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Endocrinology, doi:10.1210/en.2005-0918
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Endocrinology Vol. 147, No. 4 1932-1940
Copyright © 2006 by The Endocrine Society

Calcitonin Gene-Related Peptide Receptor Activation by Receptor Activity-Modifying Protein-1 Gene Transfer to Vascular Smooth Muscle Cells

Zhongming Zhang, Ian M. Dickerson and Andrew F. Russo

Department of Physiology and Biophysics, University of Iowa (Z.Z., A.F.R.), Iowa City, Iowa 52242; and Department of Neurobiology and Anatomy, University of Rochester (I.M.D.), Rochester, New York 14642

Address all correspondence and requests for reprints to: Dr. Andrew F. Russo, Department of Physiology and Biophysics, 51 Newton Road, University of Iowa, Iowa City, Iowa 52242. E-mail: andrew-russo{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator that plays a protective role in the cardiovascular system. The receptor for CGRP is an unusual complex of the G protein-coupled calcitonin-like receptor and an obligate receptor activity modifying protein-1 (RAMP1). In this report we provide the first evidence that RAMP1 is rate limiting in vascular smooth muscle cells. Although cultured rat aorta smooth muscle cells express calcitonin like-receptor and RAMP1, we found that CGRP is not a potent activator of the receptor. After overexpression of RAMP1 by adenoviral gene transfer, there was a striking increase in CGRP-induced production of cAMP, with a 75-fold decrease in the EC50 and a 1.5-fold increase in the maximal response. The biological consequence of this increased receptor activity was observed in three different paradigms. First, RAMP1 gene transfer caused a CGRP-dependent decrease in cell proliferation. Second, RAMP1 and CGRP treatment led to a 3-fold greater free radical-induced reduction in cell number. Finally, RAMP1 gene transfer resulted in a 5-fold CGRP-dependent increase in terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive apoptotic cells upon serum withdrawal. The mechanisms underlying these effects involved cAMP-dependent pathways. We propose that RAMP1 gene transfer may be an effective strategy for increasing the effectiveness of CGRP-induced decrease in restenosis after aortic angioplasty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CARDIOVASCULAR system is richly innervated by perivascular calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers (1, 2). CGRP plays multiple roles in the cardiovascular system, where it is recognized as the most potent vasodilatory peptide (3, 4). CGRP-mediated vasodilation occurs by relaxation of vascular smooth muscle, either by direct action on the vascular smooth muscle or indirectly by an endothelium-dependent induction of nitric oxide. In addition to vasodilation, CGRP might play a protective role after myocardial infarction and vascular damage (4, 5, 6). Coincident with these vascular effects, CGRP is a key player in neurogenic inflammation and nociception (4, 7, 8).

CGRP actions are mediated by an unusual multimer of a core G protein-coupled receptor, calcitonin like-receptor (CLR), and two accessory proteins, receptor activity-modifying protein-1 (RAMP1) and receptor component protein (RCP) (9, 10). CLR was initially described as an orphan receptor until RAMP1 was shown to enable CGRP binding and signaling at the CLR (11). RAMP1 is a small, 148-amino acid, single-pass transmembrane protein with a large extracellular amino terminus and a short intracellular carboxyl tail. In addition to pharmacological specificity, RAMP1 influences glycosylation and trafficking of CLR to the cell surface (11, 12, 13), although its requirement as a chaperone is controversial (14, 15). Two additional RAMPs (RAMP2 and RAMP3) share approximately 30% identity with RAMP1. Coexpression of RAMP2 or -3 with CLR yields adrenomedullin receptors (11, 16, 17). Adrenomedullin and CGRP have overlapping activities, including blood vessel dilation (4). The CGRP receptor in vascular smooth muscle is coupled to cAMP production (18, 19), and this coupling is facilitated by RCP, an intracellular protein that coimmunoprecipitates with the CLR/RAMP complex (20).

CGRP can inhibit vascular hypertrophy in addition to acting as a vasodilator. Hypertrophy of vascular smooth muscle occurs in response to arterial injury, angioplasty, and atherosclerosis (21, 22). CGRP can inhibit vascular smooth muscle cell proliferation in vitro (23, 24). Most notably, overexpression of CGRP by gene transfer showed a similar inhibitory effect in vivo and increased apoptosis in the neointima after angioplastic balloon injury (6). These observations suggest that modulation of CGRP activity may be a useful strategy for minimizing the restenosis that frequently occurs after angioplasty.

