopy, RT-PCR, and
several measures of functional activation. The results of both the
immunofluorescence microscopy and RT-PCR indicated that all three
2-AR subtypes were expressed in
RASM cells, although with increasing passage number, expression was
lost. Functionally, the activation of RASM cell
2-ARs stimulated MAP kinase
activity in a dose-dependent and PTx-sensitive manner, increased cell
migration, and decreased the intensity of F-actin labeling. These
results are consistent with a possible role of
2-ARs in vascular wound healing
as well as in pathologies such as atherosclerosis and restenosis.
It was interesting to note the difference in immunofluorescent
reactivity among the receptor subtypes. Although all three receptors
localize diffusely along the plasma membrane, the
2A-AR appears to also exhibit a
nuclear staining pattern and the
2C-AR exhibits a prominent
perinuclear staining pattern. The identification of differential
receptor subcellular localization is not novel and may be explained a
number of ways. For example, the receptors may exhibit different
degrees of targeting, turnover, and sequestration. Daunt et al. (7) and
Keefer et al. (15), with
2-ARs,
and Tarasova et al. (29) with CCK receptors have shown that G
protein-coupled receptors are able to both differentially localize to
specific plasma membrane domains (e.g., apical vs. basolateral) as well as subcellular domains. In addition, differential subcellular localization may also be a function of cell type (7).
Previous studies by Ping and Faber (23) using RT-PCR identified only
the presence of the 2A-AR in
both vascular tissue and cultured vascular smooth muscle cells from
rat. Perhaps the differences in the results of the present study derive
from the use of different methods of cell isolation (i.e., enzymatic
dispersal vs. tissue explant) or differences in culture conditions,
passage number, or primers and RT-PCR conditions. In addition, studies with transgenic mice lacking the
2B-AR suggest that this subtype is normally expressed in vascular smooth muscle and that its activation produces vasoconstriction (18). The knockout studies are limited, however, in that they do not look at isolated arteries or veins and
cannot rule out compensation by a nontargeted subtype. The present
study is the first to indicate that the
2C-AR is also expressed in
vascular smooth muscle. Interestingly, studies with transgenic mice
lacking the
2C subtype showed
no changes in hemodynamic parameters, which raises the question of
their function in vascular tissues.
Generally, the activation of
2-ARs is known to have effects
on a number of cellular pathways. For example, it has been shown that
the
2-ARs mediate release of
Ca2+ from intracellular stores
(17), stimulate the secretion of prostaglandins (most likely an
2B-AR response) (21), promote preadipocyte proliferation (3), and stimulate MAP kinase activation in
stably transfected CHO cells (10). In the present studies,
2-AR activation was also found
to stimulate MAP kinase activity; however, this did not appear to be
associated with any proliferative effects on the RASM cells. Besides
the association of MAP kinase with proliferation, the activation of
this pathway has also been associated with effects on vascular
contraction, cardiac hypertrophy, and migration. To examine if there
might be an effect of
2-AR activation on cell migration, we performed an assay that determined the
linear dispersion of a cell population from a defined source and
provided a stochastic measure of cell migration, known as the random
motility coefficient (12, 28). We found that
2-AR activation stimulated cell
migration and that this was blocked in the presence of the
2-AR antagonist RW.
Previously, in wound-healing studies of the corneal endothelium, it has
been found that the migration and spreading of cells is associated with
a decrease in F-actin in the cortical region of the cells (14, 22). We
examined changes in RASM cell F-actin and found that
2-AR activation produced a
dramatic decrease in TRITC-phalloidin labeled F-actin throughout the
cell.
These results illustrate that ARs mediate functions beyond
vasoconstriction. The fact that
2-AR stimulation had an effect on cell migration implies that they may play a role in vascular wound
healing and may potentially contribute to pathologies such as
atherogenesis or hypertension. In this sense, a model may be developed
in which, under normal conditions,
2A-ARs in the endothelium facilitate the release of nitric oxide to produce a net hypotensive response (in conjunction with a centrally mediated decrease in sympathetic tone), whereas the
2-ARs in the medial smooth
muscle compete by modulating vasoconstriction. Additionally, after a lesion to the intima, catecholamines from the blood would have greater
access to the medial smooth muscle
2-ARs, which could cause
cellular migration and thereby contribute to the wound-healing process
or, if uncontrolled, contribute to atherogenesis. An important question
that still needs to be addressed is whether these effects on cell
migration and cytoskeletal F-actin can be attributed to the activation
of a specific
2-AR subtype.
This is particularly interesting in terms of the apparent differences
in the desensitization and downregulation of the receptor subtypes. In
studies by Kobilka and colleagues (7, 31) and in our preliminary
studies with RASM cells, it appears that there are differences between
the subtypes with respect to the ability of agonists to cause receptor internalization, downregulation, and/or desensitization.
