Basal cAMP production was not increased by infection with the AdCMV-RAMP1 virus, and the CGRP receptor antagonist blocked the increased cAMP production. Two control viruses were used. 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. 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. Expression of RAMP1 after adenoviral gene transfer. 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). To date relatively little is known about regulation of RAMP1 levels. Images were captured using a Zeiss LSM 510 confocal microscope (New York, NY). Incubation of rat aorta smooth muscle cultures with 100 nm CGRP induced a 2.5-fold induction of intracellular cAMP (Fig. International Union of Pharmacology: XXXII: the mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. cAMP levels were measured using a RIA kit as recommended by the manufacturer for the overnight protocol (Amersham Biosciences, Arlington Heights, IL). Its downregulation in patients with heart failure is well established. GeneCards Summary for CALCRL Gene. The efficacy of CGRP signaling in RASM cells was increased in cells infected with RAMP1-expressing virus. © American Heart Association, Inc. All rights reserved. Receptor for calcitonin-gene-related peptide (CGRP) together with RAMP1 and receptor for adrenomedullin together with RAMP3 (By similarity). Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure. CLR- and RAMP1-immunoreactive material was observed predominantly in a cytoplasmic and perinuclear pattern. The majority of adults with migraine use medications for their migraine attacks. Calcitonin gene-related peptide (CGRP) inhibitors block the effect of CGRP, which is a small protein that is highly prevalent in the sensory nerves that supply the head and the neck. Nevertheless, it might simultaneously induce cerebrovascular dilation and pain, ie, migraine-like symptoms, and for this reason, antibodies currently directed against either the CGRP receptor or CGRP itself are being evaluated as a prophylactic tool in patients with migraine. Cells were cultured overnight before assays. The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator that plays a protective role in the cardiovascular system. Rimegepant is an orally administered, small-molecule, calcitonin gene–related peptide receptor antagonist that may be effective in acute migraine treatment. Coincident with these vascular effects, CGRP is a key player in neurogenic inflammation and nociception (4, 7, 8). The mean cAMP levels (Fig. We observed that the combination of CGRP and RAMP1 could inhibit rat aortic smooth muscle cell proliferation. Nevertheless, it might simultaneously induce cerebrovascular dilation and pain, ie, migraine-like symptoms, and for this reason, antibodies currently directed against either the CGRP receptor or CGRP itself are being evaluated as a prophylactic tool in patients with migraine. By binding to this receptor, αCGRP will suppress deleterious effects mediated by the angiotensin II type 1 (AT 1) and endo- International Union of Pharmacology. HRS mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. In the second condition, we examined the effect of CGRP on cell number after a free radical stress. An ongoing role of α-calcitonin gene-related peptide as part of a protective network against hypertension, vascular hypertrophy, and oxidative stress. The staining patterns were qualitatively similar in freshly isolated cells (passage 1) and late cultures (passage 13; not shown). This α-analogue, which is not orally active, has an albumin-binding fatty acid moiety in the N-terminus, allowing (reversible) binding to albumin, thereby preventing rapid metabolism. C, Western blots of lysates prepared from uninfected, AdCMV-GFP-infected, and AdCMV-RAMP1-infected aorta smooth muscle cultures (passage 4). The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction. Thus, an interaction with multiple deleterious hormonal systems is indeed feasible (Figure). Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors. The monomer form of RAMP1 migrated close to the dye front of the gel. Clearly, future studies should critically investigate the blood pressure dependency of the beneficial end-organ effects of the α-analogue. The CGRP-dependent increase in cAMP was specific, because it was blocked by the receptor antagonist CGRP-(8–37) (500 nm). NY1020 is a rabbit polyclonal antibody raised against synthetic peptide MVTACRDPDYGTL of mouse RAMP1 (Covance, Inc., Princeton, NJ). Aorta smooth muscle cultures were either uninfected or infected with the control AdCMV-empty virus or AdCMV-RAMP1. A receptor activity modifying protein (RAMP) 2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP 1. We propose that RAMP1 gene transfer may be an effective strategy for increasing the effectiveness of CGRP-induced decrease in restenosis after aortic angioplasty. B, A representative figure (passage 2 cells) of four experiments shows decreased EC50 and increased maximal response in the cultures containing recombinant RAMP1. This demonstrates that cAMP is the main mediator of CGRP inhibition of cell proliferation. As a control, pretreatment with the CGRP-(8–37) antagonist prevented the reduction in cell number (Fig. These observations suggest that modulation of CGRP activity may be a useful strategy for minimizing the restenosis that frequently occurs after angioplasty. The cell numbers of the noninfected and control virus-induced groups were not significantly different among groups. Isoform 2: Receptor for calcitonin but is unable to couple to G proteins and activate adenylyl cyclase (PubMed: 7476993 ). 