As a model system to study the regulation of CGRP receptor activity, we used primary cultures of rat aorta smooth muscle. Previous studies have reported that these cells express functional CGRP receptors that are coupled to cAMP production (18, 19). We asked whether gene transfer of the RAMP1 subunit into cultured rat aorta smooth muscle cells would increase CGRP efficacy. We found that RAMP1 overexpression greatly decreased the CGRP receptor EC50 and increased the maximal production of cAMP, suggesting increased receptor affinity and more efficient signaling in the presence of heterologous RAMP1. After RAMP1 gene transfer, the proliferating muscle cells were more sensitive to free radicals, and there was increased CGRP-induced apoptosis even in the absence of exogenous free radicals. We suggest that the RAMP1 subunit may be rate limiting in certain tissues, and that RAMP1 gene transfer may provide a potential therapeutic method of preventing deleterious vascular hypertrophy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat aorta smooth muscle cells were obtained from thoracic aorta from 4-wk-old adult male Sprague Dawley rats, weighing 175–200 g. Pieces of aortic muscle were carefully removed from the endothelium and isolated by incubation with DMEM high-glucose medium (Invitrogen Life Technologies, Inc., Carlsbad, CA) containing collagenase IV (2 mg/ml) for 30 min, then triturated vigorously until the solution was cloudy. Cells were collected by centrifugation. The cells were cultured with DMEM high-glucose medium containing 0.1 mM MEM nonessential amino acids, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin-streptomycin, 0.01 M HEPES (pH 7.2), 20% fetal bovine serum, and 1x Basal Medium Eagle vitamins (Sigma-Aldrich Corp., St. Louis, MO) for 24 h in a humidified 5% CO2 incubator. After the first passage by trypsinization, the cells were cultured with the above medium, except with 10% fetal bovine serum. Cells from passages 1–5 were used unless otherwise indicated. Cell culture reagents were purchased from Invitrogen Life Technologies, Inc., unless otherwise stated. HEK293 cells were maintained in DMEM high-glucose medium with 0.01 M HEPES (pH 7.2), 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin.

Adenoviral (Ad) vectors and infections
Human RAMP1 cDNA (4) in pcDNA 3 was a gift from Steve Foord (GlaxoSmithKline, Inc., Research Triangle Park, NC). The cDNA was cloned into the pacAd5 cytomegalovirus (CMV)-K-N pA shuttle vector containing the CMV promoter at the BamHI site. The 460-bp insert extended from 6 bp upstream of the start codon to 7 bp downstream of the stop codon. The fidelity and orientation of the insert were confirmed by sequencing. Replication-deficient recombinant adenovirus (Ad5 serotype), AdCMV-RAMP1, was generated by the University of Iowa Gene Transfer Vector Core. Two control viruses were used. The parental pacAd5 CMV-K-N pA vector was used to generate AdCMV-empty, and the same vector with enhanced green fluorescent protein (GFP) was used to generate AdCMV-GFP. Adenovirus titers were determined by plaque assays on HEK293 cells. RAMP1 virus titer was 7.2 x 1010 plaque-forming units (pfu)/ml, and the control viruses had similar titers of 4–5 x 1010 pfu/ml. The purified viruses were aliquoted in Dulbecco’s PBS (2.7 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, and 4.3 mM Na2HPO4) with 3% sucrose and stored at –80 C.

Cells were plated at a density of 6 x 104 cells/well in a six-well cell culture plate (Costar, Cambridge, MA) After 2 d, cells were 70% confluent and infected with 0.6 µl virus (4 x 107 pfu at a multiplicity of infection estimated at 150) in 1 ml serum-free medium for 5 h, followed by addition of another 1 ml medium with 20% fetal bovine serum (final 10% concentration). Cells were cultured overnight before assays. For control infections of HEK293 cells, the cells were plated at 6 x 105 cells/well and infected with 0.6 µl AdCMV-RAMP1 or an equal amount of AdCMV-GFP virus or vehicle on the next day when cells were 70% confluent.

