alpha 2-Adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells

Jeremy G. Richman and John W. Regan

Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721-0207

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 2-adrenergic receptors (alpha 2-ARs) might have in these cellular responses, we characterized the alpha 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 alpha 2-AR subtypes (alpha 2A, alpha 2B, and alpha 2C) were initially present. Mitogen-activated protein kinase activity in the RASM cells was stimulated fivefold over basal by the alpha 2-selective agonist dexmedetomidine (Dex) and was blocked by coincubation with the alpha 2-selective antagonist rauwolscine (RW) or by preincubation of the cells with the Gi/Go-protein inhibitor pertussis toxin. alpha 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 alpha 2-ARs in RASM cells. The involvement of alpha 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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha 2A/D, alpha 2B, and alpha 2C-AR. The alpha 2D is a homologue of the human alpha 2A-AR. Traditionally, alpha 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, alpha 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 alpha 2-AR subtypes have either been deleted (18) or mutated (20), show that the alpha 2A subtype is involved in the central control of blood pressure, whereas the alpha 2B subtype is involved with peripheral effects on blood pressure. These results compliment other studies that have shown the involvement of alpha 2-AR activation in the contraction of vascular smooth muscle (18, 23, 24) as well as studies indicating that alpha 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 alpha 2-ARs in the cellular biology of vascular tissue, we characterized the alpha 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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha 2-AR subtypes were as follows: alpha 2A amino terminus (amino acids 4-26), alpha 2A sense primer 5'-CCCATGGGATCCCTGCAGCCGGACGCGGGC-3', alpha 2A antisense primer 5'-CAGGGAGAATTCGGTGGCCCGGGCGCCGCC-3'; alpha 2B second extracellular loop (amino acids 150-177), alpha 2B sense primer 5'-CCGCCCGGATCCTACAAGGGCGACCAGGGC-3', alpha 2B antisense primer 5'-GAAAGAGAATTCGCTGGAGGCCAGGATGTACCA-3'; alpha 2C amino terminus (amino acids 3-45), alpha 2C sense primer 5'-ACCATGGGATCCCCGGCGCTGGCGGCGGCG-3', alpha 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 alpha 2-AR subtypes (13). It was determined that each antibody was specific for its respective alpha 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.


View larger version (104K):
[in this window]
[in a new window]
 
Fig. 1.   Characterization of alpha 2-adrenergic receptor (AR) subtype-selective polyclonal antibodies. COS-7 cells were transiently transfected with the alpha 2A-ARs (A-C), alpha 2B-ARs (D-F), or alpha 2C-ARs (G-I). Immunofluorescent microscopy was then done as described in MATERIALS AND METHODS. A, D, and G show fluorescence of cells after incubation with anti-alpha 2A-NH2 polyclonal antibodies. B, E, and H show fluorescence of cells after incubation with anti-alpha 2B-2ECL polyclonal antibodies. C, F, and I show fluorescence of cells after incubation with anti-alpha 2C-NH2 polyclonal antibodies. Secondary antibody was a fluorescein isothiocyanate-rabbit anti-chicken immunoglobulin. Images are representative of at least 3 coverslips and 5 random fields/coverslip. Experiments have been repeated at least 4 times with similar results.

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. alpha 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: alpha 2A sense primer 5'-CTCCCTGCAGCCGGATGCC-3', alpha 2A antisense primer 5'-CCAGCGCCCTTCTTCTCTATG-3', alpha 2A expected product size 528 base pairs (bp); alpha 2B sense primer 5'-CGCCATCGCGTCGGCCATC-3', alpha 2B antisense primer 5'-GAGACCTCTGCAGTGGCTG-3', alpha 2B expected product size 583 bp; alpha 2C sense primer 5'-CTGGCGGCGGCGGCGGCTGA-3', alpha 2C antisense primer 5'-TCGGGCCGGCGGTAGAAAG-3', alpha 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 beta -glycerophosphate, 1 mM ethylene glycol-bis(beta -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 [gamma -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).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 2-AR-specific antibodies. COS-7 cells were transiently transfected with the alpha 2A-ARs (A-C), alpha 2B-ARs (D-F), or alpha 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-alpha 2A-NH2 polyclonal antibodies. Figure 1, B, E, and H show fluorescence of cells after incubation with anti-alpha 2B-2ECL polyclonal antibodies. Figure 1, C, F, and I show fluorescence of cells after incubation with anti-alpha 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.

