Beta-calcitonin Gene-related Peptide Research Articles

P177 Beta-CGRP in inflammatory bowel disease: a protective neuropeptide of the gut

Abstract Background Experimental models indicate that beta calcitonin gene-related peptide (beta CGRP), libertated by enteric neurons, has a potential role on gastrointestinal mucosa homeostasis, but its real role is not well profiled. We measured beta-CGRP circulating levels in a cohort of subjects with a recent diagnosis of inflammatory bowel disease (IBD), to assess the potential role of this neuropeptide in its pathogenesis. Methods Morning serum beta-CGRP levels were measured by ELISA (CUSABIO, China) in 96 consecutive patients recently diagnosed of IBD patients (< 1 year) and compared with those belonging to 50 matched healthy controls (HC) and 50 chronic migraine (CM) patients matched by age and sex. We only allowed treatment with either steroids or mesalazine, and registered cardiovascular comorbidities, autoimmune concomitant diseases or extraintestinal manifestations. Statistical analysis was made using SPSS®. Results Beta CGRP concentrations were lower in IBD patients (3.1±1.9 pg/mL; 2.9 [2.4-3.4] pg/mL) when comparted to HC (4.7±2.6; 4.9 [4.0-5.8] pg/mL; p<0.001) and CM patients (4.6±2.6; 4.7 [3.3-6.2] pg/mL; p<0.001). Respecting IBD subtypes, beta-CGRP levels were lower in ulcerative colitis patients (3.0±1.9pg/mL; 2.5 [2.1-3.4] pg/mL) as well as Crohn’s disease patients (3.3±2.0 pg/mL; 3.2 [2.4-3.9] pg/mL) to those of HC (p<0.01 and p<0.05, respectively) and CM (p<0.01 and p<0.05, respectively). Beta-CGRP levels in CM did not differ to those with HC (p=0.92). There were no significant differences when patients were classified by presence/absence of cardiovascular risk factors (yes: 3.1±1.7 pg/mL; no: 3.2±2.1 pg/mL; p=0.93); autoimmune comorbidities (yes: 3.4±1.4 pg/mL; no: 3.1±2.0 pg/mL; p=0.24); mesalazine treatment (yes: 3.1±1.9 pg/mL; no: 3.2±1.9 pg/mL; p=0.91); or steroid treatment (yes: 3.0±1.9 pg/mL; 3.2±1.9 pg/mL; p=0.52). Main characteristics and main results are exposed in table 1 and figure 1. Conclusion The reduction in serum beta-CGRP concentrations y patients with recent diagnosis of IBD, compared to two control groups, strongly supports a role for this neuropeptide in the pathophysiology of the disease since its early stages. This decrease was found in both IBD subtypes. Our data indicate a protective role of beta-CGRP in the homeostasis of the gastrointestinal tract. This study has been co-funded by Instituto de Salud Carlos III (ISCIII) through the project PI20/01358 and by Fondos Europeos de Desarrollo Regional (FEDER), "Una manera de hacer Europa".

P087 Calcitonin gene related peptide beta (CGRPβ): a new player in inflammatory bowel disease pathophysiology

Abstract Background Calcitonin gene related peptide (CGRP) is mainly present in the central nervous system (α isoform) and enteric nervous system (β isoform). CGRP is known to be the most potent vasodilator of the body, and it promotes migration of inflammatory cells. Although its function in the gut is not well established yet, CGRPβ is postulated to have a protective role against inflammation in the gut, promoting migration of inflammatory cells, regulating nociception, motility and gut microbioma. The aim of the study is to analyse serum levels of CGRPβ in patients newly diagnosed with inflammatory bowel disease (IBD), to unravel its potential role in the disease. Methods CGRPβ levels were measured by ELISA (CUSABIO, China) in early morning samples of newly diagnosed patients with IBD. Classification of the disease (ulcerative colitis- UC, Crohn’s disease- CD or unclassified inflammatory bowel disease- U-IBD), demographic data and treatment during sample collection were also collected. The results were compared with healthy controls (HC) stratified by age and sex. Statistical analysis was performed using SPSS and CGRPβ levels were compared with Mann-Whitney and Dunn’s tests. Results Seventy-one patients with newly diagnosed IBD (mean age 48.3±16.5 years, 63.4% females) were matched with 71 HC (mean age= 48.2±16.7 years-64.8% females). Samples were collected between 0 and 249 days after IBD diagnosis was established (median 49 days, interquartile range 29-66 days). Thirty patients were diagnosed as CD, 36 as UC and 5 as U-IBD. Most patients with CD had ileal location and inflammatory behaviour. One of these patients was on methotrexate due to a prior diagnosis of rheumatoid arthritis; the rest were either with no treatment, aminosalicylates, steroids or budesonide, or some combination of these. Half of the patients with UC had proctitis. Six had no treatment, and the rest were prescribed aminosalicylates, seven of them in combination with steroids. These characteristics are shown in table 1. Serum CGRPβ levels in patients with IBD were significantly decreased (2.95±1.68 pg/mL) compared to HC (4.65±2.59 pg/mL), (p<0.0001). These results stayed significant, in both CD (3.15±2.08 pg/mL; p<0.05), and UC (2.83±12.7 pg/mL; p <0.01), but not in IBD-U (2.56±1.73 pg/mL), though the number of U-IBD cases was low. CGRPβ concentrations did not differ significantly in patients with CD and UC. The results are illustrated in figures 1 and 2. Conclusion The consistent decrease in CGRPβ levels in IBD shown here strongly supports a protective role of this peptide in the homeostasis of the intestinal mucosa. This work was supported by a grant from the Instituto de Salud Carlos III (PI20/01358).

