The multiple RAMP1 species are indicated with arrows. Figure. Isoform 2: Receptor for calcitonin but is unable to couple to G proteins and activate adenylyl cyclase (PubMed: 7476993 ). Calcitonin Gene-Related Peptide Receptor Agonism, Global Impact of the 2017 ACC/AHA Hypertension Guidelines. 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). Calcitonin gene-related peptide innervation and binding sites in rat aorta during development. 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. On d 1–5, there were significantly fewer cells in the Ad-RAMP1 plus CGRP cultures (*, P < 0.01; **, P < 0.05). The endogenous receptor EC50 of CGRP was 1.2 ± 1.0 × 10 −7m (Fig. 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. Coexpression of RAMP2 or -3 with CLR yields adrenomedullin receptors (11, 16, 17). A final issue that remains is whether the α-analogue would trigger migraine attacks. Aorta smooth muscle cultures were either uninfected or infected with the control AdCMV-GFP virus or AdCMV-RAMP1. Effect of olmesartan medoxomil on number and survival of circulating endothelial progenitor cells and calcitonin gene related peptide in hypertensive patients. A previous study showed increased vascular smooth muscle cell migration after gene transfer of CLR and RAMP2 and -3, but not RAMP1 (27). Within the heart, RAMP1 and RAMP3 levels appear to change in the absence of changes in CLR expression after aorta banding (41). An ongoing role of α-calcitonin gene-related peptide as part of a protective network against hypertension, vascular hypertrophy, and oxidative stress. To our knowledge, this is the first study demonstrating an effect of RAMP1 gene transfer in vascular smooth muscle. 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. PMID 11298188. It is clear that both CLR and RAMP1 are required for CGRP receptor activity. 5). This demonstrates that cAMP is the main mediator of CGRP inhibition of cell proliferation. Receptors mediating CGRP-induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary artery differentiated by h(α) CGRP(8–37). 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. 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 monomer form of RAMP1 migrated close to the dye front of the gel. There is increasing evidence that free radical generation by oxidative stress is a component of cardiovascular pathologies, such as hypertension, diabetes, and arterial thrombosis (47–49). 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. Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells. A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. Calcitonin gene–related peptide receptor has been implicated in the pathogenesis of migraine. Sarcoplasmic reticulum Ca2+ ATPase2 is responsible for the transfer of Ca2+ from the cytosol to the lumen of the sarcoplasmic reticulum, thereby facilitating relaxation. Reactive oxygen species induce apoptosis of vascular smooth muscle cell. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. The cDNA was cloned into the pacAd5 cytomegalovirus (CMV)-K-N pA shuttle vector containing the CMV promoter at the BamHI site. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. 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). 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. The efficacy of CGRP signaling in RASM cells was increased in cells infected with RAMP1-expressing virus. The 460-bp insert extended from 6 bp upstream of the start codon to 7 bp downstream of the stop codon. Nearly 95% of the cells expressed the enhanced GFP reporter when infected at a multiplicity of infection of 150 (Fig. The discovery of RAMP1 has opened a new perspective on potential ways to regulate CGRP receptor activity. 3B). The calcitonin receptor (CTR) is a seven-transmembrane class II G-protein-coupled receptor for the 32-amino-acid calcitonin, and is highly expressed on the osteoclast plasma membrane. 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. C, Western blots of lysates prepared from uninfected, AdCMV-GFP-infected, and AdCMV-RAMP1-infected aorta smooth muscle cultures (passage 4). 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. 1). In the AdCMV-RAMP1-infected cultures, there were 9% TUNEL-positive cells after CGRP treatment. An adenoviral vector with the CMV promoter expressing enhanced GFP (AdCMV-GFP) was used as a control virus. 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. • Pondel M (December 2000). AdCMV-RAMP1 gene transfer increases CGRP-induced cAMP production. 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]. A, Schematic of the AdCMV-RAMP1 vector. Two control viruses were used. 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. CGRP treatment of AdCMV-RAMP1-infected cells caused a significant decrease in the number of cells (Fig. After thorough washing with PBST, immunocomplexes were visualized using enhanced chemiluminescence detection (Amersham Biosciences). The calcitonin receptor is encoded by the CALCR gene and comprises >90 kb and 14 exons. Cells were collected by centrifugation. However, given that neprilysin degrades multiple substrates in addition to natriuretic peptides, including amyloid β and endothelin-1, concerns exist about a link between neprilysin inhibition and Alzheimer’s disease1 and the detrimental consequences of endothelin-1 upregulation.2 Enhancing the nitric oxide-cyclic guanosine monophosphate pathway resulted in high rates of hypotension (cinaciguat), and heart failure trials investigating drugs interfering with this pathway were either inconclusive (vericiguat) or still need to be done on a sufficiently large scale (sildenafil).1. Dallas, TX 75231 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.). Circulation is available at http://circ.ahajournals.org. Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB), Journal of the American Heart Association (JAHA), Customer Service and Ordering Information, Basic, Translational, and Clinical Research. 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. 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). 2C). The study by Aubdool et al5 shows that αCGRP lowers blood pressure, reduces cardiac hypertrophy, and is renoprotective. Summary. The mean intracellular cAMP levels ± sd of three experiments performed in duplicate are shown. Adenovirus titers were determined by plaque assays on HEK293 cells. 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. Data are given as the mean ± sd. Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies. Its pharmacology is complicated by the existence of several splice variants. Indeed, elevation of cAMP was sufficient to inhibit proliferation and induce apoptosis of human aortic vascular smooth muscle cells (38). 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. 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). In addition to the decreased EC50, RAMP1 overexpression also increased the Rmax. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Vascular actions of calcitonin gene-related peptide and adrenomedullin. 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%). 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. Wiping out CGRP: potential cardiovascular risks. Calcitonin gene-related peptide is a potent vasodilator. The fraction of TUNEL-positive cells was determined from three to five independent experiments performed in triplicate. 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. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. The staining patterns were qualitatively similar in freshly isolated cells (passage 1) and late cultures (passage 13; not shown). Furthermore, there appear to be relatively few CGRP-binding sites (54). Dexamethasone increases RAMP1 and CRLR mRNA expressions in human vascular smooth muscle cells. Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter. Cell culture reagents were purchased from Invitrogen Life Technologies, Inc., unless otherwise stated. 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. The calcitonin receptor is thought to couple to the heterotrimeric guanosine triphosphate-binding protein that is sensitive to cholera toxin.
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