cAMP assay
Cells were washed with serum-free medium twice and incubated with serum-free medium containing 0.22 mg/ml 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corp.) for 15 min. The samples were then treated with the indicated concentrations of human CGRP (Sigma-Aldrich Corp.), the combination of CGRP (100 nM) and CGRP-(8–37) antagonist (500 nM; Sigma-Aldrich Corp.), or the PBS vehicle for 10 min. The medium was removed, and the cells were lysed by addition of 1 ml ice-cold 0.5 N perchloric acid containing 180 µg/ml theophylline (Sigma-Aldrich Corp.). The lysates was neutralized by the addition of 500 µl 0.72 M KOH/0.6 M KHCO3 for 10 min at room temperature. Samples were centrifuged at 3000 x g for 10 min at 4 C. Supernatants were saved at –20 C until assayed. cAMP levels were measured using a RIA kit as recommended by the manufacturer for the overnight protocol (Amersham Biosciences, Arlington Heights, IL). Assays were performed in duplicate in at least three independent experiments. Results were expressed as femtomoles of cAMP per well, and EC50 and maximal response (Rmax) values were determined using a nonlinear regression curve-fitting computer program within PRISM version 2.0 (Graph-Pad, Inc., San Diego, CA). Data are given as the mean ± SD. The mean cAMP levels (Fig. 3AGo) were compared by ANOVA and Tukey’s test. The –logEC50 and Rmax values from four independent experiments were analyzed using paired Student’s t test (two-tailed). Statistical analyses were performed using PRISM and SPSS software (SPSS, Inc., Chicago, IL).


Figure 3
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  		FIG. 3. AdCMV-RAMP1 gene transfer increases CGRP-induced cAMP production. A, Rat aorta smooth muscle cells were infected with either the control virus AdCMV-GFP or AdCMV-RAMP1 or were not infected. After 24 h, the cultures were treated for 10 min with vehicle (Con), 100 nM CGRP, or the combination of 100 nM CGRP and 500 nM CGRP-(8–37) antagonist (CGRP + 8–37). The mean intracellular cAMP levels ± SD of three experiments performed in duplicate are shown. CGRP significantly increased cAMP levels relative to control for all three conditions (*, P < 0.05; ***, P < 0.001). In the presence of recombinant RAMP1, there was an even greater increase in CGRP-stimulated cAMP levels compared with the cells without viral infection (**, P < 0.01). For all conditions, there was no significant increase in the presence of CGRP-(8–37). B, A representative figure (passage 2 cells) of four experiments shows decreased EC50 and increased maximal response in the cultures containing recombinant RAMP1. The mean and range of duplicate samples are shown. The maximal responses in this example are greater than those in A, because cell density was greater.

 
Immunocytochemistry
Cultures were briefly rinsed in PBS and fixed in 2% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) in PBS for 30 min at 4 C. Cells were washed twice with PBS and permeabilized with 0.1% Triton X-100 and 0.1% Tween 20 (diluted in PBS) for 45 min at room temperature. The fixed cells were blocked with 0.1% goat serum in PBS for 1 h at room temperature, then incubated overnight 4 C with either CLR chicken antiserum OCA-910 (25) (diluted 1:100), RAMP1 rabbit antiserum (OA-350) (20, 25) (diluted 1:200) or RCP rabbit polyclonal antiserum R83 (20) (diluted 1:200). Both the CLR and RAMP1 sera were provided by Kevin Oliver (Merck, Sharp, and Dohme Neuroscience Research Center, Harlow, Essex, UK). Cells were washed and blocked for an additional 30 min before incubation with the appropriate secondary antibodies rhodamine (tetramethylrhodamine isothiocyanate)-conjugated rabbit antichicken (Chemicon International, Temecula, CA; diluted 1:1000) or rhodamine (tetramethylrhodamine isothiocyanate)-conjugated donkey antirabbit (The Jackson Laboratory, Bar Harbor, ME; diluted 1:1000) for 1 h at room temperature. Nuclei were labeled with TO-PRO-3 (Molecular Probes, Inc., Eugene, OR) diluted 1:1000 in dimethylsulfoxide (Sigma-Aldrich Corp.; data not shown). Images were captured using a Zeiss LSM 510 confocal microscope (New York, NY). Images shown are representative of three separate experiments.