CALCB rs3829222 T/T Genotype and Low Expression of CALCB Are High-Risk Factors for Adenoid Cystic Carcinoma of Salivary Gland.

Objectives To investigate the relationship between polymorphisms of calcitonin-related peptide gene II (beta-calcitonin gene-related peptide (βCGRP), CALCB) and serum CGRP levels in salivary adenoid cystic carcinoma. Materials and Methods Using the polymerase chain reaction (PCR) technique, the full-length amplification and genotype analysis of CALCB genes were performed in 39 patients with adenoid cystic carcinoma of salivary gland and 158 normal controls. The gene frequencies of major genotype of CALCB in adenoid cystic carcinoma of salivary gland and normal control group were analyzed. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate serum calcitonin gene-related peptide (CGRP) and its concentration of alpha and beta subtypes. Results Univariate logistic regression analysis showed that the CALCB rs2839222 T/T genotype was closely related to the occurrence of salivary adenoid cystic carcinoma, with a correlation coefficient of 3.89. Conclusions The serum CGRP concentration in the salivary adenoid cystic carcinoma group was 1.56 times that of the normal control group. The αCGRP subtype was significant, which was 3.02 times that of the normal control. The polymorphism of βCGRP gene is associated with genetic susceptibility to salivary adenoid cystic carcinoma, and serum CGRP and βCGRP can be used as novel markers of salivary adenoid cystic carcinoma.

BIBN4096BS and CGRP8-37 antagonize the relaxant effects of α-CGRP more than those of β-CGRP in human extracranial arteries

We hypothesize that dilatation of extracranial arteries during migraine could be caused by CGRP. We compared the relaxant effects of alpha-calcitonin gene-related peptide (alpha-CGRP) and beta-calcitonin gene-related peptide (beta-CGRP) and the antagonism by BIBN4096BS and CGRP(8-37) on rings of human temporal and occipital arteries precontracted with KCl. beta-CGRP relaxed temporal (-logEC50M = 8.1) and occipital arteries (-logEC50M = 7.6) with 19-fold and 29-fold lower potencies respectively than alpha-CGRP. Nearly maximal effective concentrations of alpha-CGRP (4 nM) and beta-CGRP (50 nM) caused stable relaxations of the temporal artery for 4 h without fading. BIBN4094BS antagonized the effects of alpha-CGRP (pK(B) = 10.1 and 9.9, respectively) more than beta-CGRP (pK(B) = 9.3 and 9.2 respectively) on both temporal and occipital arteries. CGRP(8-37) antagonized the effects of alpha-CGRP (pK(B) = 6.6 and 6.4 respectively) more than beta-CGRP (pK(B) = 5.7 and 5.5 respectively) on both temporal and occipital arteries. Antagonism of the relaxant effects of alpha-CGRP (4 nM) and beta-CGRP (50 nM) by BIBN4096BS (10 and 100 nM) was reversible for beta-CGRP, but irreversible for alpha-CGRP, 1 h after BIBN4096BS washout. We conclude that alpha-CGRP and beta-CGRP interact either at different binding sites of the same CGRP receptor system or all together with different receptor systems in human extracranial arteries. BIBN4096BS binds more firmly to the receptor activated by alpha-CGRP than to the receptor activated by beta-CGRP.