Primary cultures of RASM cells were prepared from explants and were initially characterized with respect to their morphology and the presence of smooth muscle-specific alpha -actin. At confluence, cell cultures displayed the typical "hill and valley" growth characteristic of vascular smooth muscle cells. Immunofluorescent labeling using a monoclonal anti-smooth muscle alpha -actin primary antibody showed positive labeling, as expected of these cells (data not shown). Immunofluorescence microscopy was then used to determine the expression of the alpha 2-ARs in RASM cells, using antibodies that were selective for either the alpha 2A-, alpha 2B-, or alpha 2C-AR subtypes. As shown in Fig. 2A, positive labeling of RASM cells was obtained, with antibodies directed against the alpha 2A-AR subtype that was blocked when the antibodies were preincubated with the fusion protein used to generate these antibodies (D). Similarly, antibodies selective for the alpha 2B- (B) and alpha 2C- (C) AR subtypes showed specific labeling that was blocked by preincubation with the corresponding fusion proteins (E and F, respectively). Thus it appears that all three alpha 2-AR subtypes are expressed in RASM cells. It should be noted, however, that with increasing passage number, alpha 2-AR immunoreactivity decreased, such that by passages 10-12 it was undetectable. For this reason, all experiments were performed with RASM cells of passages 2-6. In addition, preliminary experiments revealed that, in early passages, neither the presence or absence of serum nor the cell density affected the expression of the receptors.


View larger version (100K):
[in this window]
[in a new window]
 
Fig. 2.   Immunofluorescent labeling of cultured rat aortic smooth muscle cells (RASM) with antibodies selective for the alpha 2A-AR (A and D), alpha 2B-AR (B and E), and alpha 2C-AR (C and F) subtypes. RASM cells were cultured and prepared as described in MATERIALS AND METHODS. A, B, and C show fluorescence after incubation of RASM cells with 1-8 ng of primary antibody followed by a secondary antibody coupled with Cy5 (1 ng). D, E, and F show background fluorescence after incubation of RASM cells with primary antibody that had been preincubated with a 10-fold excess of its corresponding fusion protein (10-80 ng) followed by the secondary Cy5 antibody (1 ng). Images are representative of at least 3 coverslips and 5 random fields/coverslip (n >=  4). Scale bar in A = 20 µm.

To further verify the presence of the three alpha 2-AR subtypes in RASM cells, subtype-selective primers were designed and RT-PCR was performed using RNA isolated from these cells. The alpha 2-ARs are very GC rich, and GC clamping is often a problem. Consequently, we used 40 PCR cycles to amplify the potential alpha 2-AR cDNA present. As shown in Fig. 3, RT-PCR products of the predicted size were obtained for all three subtypes. These products were absent when reactions were done with RNA that had been treated with RNase, indicating that they arose from RNA and not from contaminating genomic DNA. Linearized plasmid DNA was also incubated with RNase to verify that there was no deoxyribonuclease present.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 3.   Reverse transcriptase-polymerase chain reaction (RT-PCR) of RASM cell RNA. Ethidium bromide-stained gels with the products obtained from RT-PCR of RASM cell RNA using primers selective for the alpha 2A-AR (lanes 1 and 2), alpha 2B-AR (lanes 3 and 4), and alpha 2C-AR (lanes 5 and 6) subtypes. For each subtype, reactions were done with RNA that was either treated with RNase inhibitor (lanes 2, 4, and 6) or treated with RNase (lanes 1, 3, and 5). Arrows at left indicate sizes of standards. Expected product sizes were 528 base pairs (bp), 582 bp, and 583 bp for the alpha 2A-, alpha 2B-, and alpha 2C-AR, respectively. M, molecular size marker (HaeIII phi x174 and HindII lambda ). RNA isolation and RT-PCR have been repeated at least 2 times for each subtype.

alpha 2-AR-stimulated MAP kinase activity. MAP kinase activity was measured in primary cultures of RASM cells after stimulation with the alpha 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 alpha 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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   alpha 2-AR stimulation of mitogen-activated protein (MAP) kinase activity. ERK 1/2 kinase activity in RASM cells was measured as described in MATERIALS AND METHODS under basal (unstimulated) conditions and after a 5-min incubation with either dexmedetomidine (Dex) at the indicated concentrations or with 100 nM Dex plus 100 nM rauwolscine (Dex + RW), 100 nM Dex after preincubation with 150 ng/ml pertussis toxin (Dex + PTX). Cells were incubated for 10 min with 100 nM phorbol myristate acetate (PMA) after preincubation with 150 ng/ml PTX (PMA + PTX) as a positive control. Values are mean multiple inductions over basal ± SE (n = 4). * P < 0.05 vs. Dex + RW treatment group.