Cell number and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assays
Cultures grown to 70% confluence were either uninfected or infected with AdCMV-RAMP1 or AdCMV-GFP (multiplicity of infection, 150). Proliferation was measured in 5% serum-containing growth medium after 100 nM CGRP treatment for the indicated times. In some experiments the cell number was measured after 24 h without serum in Hanks’ balanced salt solution (HBSS; Invitrogen Life Technologies, Inc.), as indicated in the text. The glucose oxidase treatment paradigm was modified from (26). The cells were pretreated with 100 nM CGRP, 100 nM CGRP plus 500 nM CGRP-(8–37) or 25 µM 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMP; Alexis Biochemicals Corp., San Diego, CA), or PBS vehicle for 6 h in serum-containing medium, then washed with HBSS twice and incubated with HBSS containing 0.02 U/ml glucose oxidase (Sigma-Aldrich Corp.) for 1.5 h at 37 C at ambient CO2, then incubated with serum-containing growth medium for 3 h, followed by trypan blue exclusion and cell counting. Viable cells were counted using a hemocytometer 5 min after mixing 0.4% trypan blue (Invitrogen Life Technologies, Inc.) with an equal volume of cells.

TUNEL assay was performed according to the manufacturer’s instructions (Molecular Probes). The cells were pretreated with 100 nM CGRP, 100 nM CGRP plus 500 nM CGRP-(8–37) or 25 µM Rp-8-Br-cAMP, or PBS vehicle for 6 h in serum-containing medium, then washed with HBSS twice and incubated in HBSS at 37 C at ambient CO2 with or without CGRP or CGRP plus CGRP-(8–37) at 37 C overnight. TUNEL-stained samples were analyzed under a Zeiss LSM 510 confocal microscope, and at least 600 cells were counted in randomly selected fields. The means in Figs. 4–6GoGoGo were statistically compared by ANOVA (two-tailed), followed by Tukey’s test, and were considered significant at P < 0.05.


Figure 4
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  		FIG. 4. CGRP-induced decrease in cell number. A, Aorta smooth muscle cultures (passage 4) were infected with AdCMV-RAMP1 (Ad-RAMP1) virus or AdCMV-GFP control (Ad-GFP) virus or were not given virus (no virus) 1 d before treatment at d 0 with vehicle (Con), 100 nM CGRP, or 100 nM CGRP plus 500 nM CGRP-(8–37) (C + 8–37) in 5% serum-containing medium. On d 0, there was no significant difference among the different cultures. On d 1–5, there were significantly fewer cells in the Ad-RAMP1 plus CGRP cultures (*, P < 0.01; **, P < 0.05). Data expressed as the mean ± SD from three experiments, each performed in duplicate. B, Cells were infected and treated for 24 h as described in A, except in serum-free HBSS. The cell numbers were normalized to the no virus control cell number. The addition of CGRP significantly decreased cell number only in the AdCMV-RAMP1-infected cells (*, P < 0.01). Data are expressed as the mean ± SD from three experiments, each performed in duplicate.

 

Figure 5
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  		FIG. 5. Combinatorial effect of CGRP and glucose oxidase free radical generation on cell number. Aorta smooth muscle cultures were either uninfected or infected with the control AdCMV-GFP virus or AdCMV-RAMP1. The cultures were pretreated with vehicle (Con), 100 nM CGRP, or the combination of 100 nM CGRP and 500 nM CGRP-(8–37) (8–37) or 25 µM Rp-8-Br-cAMP (RpcA) for 6 h. CGRP was removed, and the cultures were treated with vehicle or 0.02 U/ml glucose oxidase for 1.5 h. The glucose oxidase was removed, and viable cells were counted after 3 h and normalized to the no virus control cell number. The addition of CGRP significantly decreased cell number only in AdCMV-RAMP1-infected cells (*, GO + CGRP vs. GO, P < 0.01), which was prevented by CGRP-(8–37) or Rp-8-Br-cAMP. Data are expressed as the mean ± SD from three to five experiments performed in duplicate.

 

Figure 6
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  		FIG. 6. CGRP-induction of TUNEL-positive cells. Aorta smooth muscle cultures were either uninfected or infected with the control AdCMV-empty virus or AdCMV-RAMP1. Cultures were treated with vehicle (Con), 100 nM CGRP, or the combination of 100 nM CGRP and 25 µM Rp-8-Br-cAMP and switched to serum-free HBSS medium overnight (~18 h). Cell nuclei were stained with propidium iodide. TUNEL-positive cells were detected by immunocytochemical staining with anti-BrdU antiserum. The fraction of TUNEL-positive cells was determined from three to five independent experiments performed in triplicate. The AdCMV-RAMP1 cultures treated with CGRP had significantly more TUNEL-positive cells than either the vehicle (PBS)-treated or CGRP- plus 25 µM Rp-8-Br-cAMP-treated AdCMV-RAMP1 cultures or the uninfected or AdCMV-GFP-infected cells with or without CGRP (P < 0.01).