Interleukin-1beta induces beta-calcitonin gene-related peptide secretion in human type II alveolar epithelial cells.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide mainly present in sensory nerve fibers, which is present in almost all organs, but it is also found in cultured rat type II alveolar epithelial cells (AEII). Our data have previously shown that CGRP may play an important role in inflammation as an immunomodulator. Proinflammatory factor IL-1beta induces CGRP release from neuron-derived sources. However, whether IL-1beta can induce CGRP secretion from a nonneural source, AEII cells, is not known. In the present study, we demonstrated that human AEII A549 cells expressed beta-CGRP, and IL-1beta (0.001-50 ng/ml) directly increased CGRP secretion from these cells in a time- and concentration-dependent manner. The mRNA level of beta-CGRP was also elevated by IL-1beta (1 ng/ml). In addition, we found that IL-1beta-induced CGRP production was mediated through the PKC-p38 mitogen-activated protein (MAP) kinase-NF-kappaB signaling pathway. Furthermore, IL-1beta-induced chemokines MCP-1 and IL-8 were partially inhibited by exogenous hCGRP (0.1-10 nM) and potentiated by hCGRP8-37 (0.1-10 nM), a CGRP1-receptor antagonist. In addition, the CGRP-inhibited chemokine effect was partially reduced by Rp-cAMP, a cAMP-PK inhibitor. These results suggest that AEII-derived CGRP may act in an autocrine/paracrine mode and play an important inhibitory role in the local area in lung inflammatory diseases.

Development and potential of non-peptide antagonists for calcitonin-gene-related peptide (CGRP) receptors: evidence for CGRP receptor heterogeneity.

Calcitonin-gene-related peptide (CGRP) is a 37-amino-acid vasodilatory peptide, of which two isoforms, alpha CGRP and beta CGRP, have been described. The use of C-terminal fragments of CGRP peptide, such as human alpha CGRP-(8-37), has led to the pharmacological subdivision of CGRP receptors into CGRP-1 [high potency for binding of human alpha CGRP-(8-37)] and CGRP-2 (lower potency) receptors. We have recently developed BIBN4096BS, the first non-peptide CGRP antagonist, which has sub-nanomolar affinity for primate CGRP receptors. The use of BIBN4096BS has led to the discovery of further functional CGRP receptor heterogeneity in rat tissues. To further exploit BIBN4096BS as a pharmacological tool, we used BIBN4096BS in pig left anterior descending coronary vessels and cerebral basilar arteries, and compared functional with molecular data, characterizing CGRP receptor components. Our data show that, apart from a subdivision into CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of alpha CGRP and beta CGRP. However, evidence for CGRP receptor heterogeneity on a molecular level is scarce.

Functional interaction of G protein-coupled receptors of the adrenomedullin peptide family with accessory receptor-activity-modifying proteins (RAMP).

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), calcitonin (CT), and amylin are homologous polypeptides with overlapping biological actions such as vasodilatation and inhibition of bone resorption. They are brought about through receptors that include the CT receptor (CTR) and an initially orphan CT receptor-like receptor (CRLR) in association with receptor-activity-modifying proteins (RAMP)1, -2, and -3. Co-expression of CRLR with RAMP1 or -2 revealed CGRP or AM receptors, respectively. The CTR interacts with CT and does not require a known RAMP for functional expression. The same CTR is a CGRP/amylin or an amylin receptor upon co-expression with RAMP1 or -3, respectively. Interactions between CRLR and RAMP are thought to be required for their delivery to the cell surface. There, heterodimeric complexes between CRLR or CTR and the corresponding RAMP reveal high-affinity receptors for AM, CGRP, and amylin. Here we review the current knowledge on interactions of G protein-coupled receptors with defined associated proteins.

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.

Role of alpha- and beta-calcitonin gene-related peptide in postoperative small bowel ileus

Ablation of α-calcitonin gene-related polypeptide (CGRP) containing neurons with the afferent neurotoxin capsaicin improves postoperative foregut transit in a rodent model. Similarly, administration of a selective α-CGRP antibody or hCGRP (8–37), a CGRP receptor antagonist, improves postoperative gastric emptying. Unlike the stomach, which contains only α-CGRP, the small bowel additionally contains β-CGRP. The role of the latter in postoperative small bowel transit is unknown. The purpose of this study was to evaluate the effect of an α-CGRP antibody and hCGRP (8–37) on postoperative small bowel transit. Male Sprague-Dawley rats underwent placement of duodenal catheters and were randomly assigned to 1 of 11 groups. Four groups were pretreated with 1% capsaicin. One week later, all animals underwent standardized laparotomy following administration of a control antibody or the α-CGRP monoclonal antibody, or during infusion of hCGRP (8–37) at varying doses. Small bowel transit was measured 25 minutes postoperatively. The α-CGRP antibody sped postoperative transit when given alone or in combination with capsaicin. In contrast, animals treated with hCGRP (8–37) showed no significant improvement in postoperative transit, and the beneficial effect of capsaicin was blocked. Unlike their similar effects on postoperative gastric emptying, we found that hCGRP (8–37) and the α-CGRP antibody had differing effects on postoperative small bowel transit. The reason for this is unknown but may be related to their differing specificities for α- and β-CGRP.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague–Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35 S -labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Effect of calcitonin gene-related peptide on sodium absorption through isolated skin of Rana esculenta.

