Positive inotropic effects and receptors of calcitonin gene-related peptide (CGRP) in porcine ventricular muscles

Calcitonin gene-related peptide (CGRP) in porcine ventricular muscles. positive inotropic effects in the isolated, electrically driven false tendon of the porcine heart. Specific CGRP-binding sites were present in solubilized membrane fractions; the dissociation constant (Kd) and the maximum binding (Bmax) were 50.4 pM and 180 fmol/mg protein, respectively. SDS-PAGE analysis of CGRP-binding sites revealed the molecular mass of 70 K and 120 K. Few CGRP-like immunoreactive nerves were present in the ventricular muscle layer. These results indicate that CGRP activates specific receptor sites on the ventricular muscles and causes positive inotropic responses. CGRP receptors in ventricles are likely to be activated by circulating CGRP.     

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal mactophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.     

Synthesis of calcitonin gene-related peptide (CGRP) by rat arterial endothelial cells

We investigated the protein and mRNA expression of calcitonin gene-related peptide (CGRP) in endothelial cells of the rat thoracic aorta and femoral artery. Light microscopic immunocytochemistry revealed that immunoreactivity for CGRP was preferentially located in the endothelium of both vessels. Immunoelectron microscopy showed that CGRP-immunoreactive gold particles were preferentially localized on cisterns of the rough endoplasmic reticulum and on the Weibel-Palade (WP) bodies in the endothelial cells. Prepro CGRP mRNA signals were also detected on the endothelium. Our results are the first to demonstrate that endothelial cells of both elastic and large muscular arteries synthesize CGRP and store it, in part, in WP bodies, implying that CGRP may act as an endothelium-derived relaxing factor in these vessels.     

降钙素基因相关肽(CGRP)mRNA在大鼠淋巴细胞中的表达

最近,我们研究发现大鼠胸腺和淋巴结淋巴细胞中存在降钙素基因相关肽（ＣＧＲＰ）样免疫反应活性。应用人工合成的可特异扩增降钙素／降钙素基因相关肽基因部分片断的寡核苷酸引物,通过逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）,检测在大鼠脊髓背根神经节、胸腺细胞及肠系膜淋巴结淋巴细胞中是否存在ＣＧＲＰ ｍＲＮＡ,进一步研究大鼠淋巴细胞能否合成ＣＧＲＰ。结果显示,通过ＲＴ－ＰＣＲ从脊髓背根神经节（阳性对照）、胸腺和淋     

Activities of calcitonin gene-related peptide (CGRP) and related peptides at the CGRP receptor.

In guinea pig pancreatic acini rat calcitonin gene-related peptide (CGRP) increased amylase release 2-fold, salmon calcitonin had an efficacy of only 44% of that of CGRP and [Tyr0]CGRP(28-37) and human calcitonin had no actions. [Tyr0]CGRP(28-37), but not human calcitonin, antagonized the actions of CGRP in pancreatic acini with an IC50 of 3 microM. [Tyr0]CGRP(28-37) produced a parallel rightward shift in the dose-response curve for CGRP-stimulated amylase secretion. The inhibition was specific for CGRP and was reversible. Studies with 125I-CGRP demonstrated that CGRP, salmon calcitonin and [Tyr0]CGRP, but not human calcitonin, interacted with CGRP receptors on pancreatic acini. These results indicate that various CGRP-related peptides demonstrate different relationships between their abilities to occupy the CGRP receptor and to affect biologic activity, with CGRP itself being a full agonist, salmon calcitonin a partial agonist, [Tyr0]CGRP(28-37) a competitive antagonist, and human calcitonin having no actions.     

The role of calcium in endotoxin-induced release of calcitonin gene-related peptide (CGRP) from rat spinal cord

In the present study, the role of calcium in endotoxin-induced CGRP release was studied. 2 .5-50 μg/mL endotoxin and 1 -10 mmol/L caffeine caused concentration-dependent increase of CGRP release from rat spinal cord in vitro. However, no additive effect could he found when caffeine and endotoxin were concomitantly incubated. By using capsaicin, Ca2 -free medium, Omega-Conotoxin, nifedipine, W-7, ryanodine, MgCl2, Tris-ATP, rutheni-um red, the results indicate that the release of CGRP evoked by endotoxin from the sensory fibers of rat spinal cord is dependent on extracellular calcium. After entering into the cell through the N-type calcium channel, calcium binds to calmodulin, and triggers calcium release from intracellular calcium store by activating the caffeine-sensitive but ryan-odine-insensitive mechanism.     