 
Western blots
Cells were rinsed once with ice-cold PBS, scraped, and collected by centrifugation. The pellet was resuspended in PTN50 buffer (50 mM PBS, 1% Triton X-100, and 50 mM NaCl) plus protease inhibitor cocktail (Sigma-Aldrich Corp.) and homogenized by sonication (model 550, Fisher Scientific, Pittsburgh, PA; setting 4; 10–15 sec). Protein was measured by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of cell lysate (30 µg) were subjected to 12.5% SDS-PAGE and transferred to Immobilon-P membranes as recommended by the manufacturer (Millipore Corp., Bedford, MA). Membranes were incubated for 3 h with anti-RAMP1 (NY1020) antiserum diluted 1:1000 in PBS containing 0.04% Tween 20 (PBST) plus 1% nonfat dry milk. After washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Biosciences, Arlington Heights, IL) diluted 1:5000 in PBST. After thorough washing with PBST, immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham Biosciences). The same filters were blocked with PBST plus 5% nonfat dry milk for 1 h before incubating with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH; V-18) goat polyclonal IgG (Santa Cruz Biotechnology) or CLR (NY1045; both diluted 1:1000) antiserum for 3 h. Membranes were washed, then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min [donkey antigoat IgG, diluted 1:10,000 (Santa Cruz Biotechnology, Inc.), or donkey antirabbit IgG, diluted 1:5,000 (Amersham Biosciences), for visualizing GAPDH and CLR, respectively].

NY1020 is a rabbit polyclonal antibody raised against synthetic peptide MVTACRDPDYGTL of mouse RAMP1 (Covance, Inc., Princeton, NJ). NY1020 recognized the expected band for a RAMP1 monomer (~14–16 kDa) in cell lines expressing CGRP receptors (NIH-3T3) or in HEK293 cells infected with the AdCMV-RAMP1 virus (1 d after infection), but not in cell lines known to lack CGRP receptors (COS7, HEK293) or in HEK293 cells either mock infected or infected with AdCMV-GFP virus (data not shown). Antibody NY1045 is a rabbit polyclonal antibody raised against a synthetic peptide, LGVTRNKIMT, from mouse CLR (Covance, Inc.). Using immunohistochemistry, NY1045-stained cells that are known to express CGRP receptors (NIH-3T3) and not cells known to lack CGRP receptors (COS7), and recognized the predicted approximately 66-kDa band by Western blot in NIH-3T3 cells (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adenoviral gene transfer of RAMP1 to rat aorta smooth muscle cells
Expression of CLR, RAMP1, and RCP was confirmed in rat aorta smooth muscle cells under our culture conditions (Fig. 1Go). The presence of mRNA encoding CLR and all three RAMP proteins has been previously reported (27). CLR- and RAMP1-immunoreactive material was observed predominantly in a cytoplasmic and perinuclear pattern. The staining patterns were qualitatively similar in freshly isolated cells (passage 1) and late cultures (passage 13; not shown). An adenoviral vector was constructed containing human RAMP1 cDNA under control of the CMV promoter (AdCMV-RAMP1; Fig. 2AGo). An adenoviral vector with the CMV promoter expressing enhanced GFP (AdCMV-GFP) was used as a control virus. Nearly 95% of the cells expressed the enhanced GFP reporter when infected at a multiplicity of infection of 150 (Fig. 2BGo). Infection of the rat aorta smooth muscle cultures with AdCMV-RAMP1 yielded an increase in the intensity of an immunoreactive protein at the expected size for RAMP1 (~14 kDa; Fig. 2CGo). Additional immunoreactive material was observed that may correspond to multimers of RAMP1, as previously reported (11). We also confirmed that overexpression of RAMP1 did not increase the level of the approximately 66-kDa CLR protein (Fig. 2CGo). The nature of the higher molecular mass band in the CLR blot is not known, although CLR complexes have been reported (13). The antibody that we used did not allow us to distinguish cell surface from intracellular CLR, so the amount of cell surface receptor could not be determined. As a control, the amount of GAPDH staining was similar in the various samples.


Figure 1
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  		FIG. 1. Detection of endogenous CLR, RAMP1, and RCP in cultured rat aorta smooth muscle cells. Confocal images of immunocytochemical staining of passage 3 cells with antisera directed against CLR (A), RAMP1 (B), and RCP (C). All sections were stained with TO-PRO-3 to confirm the location of nuclei (not shown). Scale bar, 20 µm.