Calcitonin gene-related peptide (CGRP) added to the internal fluid bathing the isolated skin of Rana esculenta strongly stimulates the active sodium absorption. This action is dose-dependent, the dose eliciting the maximal effect being 2 . 10(-7) M; alpha and beta CGRP exhibit the same potency. The CGRP action on sodium transport is mainly due to its interaction with CGRP1 receptors, since it is inhibited by CGRP8-37, its specific antagonist. The second messengers probably involved in the action of CGRP are cAMP and Ca+2, since this action is reduced by SQ22536 and W7, which are inhibitors of adenyl cyclase and calmodulin respectively. On the contrary, inhibitors of protein kinase C (1-O-hexadecyl-2-O-methyl-sn-glycerol) and nitric oxide synthase (L-NAME) do not modify the action on sodium transport. ETYA, an inhibitor of all the metabolic pathways of arachidonic acid, decreases the CGRP action by 38%. In order to search for the arachidonic acid metabolites involved in the CGRP action, the effect of the following inhibitors was tested: aspirin and naproxen (for cyclooxygenases), NDGA (for cyclooxygenases), NDGA (for lipoxygenases) clotrimazole (for epoxygenases). None of these substances is able to inhibit the CGRP action on sodium transport. Moreover, adding arachidonic acid inhibits the CGRP action, but this effect was also obtained by another unsaturated fatty acid, oleic acid. Since unsaturated fatty acids are able to inhibit the protein kinase A, these results indirectly support the role of cAMP as a second messenger of the CGRP action on sodium transport.

Amylin/islet amyloid polypeptide expression in medullary carcinoma of the thyroid: correlation with the expression of the related calcitonin/CGRP genes*

The amylin/islet amyloid polypeptide (IAPP) gene is the third member of the calcitonin (CT)/calcitonin gene related peptide (CGRP) gene family, which includes the alpha CT/CGRP (CALC-I) and beta CGRP (CALC-II) genes. CT is predominantly expressed in thyroid C cells, alpha and beta CGRP (CGRP-I and II) in neural tissue and amylin/IAPP in pancreatic beta cells. Both the detailed tissue distribution and the physiological role of amylin are subjects of current research. We sought to characterize the RNAs transcribed from the IAPP gene in normal human pancreas and to investigate possible ectopic expression of this gene in neuroendocrine tumours. DESIGN AND TISSUES: RNA was extracted from normal human pancreas, five phaeochromocytomas and 12 medullary thyroid carcinomas (MTCs) and studied by Northern blotting. We found that in normal human pancreas the IAPP gene transcripts differ in size from those reported for human insulinoma. Expression of the amylin/IAPP gene was detected in seven of the MTCs, while it was not detected in phaeochromocytomas. There were no apparent clinical or histological differences between IAPP positive and IAPP negative MTCs. The relative expression levels of the four mRNAs of the CT/CGRP gene family varied between the different tumours. Our findings are consistent with the view that ectopic hormone production may occasionally be causally related to the common origin of related genes. The possibility that IAPP may constitute a minor component of MTC amyloid should be considered.

GAP-43, aFGF, CCK and alpha- and beta-CGRP in rat spinal motoneurons subjected to axotomy and/or dorsal root severance.