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). Summary. Indeed, not only did the α-analogue reverse the upregulation of transforming factor β1, a major driver of fibrosis (by the induction of matrix metalloproteinase-2), but it also normalized the upregulated inflammatory markers nuclear factor kappa B and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) and the oxidative stress marker hypoxia-inducible factor 1α. 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. Cultures grown to 70% confluence were either uninfected or infected with AdCMV-RAMP1 or AdCMV-GFP (multiplicity of infection, 150). Long-lasting physiological antagonism of calcitonin gene-related peptide towards endothelin-1 in rat mesenteric arteries and human coronary arteries. 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. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Patients with heart failure experience high rates of hospitalization and death, combined with a poor quality of life. CGRP blocking medications … 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. 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 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 × 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 × 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). 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]. The cell numbers were normalized to the no virus control cell number. Indeed, its antihypertensive, anti-remodeling, and ejection fraction-improving effects provide a powerful combination for its application in heart failure. 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. 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. 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). Model of the calcitonin gene-related peptide receptor (CGRP) receptor, consisting of the calcitonin-like receptor (CLR), receptor activity-modifying protein type 1 (RAMP1), and receptor component protein (RCP). 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. Our findings demonstrate that RAMP1 levels are rate limiting in cultured aorta smooth muscle. Wiping out CGRP: potential cardiovascular risks. There is an exciting new class of medications for migraine targeting the calcitonin gene-related peptide (CGRP). This cAMP-dependent mechanism is consistent with previous studies (6, 23, 35–37). CLR was initially described as an orphan receptor until RAMP1 was shown to enable CGRP binding and signaling at the CLR (11). The authors thank Alexander Góes Martini for generating the Figure. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. One of the concerns is whether such antibodies might have cardiovascular side effects.3 Paradoxically, long-term CGRP receptor blockade is associated with a reduction in age-related diseases.10, Last year, Nilsson et al11 described an αCGRP analogue with a prolonged plasma half-life (>7 hours). Modification of the rat aortic wall during ageing; possible relation with decrease of peptidergic innervation. A second trigeminal CGRP receptor: function and expression of the AMY1 receptor. In addition to vasodilation, CGRP might play a protective role after myocardial infarction and vascular damage (4–6). For all conditions, there was no significant increase in the presence of CGRP-(8–37). The presence of mRNA encoding CLR and all three RAMP proteins has been previously reported (27). Peptides are chains of amino acids which are shorter than proteins. The reason for this difference is not known. The calcitonin receptor is encoded by the CALCR gene and comprises >90 kb and 14 exons. • Pondel M (December 2000). The receptors for the other members the family are made up of subunits. In addition to the decreased EC50, RAMP1 overexpression also increased the Rmax. The authors are to be complimented for their carefully performed proof-of-concept study, revealing that the protective effects of the α-analogue are most likely related to a reduction of the angiotensin II-induced inflammation and oxidative stress in the heart, vascular wall, and kidney. Sacubitril, when combined with angiotensin II type 1 receptor blockade, turned out to be highly successful in heart failure. AdCMV-GFP had no significant effect on cell numbers over the 5-d period compared with wild-type group. 