 

Figure 2
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  		FIG. 2. Expression of RAMP1 after adenoviral gene transfer. A, Schematic of the AdCMV-RAMP1 vector. B, Fluorescence image of AdCMV-GFP-infected rat aorta smooth muscle cells. Scale bar, 50 µm. C, Western blots of lysates prepared from uninfected, AdCMV-GFP-infected, and AdCMV-RAMP1-infected aorta smooth muscle cultures (passage 4). The same filter was sequentially incubated with antisera to detect RAMP1, CLR, and GAPDH, as indicated. The multiple RAMP1 species are indicated with arrows. The monomer form of RAMP1 migrated close to the dye front of the gel. An unknown high molecular mass band is seen with both the RAMP1 and CLR blots. Protein size standards are shown.

 
Increased CGRP receptor coupling to cAMP production after RAMP1 gene transfer
Incubation of rat aorta smooth muscle cultures with 100 nM CGRP induced a 2.5-fold induction of intracellular cAMP (Fig. 3AGo). The CGRP-dependent increase in cAMP was specific, because it was blocked by the receptor antagonist CGRP-(8–37) (500 nM).

Infection of cultured muscle cells with the AdCMV-RAMP1 virus greatly enhanced CGRP receptor activity (Fig. 3AGo). CGRP induced an approximately 8-fold increase in intracellular cAMP after RAMP1 gene transfer. Basal cAMP production was not increased by infection with the AdCMV-RAMP1 virus, and the CGRP receptor antagonist blocked the increased cAMP production. As a control, cultures infected with the AdCMV-GFP virus did not show enhanced CGRP-signaling and were similar to the noninfected cells (Fig. 3AGo).

The efficacy of CGRP signaling in RASM cells was increased in cells infected with RAMP1-expressing virus. The endogenous receptor EC50 of CGRP was 1.2 ± 1.0 x 10 –7 M (Fig. 3BGo). This is higher than the values in the 10–9 range reported in the literature for the high-affinity CGRP receptor (10), but comparable to a previous study with rat aorta smooth muscle cultures (EC50, 6.6 x 10–8) (28). After AdCMV-RAMP1 gene transfer, the EC50 was decreased to 3.0 ± 0.4 x 10–9 M (Fig. 3BGo). From four independent experiments, we observed a 75-fold decrease (P < 0.001) in the EC50 from 2.6 ± 1.0 x 10–7 to 3.2 ± 0.2 x 10–9 M.

In addition to the decreased EC50, RAMP1 overexpression also increased the Rmax. In Fig. 3BGo, there was 473.0 ± 70.2 fmol cAMP/well in the control cultures and 729.6 ± 100.3 fmol in the cultures containing overexpressed RAMP1. In four independent experiments, there was a 1.5 ± 0.17-fold (SD) increase in the maximal response (P < 0.01).

Potentiation of CGRP inhibition of proliferation and CGRP-induced cell death by RAMP1 gene transfer
To address the functional significance of the increased CGRP receptor activity, we examined the effect of CGRP on cell proliferation and apoptosis in three different paradigms.

In the first condition, the effect of CGRP and RAMP1 expression on cell proliferation was measured. The number of viable cells was scored after CGRP treatment of either noninfected, AdCMV-GFP-infected, or AdCMV-RAMP1-infected cultures in serum-containing growth medium. CGRP treatment of AdCMV-RAMP1-infected cells caused a significant decrease in the number of cells (Fig. 4AGo). The decrease in cell number was detectable as early as 24 h after treatment, and CGRP significantly decreased cell proliferation compared with all noninfected and AdCMV-GFP-infected groups for up to 4 d. The antagonist CGRP-(8–37) blocked the CGRP inhibitory effect. AdCMV-GFP had no significant effect on cell numbers over the 5-d period compared with wild-type group. There was no significant change in the number of noninfected and control virus-infected cells, either with or without CGRP (Fig. 4AGo). To match the treatment conditions of the second and third paradigms, cell number was also measured after 24 h in the absence of serum. There was a significant decrease in vascular smooth muscle cell number after CGRP treatment of the AdCMV-RAMP1-infected cells, but not under the other conditions (Fig. 4BGo).