The receptor of calcitonin gene-related peptide (CGRP) is present on the membranes of insulinoma

We demonstrate here that membranes prepared from beta cells which release insulin contain specific binding sites for calcitonin gene-related peptide (CGRP). The binding of 125I(His) human CGRP to beta cell membranes was protein concentration, time, temperature and pH dependent. Scatchard analysis of the data revealed a single class of binding sites with an apparent dissociation constant (Kd) of 1.5 nM and a maximal binding capacity of 19 fmol/mg of protein. Chicken CGRP inhibited the label binding whereas salmon calcitonin had only a weak effect. These results suggest that the effect of CGRP on insulin secretion is due to a direct action on beta cells.     

Preparation of imidazoles as potent calcitonin gene-related peptide (CGRP) antagonists

Several new potent CGRP receptor antagonists have been prepared in which the amide bond of lead compound 1 has been replaced by bioisosteric imidazole moieties. Substitution at N-1 of the imidazole was optimized to afford compounds with comparable potency to that of lead 1. Conformational restraint of the imidazole to form tetrahydroimidazo[1,5-a]pyrazine 43 gave substantially improved permeability.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.     

Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects

The distribution of perivascular nerve fibers displaying calcitonin gene-related peptide (CGRP) immunoreactivity and the effect of CGRP on vascular smooth muscle were studied in the guinea-pig. Perivascular CGRP fibers were seen in all vascular beds. Generally, they were more numerous around arteries than veins. Small arteries in the respiratory tract, gastrointestinal tract and genitourinary tract had numerous CGRP fibers. The gastroepiploic artery in particular received a rich supply of such fibers. Coronary blood vessels had a moderate supply of CGRP fibers. In the heart, a moderate number of CGRP fibers was seen running close to myocardial fibers. The atria had a richer supply than the ventricles. Numerous CGRP immunoreactive nerve cell bodies and nerve fibers were seen in sensory (trigeminal, jugular and spinal dorsal root) ganglia. Sequential or double immunostaining with antibodies against substance P and CGRP suggested co-existence of the two peptides in nerve cell bodies in the ganglia and in perivascular fibers. In agreement with previous findings CGRP turned out to be a strong vasodilator in vitro as tested on several blood vessels (e.g. basilar, gastroepiploic and mesenteric arteries). Conceivably, perivascular CGRP/SP fibers have a dual role as regulator of local blood flow and as carrier of sensory information.     

Benzodiazepine calcitonin gene-related peptide (CGRP) receptor antagonists: optimization of the 4-substituted piperidine

In our continuing effort to identify CGRP receptor antagonists for the acute treatment of migraine, we have undertaken a study to evaluate alternative 4-substituted piperidines to the lead dihydroquinazolinone 1. In this regard, we have identified the piperidinyl-azabenzimidazolone and phenylimidazolinone structures which, when incorporated into the benzodiazepine core, afford potent CGRP receptor antagonists (e.g., 18 and 29). These studies produced a potent analog (18) which overcomes the instability issues associated with the lead structure 1. A general pharmacophore for the 4-substituted piperidine component of these CGRP receptor antagonists is also presented.     

Pharmacological evidence for the involvement of multiple calcitonin gene-related peptide (CGRP) receptors in the antisecretory and antiulcer effect of CGRP in rat stomach.

