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.     

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.     

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     

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.     

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.     

Role of calcitonin gene-related peptide in the phenol-induced neurogenic hypertension in rats

Previous investigations have demonstrated that capsaicin-sensitive sensory nerves are involved in the development of hypertension in some rat models of hypertension. To determine the role played by calcitonin gene-related peptide (CGRP; the predominant neurotransmitter in capsaicin-sensitive sensory nerves) in a rat model of neurogenic hypertension, in which hypertension was induced by injecting 50 microl of 10% phenol in the lower pole of the left kidney, systolic blood pressure (SBP) was monitored by the tail-cuff method throughout the experiment. Fifteen days after injection of phenol, mean arterial pressure (MAP), concentrations of CGRP in the plasma, the expression of CGRP mRNA in dorsal root ganglia (DRG) and CGRP content in laminae I and II of the spinal cord were measured. SBP was significantly increased 5 days after the intrarenal injection of phenol (164+/-7 mm Hg, p<0.01). At the end of experiment, blood pressure (BP) was significantly elevated in the phenol-injected rats compared with the controls (SBP: 187+/-6 vs. 122+/-4 mm Hg, p<0.01; MAP: 157.56+/-3.02 vs. 103.80+/-2.04 mm Hg, p<0.01). Treatment with capsaicin, which selectively depletes neurotransmitters from the capsaicin-sensitive nerves, failed to enhance the development of hypertensive responses to the intrarenal injection of phenol. Intravenous administration of CGRP(8-37), the specific CGRP receptor antagonist, also failed to increase the already elevated MAP. The expression of CGRP mRNA (both alpha- and beta-CGRP isoforms), the content of CGRP in laminae I and II of the dorsal horn of the spinal cord and the concentration of CGRP in the plasma was decreased in the rats treated with phenol. These results suggest that CGRP does not play a counterregulatory role in the phenol-induced hypertensive rats, and support the hypothesis that reduction of CGRP (alpha and beta isoforms) could contribute to a blood pressure elevation in this setting.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Adrenomedullin inhibits neurotransmission of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves in rat mesenteric resistance arteries.

We have reported that the rat mesenteric resistance artery has innervation of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves). We also demonstrated that adrenomedullin (AM) causes mesenteric vasodilation through activation of CGRP receptors. The present study was designed to examine the effect of AM on neurotransmission of CGRPergic nerves in rat mesenteric arteries. In preconstricted preparations without endothelium, periarterial nerve stimulation (PNS, 1 and 2 Hz) induced a frequency-dependent vasodilation. A bolus injection of CGRP (10 pmol) into the perfusate also caused a vasodilation. AM (0.1 to 10 nM) concentration-dependently caused 40% to 60% inhibition of the PNS-induced vasodilation, but AM did not attenuate vasodilation induced by exogenous CGRP injection. The inhibitory effect of AM (10 nM) on PNS-induced vasodilation was further potentiated by CGRP [8-37] (CGRP receptor antagonist, 50 nM), which attenuated the vasodilator response to the CGRP injection. Combined perfusion of AM [22-52] (AM receptor antagonist, 10 to 100 nM) resulted in further inhibition of PNS-induced neurogenic vasodilation without affecting the vasodilator response to the CGRP injection. CGRP [8-37] but not AM [22-52] antagonized vasodilation induced by AM perfusion. These findings suggest that AM presynaptically inhibits neurotransmission of CGRPergic nerves, probably decreasing CGRP release, via receptors different from CGRP receptors.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

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.     

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.     

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).     

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.