In the second condition, we examined the effect of CGRP on cell number after a free radical stress. Cells were treated with the enzyme glucose oxidase, which has been shown to generate hydrogen peroxide and cause vascular smooth muscle cell apoptosis (29). Cells were infected with either AdCMV-RAMP1, a control AdCMV-GFP virus, or noninfected cells in the presence or absence of CGRP. Cells were treated for 6 h with 100 nM CGRP, the medium was then replaced with fresh medium containing glucose oxidase (0.025 U/ml; without CGRP) for 1.5 h, then changed to fresh medium without glucose oxidase or CGRP for a final 3 h. Treatment with glucose oxidase reduced the number of viable cells in all three groups (Fig. 5Go). Treatment with higher concentrations of glucose oxidase (0.1–0.4 U/ml) for the entire duration of the experiment caused a nearly complete loss of viable cells (data not shown). The cell numbers of the noninfected and control virus-induced groups were not significantly different among groups. However, there were significantly fewer cells after CGRP treatment of the cultures containing the RAMP1 virus compared with the CGRP-treated noninfected or control virus-infected cultures (P < 0.01). As a control, pretreatment with the CGRP-(8–37) antagonist prevented the reduction in cell number (Fig. 5Go). Addition of a cell-permeable inhibitor of cAMP-mediated pathways, 8-Br-Rp-cAMP, also prevented the effect of CGRP on cell number (Fig. 5Go). This demonstrates that cAMP is the main mediator of CGRP inhibition of cell proliferation.

In the third condition, we asked whether the CGRP-induced reduction in cell number was due to apoptosis. Because free radicals alone can induce apoptosis (29, 30, 31), we tested the effect of CGRP and RAMP1 overexpression after serum withdrawal. As an indicator of apoptosis, the cells were analyzed by the TUNEL assay, which detects nicked DNA (Fig. 6Go). In the AdCMV-RAMP1-infected cultures, there were 9% TUNEL-positive cells after CGRP treatment. There were very few TUNEL-positive cells in the noninfected (<1%) or AdCMV-empty infected (<2%) cultures either with or without CGRP or in the AdCMV-RAMP1 cultures in the absence of CGRP (<2%). Treatment with 8-Br-Rp-cAMP prevented the CGRP-induced apoptosis (Fig. 6Go). These results demonstrate that CGRP treatment in combination with RAMP1 gene transfer is sufficient to induce apoptosis of vascular smooth muscle by a cAMP-mediated mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of RAMP1 has opened a new perspective on potential ways to regulate CGRP receptor activity. It is clear that both CLR and RAMP1 are required for CGRP receptor activity. However, most of these studies have been performed in immortalized cells that do not necessarily express endogenous CGRP receptor proteins. To our knowledge, this is the first study demonstrating an effect of RAMP1 gene transfer in vascular smooth muscle. A previous study showed increased vascular smooth muscle cell migration after gene transfer of CLR and RAMP2 and -3, but not RAMP1 (27). Because CLR and all three RAMP isoforms are present in cultured rat aortic smooth muscle (27), this raises the possibility of competition between RAMPs for CLR, as reported by Muff and colleagues (32) between RAMP1 and RAMP3 in vascular endothelial cells. CGRP can also interact with the amylin receptor composed of RAMP1 and the calcitonin receptor (33, 34). However, previous studies have demonstrated that rat aortic smooth muscle does not have calcitonin receptor activity (19), and thus, the effects we observed are most likely due to interactions between RAMP1 and CLR. In addition, the ability of the CGRP-(8–37) antagonist to block receptor activity is consistent with involvement of the CLR/RAMP1 protein complex.

Our findings demonstrate that RAMP1 levels are rate limiting in cultured aorta smooth muscle. At the cellular level, RAMP1-induced CGRP action was manifested as increased production of cAMP, which mediated an inhibition of proliferation and induction of apoptosis. This cAMP-dependent mechanism is consistent with previous studies (6, 23, 35, 36, 37). Indeed, elevation of cAMP was sufficient to inhibit proliferation and induce apoptosis of human aortic vascular smooth muscle cells (38). Recently, in vitro CGRP gene transfer was also shown to inhibit aortic and pulmonary artery smooth muscle cell proliferation (35). The RAMP1-induced potentiation of these biological effects illustrates that RAMP1 is rate limiting not only for cAMP generation, but also for downstream effects on cell proliferation and viability.