Because the
2-agonist-stimulated effects on
cell migration and on F-actin typically have long time courses, it
might be significant if these effects were mediated by a receptor that
does not desensitize.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ronald Heimark for help and advice and Dr. Stuart Williams and Dennis Salzmann for use of equipment and generous assistance with the migration assays. In addition, we thank Drs. Henry I. Yamamura and William Roeske for generous guidance and advice.
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FOOTNOTES |
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This work was supported by grants to J. W. Regan from the National Eye Institute (EY-D9355 and EY-11291) and from the Robert Wood Johnson Pharmaceutical Research Institute, by a Core Center Grant to the Southwest Environmental Health Sciences Center (ES-06694), and by a fellowship awarded to J. G. Richman by the National Institute on Drug Abuse (DA-05613).
Present address of J. G. Richman and J. W. Regan: College of Pharmacy, University of Arizona, Tucson, AZ 85721-0207.
Address reprint requests to J. G. Richman.
Received 17 July 1997; accepted in final form 21 November 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Blaes, N.,
and
J. P. Boissel.
Growth-stimulating effect of catecholamines on rat aortic smooth muscle cells in culture.
J. Cell. Physiol.
116:
167-172,
1983[Medline].
2.
Bockman, C. S.,
I. Gonzalez-Cabrera,
and
P. W. Abel.
-2 Adrenoceptor subtype causing nitric oxide-mediated vascular relaxation in rats.
J. Pharmacol. Exp. Ther.
278:
1235-1243,
1996[Abstract].
3.
Bouloumié, A.,
V. Planat,
J. Devedijian,
P. Valet,
J. Saulnier-Blanche,
M. Record,
and
M. Lafontan.
2-Adrenergic stimulation promotes preadipocyte proliferation.
J. Biol. Chem.
269:
30254-30259,
1994
4.
Burkey, T. H.,
and
J. W. Regan.
Activation of mitogen-activated protein kinase by the human prostaglandin EP3A receptor.
Biochem. Biophys. Res. Commun.
211:
152-158,
1995[Medline].
5.
Bylund, D.,
D. Eikenberg,
J. Hieble,
S. Langer,
R. J. Lefkowitz,
K. Minnemann,
P. Molinoff,
R. Ruffolo,
and
U. Trendelenburg, IV.
International union of pharmacology nomenclature of adrenoceptors.
Pharmacol. Rev.
46:
121-136,
1994[Medline].
6.
Campbell, G. R.,
J. H. Campbell,
J. A. Manderson,
S. Horrigan,
and
R. E. Rennick.
Arterial smooth muscle. A multifunctional mesenchymal cell.
Arch. Pathol. Lab. Med.
112:
977-986,
1988[Medline].
7.
Daunt, D. A.,
C. Hurt,
L. Hein,
J. Kallio,
F. Feng,
and
B. K. Kobilka.
Subtype-specific intracellular trafficking of 2-adrenergic receptors.
Mol. Pharmacol.
51:
711-720,
1997
8.
Docherty, J. R.,
and
G. Brady.
Prejunctional actions of K+ channel blockers in rat vas deferens.
Eur. J. Pharmacol.
287:
287-293,
1995[Medline].
9.
Eguchi, S.,
Y. Hirata,
T. Imai,
and
K. Kanno.
Phenotypic change of endothelin receptor subtype in cultured rat vascular smooth muscle cells.
Endocrinology
134:
222-228,
1994[Abstract].
10.
Flordellis, C. S.,
M. Berguerand,
P. Gouache,
V. Barbu,
H. Gavras,
D. E. Handy,
G. Bereziat,
and
J. Masliah.
2 Adrenergic receptor subtypes expressed in chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21Ras independent pathway.
J. Biol. Chem.
270:
3491-3494,
1995
11.
Hedin, U.,
B. Bottger,
E. Forsberg,
S. Johansson,
and
J. Thyberg.
Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells.
J. Cell Biol.
107:
307-319,
1988[Abstract].
12.
Hoying, J. B.,
and
S. K. Williams.
Measurement of endothelial cell migration using an improved linear migration assay.
Microcirculation
3:
167-174,
1996[Medline].
13.
Huang, Y.,
D. W. Gil,
P. Vanscheeuwijck,
D. W. Stamer,
and
J. W. Regan.
Localization of 2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies.
Invest. Ophthalmol. Visual Sci.
36:
2729-2739,
1995.[Abstract]
14.
Ichijima, H.,
W. M. Petroll,
P. A. Barry,
P. M. Andrews,
M. Dai,
H. D. Cavanagh,
and
J. V. Jester.
Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.
Invest. Ophthalmol. Vis. Sci.
34:
2803-2812,
1993[Abstract].
15.