[3H]thymidine incorporation assay. Because the stimulation of MAP kinase activity is frequently associated with cellular proliferation, we examined the effects of alpha 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.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of alpha 2-AR stimulation on [3H]thymidine incorporation. RASM cells were seeded in 96-well plates under subconfluent conditions (150 cells/mm2) in DMEM with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Pen/Strep). After 2.5 h, cells were rinsed with phosphate-buffered saline and incubated in serum-free DMEM with insulin, transferrin, selenium, and Pen/Strep overnight. Cells were then incubated for 24 h alone (basal) or in the presence of increasing concentrations of Dex or 2% FBS. Cells were then lysed in H2O and filtered onto Whatman GF/B filters using a Brandel cell harvester. The last 2 bars represent multiple-proliferation of cells seeded under same conditions as used for cell migration assays (in strip) and treated for 48 h with 1 µM Dex or 2% FBS. Values are mean multiple [3H]thymidine incorporations over basal ± SE (n = 6 for assays in 96-well plates and n = 3 for assays done from cell strips). * P < 0.05 vs. cells treated with 1 µM Dex.

alpha 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 alpha 2-ARs.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   alpha 2-AR stimulation of RASM cell migration. Cell migration was determined as described in MATERIALS AND METHODS and was expressed in terms of multiple increase in random motility coefficient (µ-coeff) over basal value. Cells were incubated for 48 h under basal conditions (untreated), with 1 µM Dex, or with 1 µM Dex + 1 µM RW (Dex + RW). Values are means ± SE (n = 4). * P < 0.05 vs. Dex + RW treatment group.

Effects of alpha 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 alpha 2-ARs.


View larger version (200K):
[in this window]
[in a new window]
 
Fig. 7.   alpha 2-AR inhibition of Texas red isothiocyanate (TRITC)-phalloidin F-actin labeling in RASM cells. Fluorescence microscopy with TRITC-labeled phalloidin (1 mg/ml) was used to examine F-actin labeling either under basal conditions (A-C, no drug treatment) or after a 6-h incubation with 1 µM Dex (D-F) or with 1 µM Dex + 1 µM RW (G-I). Scale bar in D = 20 µm. All images were acquired under identical conditions (n = 3).

To see if a pulse of Dex would initiate a migratory response or a change in actin labeling, experiments were performed in which cells were incubated with Dex (0.1 and 1.0 µM) for 5 min (as in the MAP kinase assays) and rinsed and then, 38 or 18 h later, changes in migration and actin, respectively, were monitored. However, in both of these cases, there were no differences between the pulse-treated cells and untreated cells. In addition, we looked for changes in actin immediately after a 5-min Dex pulse and saw no differences. It appears that constant Dex exposure is necessary.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha 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 alpha 2-AR subtypes were expressed in RASM cells, although with increasing passage number, expression was lost. Functionally, the activation of RASM cell alpha 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 alpha 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 alpha 2A-AR appears to also exhibit a nuclear staining pattern and the alpha 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 alpha 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 alpha 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 alpha 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 alpha 2C-AR is also expressed in vascular smooth muscle. Interestingly, studies with transgenic mice lacking the alpha 2C subtype showed no changes in hemodynamic parameters, which raises the question of their function in vascular tissues.

Generally, the activation of alpha 2-ARs is known to have effects on a number of cellular pathways. For example, it has been shown that the alpha 2-ARs mediate release of Ca2+ from intracellular stores (17), stimulate the secretion of prostaglandins (most likely an alpha 2B-AR response) (21), promote preadipocyte proliferation (3), and stimulate MAP kinase activation in stably transfected CHO cells (10). In the present studies, alpha 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 alpha 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 alpha 2-AR activation stimulated cell migration and that this was blocked in the presence of the alpha 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 alpha 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 alpha 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, alpha 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 alpha 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 alpha 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 alpha 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 alpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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. alpha -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. alpha 2-Adrenergic stimulation promotes preadipocyte proliferation. J. Biol. Chem. 269: 30254-30259, 1994[Abstract/Free Full Text].

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 alpha 2-adrenergic receptors. Mol. Pharmacol. 51: 711-720, 1997[Abstract/Free Full Text].

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. alpha 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[Abstract/Free Full Text].

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 alpha 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 alpha 2A-adrenergic receptor on the basolateral membrane of Madin-Darby canine kidney cells. J. Biol. Chem. 269: 16425-16433, 1994[Abstract/Free Full Text].

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 alpha 2-adrenergic receptor. Science 238: 650-656, 1987[Medline].

17.   Lepretre, N., J. Mironneau, and J. L. Morel. Both alpha 1a- and alpha 2a-adrenoceptor subtypes stimulate voltage-operated L-type calcium channels in rat portal vein myocytes. J. Biol. Chem. 269: 29546-29552, 1994[Abstract/Free Full Text].

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 alpha 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 alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 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 alpha 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 alpha -1 and alpha -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 alpha -adrenoceptor gene expression in arterial and venous smooth muscle. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H1501-H1509, 1993[Abstract/Free Full Text].

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 alpha 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[Abstract/Free Full Text].

27.   Schwartz, S. M., R. L. Heimark, and M. W. Majesky. Developmental mechanisms underlying pathology of arteries. Physiol. Rev. 70: 1177-1209, 1990[Abstract/Free Full Text].

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[Abstract/Free Full Text].

30.   Vanscheeuwijck, P., Y. Huang, D. Schullery, and J. W. Regan. Antibodies to a human alpha 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[Abstract/Free Full Text].


AJP Cell Physiol 274(3):C654-C662
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society