To date relatively little is known about regulation of RAMP1 levels. RAMP1 appears to be selectively induced by dexamethasone treatment of cultured human coronary artery muscle cells (39), and there is a complex regulation of coronary artery CLR and RAMP levels during hypoxia (40). Within the heart, RAMP1 and RAMP3 levels appear to change in the absence of changes in CLR expression after aorta banding (41). Recently, the cytokine TNF-{alpha} was reported to decrease CLR and RAMP1 and -2 in cultured coronary artery smooth muscle (42). Sex steroids appear to regulate CLR and RAMP levels during pregnancy (43). Decreased CLR and RAMP1 proteins were observed in vascular tissues in preeclamptic placentas along with decreased CGRP-binding sites (44), whereas the expression of all RAMPs and CLR were increased in the uterus of pregnant rats (45). The RAMP1 promoter has recently been cloned, and future studies will undoubtedly reveal other regulators of RAMP1 expression (46).

We observed that the combination of CGRP and RAMP1 could inhibit rat aortic smooth muscle cell proliferation. In contrast to other studies (23, 24), there was not a significant effect of CGRP in the absence of exogenous RAMP1, possibly due to the use of serum or angiotensin II to stimulate proliferation in those studies. The effect of CGRP and RAMP1 was particularly striking in combination with free radicals. There is increasing evidence that free radical generation by oxidative stress is a component of cardiovascular pathologies, such as hypertension, diabetes, and arterial thrombosis (47, 48, 49). Oxidative stress has been reported to cause vascular smooth muscle apoptosis (29, 30, 31), but it can also have paradoxical stimulation or inhibition of proliferation (50, 51). Our observation of decreased cell numbers after glucose oxidase treatment with CGRP and RAMP1 is consistent with in vivo studies after arterial injury (6), but differ from culture studies that reported a small, but significant, CGRP rescue of glucose oxidase-induced cell loss (26). The reason for this difference is not known.

What might be the significance of CGRP action on the aorta smooth muscle in vivo? One consideration is that there are conflicting accounts whether aortic smooth muscle is responsive to CGRP in vivo. Studies by Marshall and colleagues (52, 53) reported that the rat aorta exhibits only endothelium-dependent vasorelaxation. Furthermore, there appear to be relatively few CGRP-binding sites (54). In contrast, CGRP has been reported to relax strips of aortic smooth muscle in the absence of endothelium (55) and exogenous CGRP can inhibit the thickening of vascular intima after injury (6). Consistent with these effects, the aorta is innervated by CGRP-containing fibers, albeit at comparatively low levels (1, 54). Interestingly, this innervation appears to decrease during ageing, and it has been proposed by Connat and co-workers (56) that loss of CGRP innervation of large conducting vessels, such as the aorta, may contribute to age-related vascular changes, including hypertrophy of the smooth muscle and intimal thickening. We suggest that the relatively weak receptor activity in the cultured cells and the conflicting in vivo data indicate that the aortic muscle is normally only minimally responsive to CGRP. However, under certain conditions, such as after injury, RAMP1 might be up-regulated, and the cells become more CGRP responsive, so that CGRP would contribute to vascular remodeling. This suggestion is supported by the studies by Garel and co-workers (39), who have shown that human coronary artery muscle can be switched from a predominantly RAMP2 to a RAMP1 phenotype. An alternative, but not mutually exclusive, hypothesis is that CGRP does not have ready access to receptors on aorta smooth muscle under normal conditions in vivo. This is supported by observations that there are predominantly CGRP (CLR/RAMP1) receptors on vascular smooth muscle and adrenomedullin (CLR/RAMP2) receptors on vascular endothelial cells (57). An exception might be the small caliber vessels that lack endothelial cells and will therefore be more likely to respond to CGRP. Future in vivo studies will be required to test the efficacy of RAMP1 gene transfer on vascular remodeling of the aorta and other large conductance vessels.


   Acknowledgments
 
We thank Kevin Oliver and Steve Foord for generously sharing reagents, Penny Dong for assistance with the immunocytochemistry, and Neal Weintraub for advice and assistance with the vascular muscle cultures and glucose oxidase treatments.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grants DK-52328, HL-14388, and DE-016511. The University of Iowa Gene Transfer Core is supported in part by the NIH and the Roy J. Carver Foundation.

First Published Online December 22, 2005

Abbreviations: Ad, Adrenoviral; CGRP, calcitonin gene-related peptide; CLR, calcitonin-like receptor; CMV, cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HBSS, Hanks’ balanced salt solution; PBST, PBS containing 0.04% Tween 20; pfu, plaque-forming unit; RAMP, receptor activity-modifying protein-1; RCP, receptor component protein; Rmax, maximal response; Rp-8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling.

Received July 21, 2005.

Accepted for publication December 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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