calcitonin receptor gene

2B). Thus, an interaction with multiple deleterious hormonal systems is indeed feasible (Figure). 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 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). Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature. In addition to the decreased EC50, RAMP1 overexpression also increased the Rmax. 4B). The Association between Morphological and Functional Characteristics of the Bicuspid Aortic Valve and Bicuspid Aortopathy. Rimegepant is an orally administered, small-molecule, calcitonin gene–related peptide receptor antagonist that may be effective in acute migraine treatment. The cDNA was cloned into the pacAd5 cytomegalovirus (CMV)-K-N pA shuttle vector containing the CMV promoter at the BamHI site. Cells were treated with the enzyme glucose oxidase, which has been shown to generate hydrogen peroxide and cause vascular smooth muscle cell apoptosis (29). B, A representative figure (passage 2 cells) of four experiments shows decreased EC50 and increased maximal response in the cultures containing recombinant RAMP1. 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. https://doi.org/10.1161/CIRCULATIONAHA.117.028955, National Center To our knowledge, this is the first study demonstrating an effect of RAMP1 gene transfer in vascular smooth muscle. Nearly 95% of the cells expressed the enhanced GFP reporter when infected at a multiplicity of infection of 150 (Fig. 112 The complexity of CALCR is shared by other class II G protein–coupled receptors, and the structural organization and size of the human gene are similar to the pig CTR gene. "Calcitonin and calcitonin receptors: bone and beyond". Dallas, TX 75231 Expression of CLR, RAMP1, and RCP was confirmed in rat aorta smooth muscle cells under our culture conditions (Fig. The authors thank Alexander Góes Martini for generating the Figure. Confocal images of immunocytochemical staining of passage 3 cells with antisera directed against CLR (A), RAMP1 (B), and RCP (C). • Pondel M (December 2000). Effect of calcitonin gene-related peptide on cytosolic free Ca. On d 0, there was no significant difference among the different cultures. Biological importance of the peptides of the calcitonin family as revealed by disruption and transfer of corresponding genes. CGRP treatment of AdCMV-RAMP1-infected cells caused a significant decrease in the number of cells (Fig. To match the treatment conditions of the second and third paradigms, cell number was also measured after 24 h in the absence of serum. Since the neuropeptide calcitonin gene-related peptide (CGRP) has been established as a key player in the pathogenesis of migraine, CGRP receptor antagonists have been considered useful compounds to block headache originating from hyperactivation of such TG neurons. A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. 3B). As a model system to study the regulation of CGRP receptor activity, we used primary cultures of rat aorta smooth muscle. Given the lack of a comparator antihypertensive drug, it cannot be concluded whether the effects of the α-analogue in the angiotensin II infusion model were the consequence of blood pressure lowering per se, ie, whether they represented physiological antagonism. Recently, in vitro CGRP gene transfer was also shown to inhibit aortic and pulmonary artery smooth muscle cell proliferation (35). The receptors for the other members the family are made up of subunits. 1). After thorough washing with PBST, immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham Biosciences). Protein was measured by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). THE CARDIOVASCULAR system is richly innervated by perivascular calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers (1, 2). The presence of mRNA encoding CLR and all three RAMP proteins has been previously reported (27). 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. Adenovirus titers were determined by plaque assays on HEK293 cells. 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. The study by Aubdool et al5 shows that αCGRP lowers blood pressure, reduces cardiac hypertrophy, and is renoprotective. Calcitonin gene-related peptide is a member of the calcitonin family of peptides, which in humans exists in two forms: α-CGRP and β-CGRP, also known as CGRP I and CGRP II. 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). The molecular pharmacology of CGRP and related peptide receptor subtypes. © American Heart Association, Inc. All rights reserved. The CGRP receptor comprises calcitonin receptor-like receptor (CLR), a GPCR, and receptor activity modifying protein 1 (RAMP1), a single transmembrane protein that imparts ligand specificity and ensures CLR targeting to the cell surface. Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Post-transcriptional regulation of CRLR expression during hypoxia. In the third condition, we asked whether the CGRP-induced reduction in cell number was due to apoptosis. Protective effects were also seen when starting α-analogue treatment only during the last week of the 2-week angiotensin II infusion, ie, when hypertension was already established. CGRP significantly increased cAMP levels relative to control for all three conditions (*, P < 0.05; ***, P < 0.001). 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). 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. Furthermore, we need to know the consequence of binding of the α-analogue to albumin. use prohibited. Human RAMP1 cDNA (4) in pcDNA 3 was a gift from Steve Foord (GlaxoSmithKline, Inc., Research Triangle Park, NC). 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. A previous study showed increased vascular smooth muscle cell migration after gene transfer of CLR and RAMP2 and -3, but not RAMP1 (27). 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. Circulation is available at http://circ.ahajournals.org. Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling. Alternatively, αCGRP may stimulate amylin-1 (AM-1) receptors. Protein size standards are shown. Scale bar, 20 μm. Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats. Samples were centrifuged at 3000 × g for 10 min at 4 C. Supernatants were saved at −20 C until assayed. Among its related pathways are Signaling by GPCR and G alpha (s) signalling events . 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. Lennerz JK, Ruhle V, Ceppa EP, Neuhuber WL, Bunnett NW, Grady EF, Messlinger K. Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: differences between peripheral and central CGRP receptor distribution. Tumor necrosis factor-α downregulates adrenomedullin receptors in human coronary artery smooth muscle cells. Migraine is a common neurological disease and one of the leading causes of disability. Receptor for adrenomedullin together with RAMP2 (PubMed:22102369, PubMed:30115739). 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 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. Consistent with these effects, the aorta is innervated by CGRP-containing fibers, albeit at comparatively low levels (1, 54). Cultures grown to 70% confluence were either uninfected or infected with AdCMV-RAMP1 or AdCMV-GFP (multiplicity of infection, 150). CGRP can also interact with the amylin receptor composed of RAMP1 and the calcitonin receptor (33, 34). The effect of CGRP and RAMP1 was particularly striking in combination with free radicals. Cells were collected by centrifugation. CGRP and its receptors provide new insights into migraine pathophysiology. Search for other works by this author on: Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye. Compelling evidence for the importance of CGRP in migraine has been provided by clinical trials with multiple small molecule CGRP receptor antagonists. The cells were cultured with DMEM high-glucose medium containing 0.1 mm MEM nonessential amino acids, 2 mml-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin-streptomycin, 0.01 m HEPES (pH 7.2), 20% fetal bovine serum, and 1× Basal Medium Eagle vitamins (Sigma-Aldrich Corp., St. Louis, MO) for 24 h in a humidified 5% CO2 incubator. Sex steroids appear to regulate CLR and RAMP levels during pregnancy (43). cAMP levels were measured using a RIA kit as recommended by the manufacturer for the overnight protocol (Amersham Biosciences, Arlington Heights, IL). The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator that plays a protective role in the cardiovascular system. Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies. This central feature is associated with migraine. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Calcitonin gene-related peptide (CGRP) is expressed throughout the nervous system . Vascular actions of calcitonin gene-related peptide and adrenomedullin. Its downregulation in patients with heart failure is well established. 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. CGRP induced an approximately 8-fold increase in intracellular cAMP after RAMP1 gene transfer. Diseases associated with CALCRL include Lymphatic Malformation 8 and Primary Angle-Closure Glaucoma . 114131 - CALCITONIN RECEPTOR; CALCR - CTR; CTR1 - CALCR In a Japanese population, Nakamura et al. 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.). It is clear that both CLR and RAMP1 are required for CGRP receptor activity. International Union of Pharmacology. Calcitonin gene-related peptide (CGRP), a member of the calcitonin family, is a peptide that so far has received little attention in heart failure.3 It exists in 2 isoforms: αCGRP and βCGRP. An adenoviral vector with the CMV promoter expressing enhanced GFP (AdCMV-GFP) was used as a control virus. Two control viruses were used. Among others, such mice displayed a higher blood pressure response to angiotensin II,7 suggesting that CGRP receptor stimulation might be a valuable option to treat cardiovascular diseases. However, in the absence of vascular studies quantifying endothelial function (rather than endothelial nitric oxide synthase expression), this conclusion seems premature. Hypertrophy of vascular smooth muscle occurs in response to arterial injury, angioplasty, and atherosclerosis (21, 22). CGRP may play a causative role in migraine. March 2020. 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. 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). Recent developments include the inhibition of neprilysin with sacubitril (to increase the levels of natriuretic peptides) and drugs that stimulate the nitric oxide-cyclic guanosine monophosphate pathway.1 The latter may be achieved by enhancing soluble guanylate cyclase nitric oxide sensitivity (with vericiguat), directly activating soluble guanylate cyclase (with cinaciguat), or inhibiting the enzyme that breaks down cyclic guanosine monophosphate, phosphodiesterase-5, with sildenafil. 3B). Figure. Dexamethasone increases RAMP1 and CRLR mRNA expressions in human vascular smooth muscle cells. 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). The mechanisms underlying these effects involved cAMP-dependent pathways. 3A) were compared by ANOVA and Tukey’s test. Its major second messenger is cyclic adenosine monophosphate. The pathogenesis of atherosclerosis: a perspective for the 1990s. Unauthorized 2C). 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%). The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. The means in Figs. The authors conclude that the α-analogue does not cross the blood-brain barrier because, unlike the nitric oxide donor glyceryl trinitrate, it did not induce light avoidance. Dr MaassenVanDenBrink received grant support from Amgen and Novartis. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Assays were performed in duplicate in at least three independent experiments. PMC 2517743. 1-800-242-8721 A final issue that remains is whether the α-analogue would trigger migraine attacks. 1999; 140 : 2883-2890 CGRP blocking medications … The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. 4A). GeneCards Summary for CALCRL Gene. This is reminiscent of the disappearance of β-adrenergic receptors in the hearts of patients with heart failure with excessive cardiac norepinephrine levels. 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. 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. The same filter was sequentially incubated with antisera to detect RAMP1, CLR, and GAPDH, as indicated. However, most of these studies have been performed in immortalized cells that do not necessarily express endogenous CGRP receptor proteins. Cells were rinsed once with ice-cold PBS, scraped, and collected by centrifugation. 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). (1997) found a RFLP in the CALCR gene by the use of an AluI restriction enzyme at nucleotide 1377, expressing either proline (CC genotype) or leucine (TT genotype) at amino acid 463.

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