We have investigated the effect of the C-terminal fragment of human calcitonin gene-related peptide (human-CGRP8-37), a CGRP antagonist, on alpha-CGRP and salmon Calcitonin (sCT)-induced inhibition of gastric acid secretion stimulated by pentagastrin (24 nmol kg-1 h-1 i.v.) and gastric lesions induced by acetylsalycilic acid (ASA; 25 mM) in rats anaesthetized with urethane. Close intra arterial infusion of alpha-CGRP (2-5 nmol kg-1) and sCT (5 nmol kg-1) produced a reduction in gastric acid hypersecretion induced by pentagastrin. The concomitant infusion with human-CGRP8-37 (10 nmol kg-1) reversed the effect of both agonists. ASA-ulcers were reduced in a dose-dependent manner by infusion of alpha-CGRP (1-2 nmol kg-1 i.a.), but not by sCT (10 nmol kg-1 i.a.). Human-CGRP8-37 at a dose of 10 nmol kg-1 i.a. was unable to reverse the alpha-CGRP antiulcer effect. An higher dose of human-CGRP8-37 (50 nmol kg-1 i.a.) showed agonistic properties reducing ASA ulcers. These results suggest that the inhibitory effects of alpha-CGRP on stimulated acid secretion and aspirin ulcers are mediated by different mechanisms and/or different receptors.     

Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP)

The biological activities of calcitonin gene-related peptide (CGRP) enzymic digest fragments, chemically modified products and beta-CGRP have been compared to that of intact alpha-CGRP on rat isolated paired atria. Tryptic and chymotryptic digests both produced inactive fragments. Acetylation of the N-terminal amino acid (Alanine) or either of Lys 24 or Lys 35, resulted in reduced, but measurable, biological activity. Destruction of the disulphide bridge between Cys 2 and Cys 7 abolished biological activity. Substitution of several amino acids, Asp 3, Val 22 and Asn 25, with Asn, Met and Ser respectively (beta-CGRP), produced a peptide with similar biological activity to alpha-CGRP.     

Optimization of azepanone calcitonin gene-related peptide (CGRP) receptor antagonists: Development of novel spiropiperidines

Several novel spiropiperidine-based CGRP receptor antagonists have been developed that maintain good potency and have reduced potential for metabolism.     

The neuropeptide calcitonin gene-related peptide differently modulates proliferation and differentiation of smooth muscle cells in culture depending on the cell type

Calcitonin gene-related peptide (CGRP) is a neuropeptide present around vasculature very early during development, when smooth muscle cells (SMC) are still proliferating and not yet totally differentiated. We investigated the effects of CGRP on proliferation and differentiation of SMC in culture; 10(-7) M CGRP added in the medium of cultured smooth muscle cells every 2 days did not significantly changed cells growth rate in 1% FCS. At the opposite, this treatment modulated proliferation of cells grown in 10% FCS medium. Two distinct populations of SMC with different growth rates were obtained from our primary cultures. SMC which proliferated slowly in the presence of 10% fetal calf serum (FCS) had growth rates positively influenced by CGRP. The quantity of alpha-smooth actin expressed by these cells was not influenced by the peptide. On the contrary, SMC which proliferated more rapidly in 10% FCS medium had growth rate inhibited by CGRP. In these cells, CGRP significantly reduced the amount of expressed alpha-smooth actin, an index of SMC differentiation. In both cases, the peptide significantly increased the level of mRNA for all the actin genes. In the light of this dual role of CGRP, it can be presumed that this peptide controls smooth muscle cells proliferation and differentiation in vivo and could thus regulate the homeostasis of the vessel wall.     

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.     

Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

125I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow.     

Orally bioavailable imidazoazepanes as calcitonin gene-related peptide (CGRP) receptor antagonists: discovery of MK-2918

In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).     

Production and characterisation of immunoreactive calcitonin gene-related peptide (CGRP) from a CGRP receptor-positive cloned osteosarcoma cell line (UMR 106.01)

A subclone of an osteoblast-like osteosarcoma cell line (UMR 106.01) has recently been shown to possess specific binding sites for calcitonin gene-related peptide (CGRP) linked to adenylate cyclase. The present study provides the first demonstration for the production of immunoreactive CGRP from CGRP-receptor positive osteosarcoma cells. Mean immunoreactive CGRP levels were 15 pmol/g and 1 pmol/l for acid extracts of cells and cell-exposed media respectively. On gel filtration and high performance liquid chromatography, a major proportion of immunoreactive CGRP was found to co-elute with synthetic rat CGRP(1-37). Only negligible quantities of calcitonin were detected in cell extracts or cell-exposed supernatant. The production of authentic CGRP from a CGRP-receptor positive tumour suggests that the peptide may have autocrine effects on its producer cell.