Keefer, J. R.,
M. E. Kennedy,
and
L. E. Limbird.
Unique structural features important for stabilization versus polarization of the 2A-adrenergic receptor on the basolateral membrane of Madin-Darby canine kidney cells.
J. Biol. Chem.
269:
16425-16433,
1994
16.
Kobilka, B. K.,
T. S. Kobilka,
T. L. Yang-Feng,
U. Franke,
H. Matsui,
M. G. Caron,
R. J. Lefkowitz,
and
J. W. Regan.
Cloning, sequencing, and expression of the gene coding for the human platelet 2-adrenergic receptor.
Science
238:
650-656,
1987[Medline].
17.
Lepretre, N.,
J. Mironneau,
and
J. L. Morel.
Both 1a- and
2a-adrenoceptor subtypes stimulate voltage-operated L-type calcium channels in rat portal vein myocytes.
J. Biol. Chem.
269:
29546-29552,
1994
18.
Link, R. E.,
K. Desai,
L. Hein,
M. E. Stevens,
A. Chruscinski,
D. Bernstein,
G. S. Barsh,
and
B. K. Kobilka.
Cardiovascular regulation in mice lacking 2-adrenergic receptor subtypes b and c.
Science
273:
803-805,
1996[Abstract].
19.
Lomasney, J.,
W. Lorenz,
L. Allen,
K. King,
J. W. Regan,
T. L. Yang-Feng,
M. G. Caron,
and
R. J. Lefkowitz.
Expansion of the 2-adrenergic receptor family: cloning and characterization of a human
2-adrenergic receptor subtype, the gene for which is located on chromosome 2.
Proc. Natl. Acad. Sci. USA
87:
5094-5098,
1990[Abstract].
20.
MacMillan, L. B.,
L. Hein,
M. S. Smith,
M. T. Piascik,
and
L. E. Limbird.
Central hypotensive effects of the 2a-adrenergic receptor subtype.
Science
273:
801-803,
1996[Abstract].
21.
Nebigil, C.,
and
K. Malik.
Prostaglandin synthesis elicited by adrenergic stimuli in rabbit aorta is mediated via -1 and
-2 adrenergic receptors.
J. Pharmacol. Exp. Ther.
254:
633-640,
1990[Abstract].
22.
Petroll, W. M.,
J. V. Jester,
P. A. Barry-Lane,
and
H. D. Cavanagh.
Effects of basic FGF and TGF beta 1 on F-actin and ZO-1 organization during cat endothelial wound healing.
Cornea
15:
525-532,
1996[Medline].
23.
Ping, P.,
and
J. E. Faber.
Characterization of -adrenoceptor gene expression in arterial and venous smooth muscle.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H1501-H1509,
1993
24.
Rajanayagam, M. A.,
and
M. J. Rand.
Differential activation of adrenoceptor subtypes by noradrenaline applied from the intimal or adventitial surfaces of rat isolated tail artery.
Clin. Exp. Pharmacol. Physiol.
20:
793-799,
1993[Medline].
25.
Regan, J. W.,
T. S. Kobilka,
T. L. Yang-Feng,
M. G. Caron,
R. J. Lefkowitz,
and
B. K. Kobilka.
Cloning and expression of a human kidney cDNA for an 2-adrenergic receptor subtype.
Proc. Natl. Acad. Sci. USA
85:
6301-6305,
1988[Abstract].
26.
Ross, R.
The smooth muscle cell.
J. Cell Biol.
50:
172-186,
1971
27.
Schwartz, S. M.,
R. L. Heimark,
and
M. W. Majesky.
Developmental mechanisms underlying pathology of arteries.
Physiol. Rev.
70:
1177-1209,
1990
28.
Stokes, C. L.,
M. A. Rupnick,
S. K. Williams,
and
D. A. Lauffenburger.
Chemotaxis of human microvessel endothelial cells in response to acidic fibroblast growth factor.
Lab. Invest.
63:
657-668,
1990[Medline].
29.
Tarasova, N. I.,
R. H. Stauber,
J. K. Choi,
E. A. Hudson,
G. Czerwinski,
J. L. Miller,
G. N. Pavlakis,
C. J. Michejda,
and
S. A. Wank.
Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein.
J. Biol. Chem.
272:
14817-14824,
1997
30.
Vanscheeuwijck, P.,
Y. Huang,
D. Schullery,
and
J. W. Regan.
Antibodies to a human 2-C10 adrenergic receptor fusion protein confirm the cytoplasmic orientation of the V-VI loop.
Biochem. Biophys. Res. Commun.
190:
340-346,
1993[Medline].
31.
Von Zastrow, M.,
D. A. Daunt,
G. Barsh,
and
B. K. Kobilka.
Subtype-specific differences in the intracellular sorting of G protein-coupled receptors.
J. Biol. Chem.
268:
763-766,
1992