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. Adrenomedullin and CGRP have overlapping activities, including blood vessel dilation (4). Furthermore, there appear to be relatively few CGRP-binding sites (54). The calcitonin receptor is thought to couple to the heterotrimeric guanosine triphosphate-binding protein that is sensitive to cholera toxin. International Journal of Experimental Pathology. 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 α-analogue induced positive metabolic effects and the release of glucagon-like peptide-1 in mice with diabetes mellitus. This work was supported by National Institutes of Health (NIH) Grants DK-52328, HL-14388, and DE-016511. Protein-protein interaction and not glycosylation determines the binding selectivity of heterodimers between the calcitonin receptor-like receptor and the receptor activity-modifying proteins. "Calcitonin and calcitonin receptors: bone and beyond". 3A). THE CARDIOVASCULAR system is richly innervated by perivascular calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers (1, 2). The biological consequence of this increased receptor activity was observed in three different paradigms. However, CGRP, which also does not cross the blood-brain barrier,14 may (like glyceryl trinitrate) still trigger migraine attacks in patients with migraine,15 suggesting that this potential side effect of CGRP receptor stimulation cannot be easily ruled out on the basis of the absence of a light-avoidance response. Its pharmacology is complicated by the existence of several splice variants. 3B, 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. Compelling evidence for the importance of CGRP in migraine has been provided by clinical trials with multiple small molecule CGRP receptor antagonists. Off-target effects of oral anticoagulants - vascular effects of vitamin K antagonist and non-vitamin K antagonist oral anticoagulant dabigatran etexilate. 1999; 140 : 2883-2890 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. Among its related pathways are Signaling by GPCR and G alpha (s) signalling events . The calcitonin gene-related peptide (CGRP) receptor is composed of the calcitonin receptor-like receptor (CLR, a class B GPCR) and a single-pass membrane protein known as receptor activity modifying protein type 1 (RAMP1). An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3. Six major drugs are included within this category: telcagepant, olcegepant, BI 44370, rimegepant (BMS-927711), MK3207, and ubrogepant. CGRP I or α-CGRP is a 37-amino acid neuropeptide and is formed from the alternative splicing of the calcitonin/CGRP gene located on chromosome 11. 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. Cell nuclei were stained with propidium iodide. Both CGRP isoforms act on the CGRP receptor, consisting of the 7-transmembrane calcitonin-like receptor, a receptor activity-modifying protein type 1, and a receptor component protein (Figure).5 All 3 components are required for optimal functioning of the receptor. 4B). Alternatively, αCGRP may stimulate amylin-1 (AM-1) receptors. The multiple RAMP1 species are indicated with arrows. Calcitonin-gene related peptide (CGRP), amylin (AMY), adrenomedullin (AM), calcitonin receptor-stimulating peptide (CRSP) and intermedin/adrenomedullin-2 (IMD/AM-2) are structurally related peptides of the same family. For control infections of HEK293 cells, the cells were plated at 6 × 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. Previous studies have reported that these cells express functional CGRP receptors that are coupled to cAMP production (18, 19). An adenoviral vector was constructed containing human RAMP1 cDNA under control of the CMV promoter (AdCMV-RAMP1; Fig. The study by Aubdool et al5 shows that αCGRP lowers blood pressure, reduces cardiac hypertrophy, and is renoprotective. The mechanisms underlying these effects involved cAMP-dependent pathways. 1-800-242-8721 CGRP inhibitors are used for the management of migraine. The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells. The American Heart Association is qualified 501(c)(3) tax-exempt Biomechanical stress-induced signaling in smooth muscle cells: an update. CGRP and its receptors provide new insights into migraine pathophysiology. CGRP induced an approximately 8-fold increase in intracellular cAMP after RAMP1 gene transfer. Figure. CGRP binds to CGRP receptors composed of receptor activity-modifying protein 1 (RAMP1) and calcitonin receptor-like receptor (CLR) to modulate various functions such as pain transmission and vasodilation. By binding to this receptor, αCGRP will suppress deleterious effects mediated by the angiotensin II type 1 (AT1) and endothelin type A (ETA) receptors, as well as reduce the activity of the sympathetic nervous system (SNS). The nature of the higher molecular mass band in the CLR blot is not known, although CLR complexes have been reported (13). Because free radicals alone can induce apoptosis (29–31), we tested the effect of CGRP and RAMP1 overexpression after serum withdrawal. In four independent experiments, there was a 1.5 ± 0.17-fold (sd) increase in the maximal response (P < 0.01). Cells were rinsed once with ice-cold PBS, scraped, and collected by centrifugation. The same filter was sequentially incubated with antisera to detect RAMP1, CLR, and GAPDH, as indicated. Infection of cultured muscle cells with the AdCMV-RAMP1 virus greatly enhanced CGRP receptor activity (Fig. They have been described as small proteins. Among its related pathways are Signaling by GPCR and G alpha (s) signalling events . In Fig. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells. 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. 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. 123 Species differences include that the pig I1 insert is generated by selective use of alternate splice sites located on exon 8, while in … B, Cells were infected and treated for 24 h as described in A, except in serum-free HBSS. One of these molecules, calcitonin gene-related peptide (CGRP) affects several immune cells including T cells, B cells, dendiritic cells and mast cells. Cell culture reagents were purchased from Invitrogen Life Technologies, Inc., unless otherwise stated. Antibody NY1045 is a rabbit polyclonal antibody raised against a synthetic peptide, LGVTRNKIMT, from mouse CLR (Covance, Inc.). An unknown high molecular mass band is seen with both the RAMP1 and CLR blots. Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies. Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects. Nuclei were labeled with TO-PRO-3 (Molecular Probes, Inc., Eugene, OR) diluted 1:1000 in dimethylsulfoxide (Sigma-Aldrich Corp.; data not shown). 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). Meningeal (dural and pial) blood vessels in rat and man are innervated by a dense network of trigeminal sensory fibres, ∼40% of which contain calcitonin gene-related peptide (CGRP) (Edvinsson et al., 1987 a, b; Keller and Marfurt, 1991; Messlinger et al., 1993 ). Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta. Circulation is available at http://circ.ahajournals.org. 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. 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). Small Molecule Calcitonin Gene-Related Peptide Receptor Antagonists for the Acute Treatment of Migraine. Cells were collected by centrifugation. αCGRP, a 37 amino-acid peptide, is formed by alternative splicing of the calcitonin gene, in particular in the central and peripheral nervous system, and acts as a potent vasodilator and modulator of cerebrovascular nociception. Treatment with 8-Br-Rp-cAMP prevented the CGRP-induced apoptosis (Fig. Nearly 95% of the cells expressed the enhanced GFP reporter when infected at a multiplicity of infection of 150 (Fig. CGRP can also interact with the amylin receptor composed of RAMP1 and the calcitonin receptor (33, 34). This relates to the fact that migraine involves activation of the trigeminal nervous system, resulting in CGRP release and subsequent cerebrovascular dilation and pain. RAMP1 virus titer was 7.2 × 1010 plaque-forming units (pfu)/ml, and the control viruses had similar titers of 4–5 × 1010 pfu/ml. Adenovirus titers were determined by plaque assays on HEK293 cells. A novel α-calcitonin gene-related peptide analogue protects against end-organ damage in experimental hypertension, cardiac hypertrophy, and heart failure. The mean intracellular cAMP levels ± sd of three experiments performed in duplicate are shown. The pathogenesis of atherosclerosis: a perspective for the 1990s. 4–6 were statistically compared by ANOVA (two-tailed), followed by Tukey’s test, and were considered significant at P < 0.05. 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. 2C). 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. 4A). 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. It is important to note that systemic coinjection of the α-analogue blunted the angiotensin II-induced rise in blood pressure, as well as the vascular and cardiac remodeling, changes in water consumption, and renal injury normally associated with angiotensin II infusion (Figure). Apoptosis in the vasculature: mechanisms and functional importance. Calcitonin gene-related peptide (CGRP) is expressed throughout the nervous system . In conclusion, the study by Aubdool et al5 sets the stage for the application of CGRP receptor agonists in cardiovascular disease. Tumor necrosis factor-α downregulates adrenomedullin receptors in human coronary artery smooth muscle cells. The endogenous receptor EC50 of CGRP was 1.2 ± 1.0 × 10 −7m (Fig. Local Info βCGRP is encoded by a second CGRP gene, predominantly expressed in the enteric sensory system.4 Although it shares 34 amino acids with αCGRP, its specific function is unknown. 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. TUNEL-stained samples were analyzed under a Zeiss LSM 510 confocal microscope, and at least 600 cells were counted in randomly selected fields. One consideration is that there are conflicting accounts whether aortic smooth muscle is responsive to CGRP in vivo.