The mRNA levels for growth-associated protein 43 (GAP-43), acidic fibroblast growth factor (aFGF), alpha- and beta-calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and choline acetyltransferase (ChAT) in rat lumbar spinal motoneurons were studied by in situ hybridization 1, 5 and 21 days and 20 weeks following unilateral peripheral nerve sectioning, ventral rhizotomy or dorsal rhizotomy. Furthermore, CGRP- and aFGF-like immunoreactivities in the ventral horn were studied using immunohistochemistry. One to 21 days after axotomy, GAP-43 and alpha-CGRP mRNAs increased in lesioned motoneurons, while the aFGF mRNA levels were marginally higher in motoneurons on the lesion side as compared to the control side. beta-CGRP, CCK and ChAT mRNA levels, on the other hand, decreased during the short-term response (1-21 days) to axotomy. After ventral rhizotomy, but not peripheral axotomy, there was complete disappearance of aFGF-like immunoreactivity in the ventral root proximal to the lesion. In animals subjected to long-term survival (20 weeks) after peripheral axotomy, the expression of all studied substances had returned to normal levels. Unilateral dorsal rhizotomy did not induce any substantial short- or long-term shifts in the cellular expression of the GAP-43, aFGF, CGRP and CCK peptides or their mRNAs in motoneurons of lesioned segments. These results indicate that peptides/proteins in motoneurons are expressed differentially after axotomy. Whereas alpha-CGRP and GAP-43 are up-regulated, CCK and beta-CGRP become down-regulated and aFGF is largely unaffected.

Amylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts

We have performed a set of independent studies on the effects of the circulating pancreatic polypeptide, amylin, on rat osteoclast function, in vitro. Time-lapse video observations, measuring cell protrusions and retraction, showed that 250 nmol l-1 amylin or 250 nmol l-1 beta-calcitonin gene-related peptide (beta-CGRP) inhibited osteoclast motility (quiescence or Q effect). Both amylin and beta-CGRP produced inhibitory responses with a significant first-order regression over time (half-times, 19 and 28 min respectively). In contrast, 250 nmol l-1 amylin or 250 nmol l-1 beta-CGRP produced no change of osteoclast spread area, whilst 300 pmol l-1 calcitonin (CT) application resulted in cell retraction (R effect). Forskolin (10 mumol l-1) mimicked amylin and CGRP in inhibiting osteoclast motility (half-time, 8.6 min), and similarly lacked an effect on cell spread area. Neither amylin nor beta-CGRP (62.5-1250 nmol l-1) elevated cytosolic free calcium levels ([Ca2+]i) in single osteoclasts whilst 300 pmol l-1 salmon calcitonin (sCT) produced a rapid phasic elevation of [Ca2+]i, confirming previous results with asusuberic (1-7) eel calcitonin. The osteoclast-bone resorption assay revealed the following potency difference in direct comparison of the area of resorption per bone slice: beta-CGRP/amylin, 0.1; sCT/amylin, 800 and human CT/amylin, 12. The potency of deamidated amylin approached that of beta-CGRP. Assay precision ranged between 0.3 and 0.8. Amylin (250 nmol l-1) also significantly (P < 0.05) reduced supernatant (tartrate-resistant) acid phosphatase in the bone-osteoclast cultures. These measures independently indicate an effect of amylin on osteoclast motility through mechanisms distinct from those of calcitonin, possibly through different selectivities for receptor subtypes, the cyclic AMP-linked 'amylin subtype' and the [Ca2+]i-linked 'calcitonin subtype'.

Involvement of multiple receptors in the biological effects of calcitonin gene-related peptide and amylin in rat and guinea-pig preparations.

1. The activity of rat alpha and beta calcitonin gene-related peptide (CGRP) as compared to the structurally related peptide, rat amylin, has been investigated in the guinea-pig isolated left atrium (electrically driven), in mucosa-free strips from the base of the guinea-pig urinary bladder and in the rat isolated vas deferens (pars prostatica). The antagonist activity of the C-terminal fragment of human alpha CGRP, alpha CGRP(8-37), was also investigated. 2. In the guinea-pig isolated left atrium the three peptides produced a concentration-related positive inotropic effect, amylin being about 16 and 31 times less potent than alpha or beta CGRP, respectively. Human alpha CGRP(8-37) produced a rightward displacement of the log concentration-response curve to the three agonists tested, without depression of maximal response attainable. Apparent pKB values calculated on the basis of the displacement produced by 1 microM human alpha CGRP(8-37) indicated an agonist-independent affinity of the antagonist (6.66 +/- 0.11 for alpha CGRP, 6.42 +/- 0.17 for beta CGRP and 6.95 +/- 0.11 for amylin). 3. In the guinea-pig isolated urinary bladder, alpha or beta CGRP or amylin produce a concentration-related inhibition of twitch contractions evoked by train electrical field stimulation (10 Hz frequency, 0.25 ms duration at 100 V for 0.5 s every 60 s). Amylin was about 100 times less potent than alpha or beta CGRP. Human alpha CGRP(8-37) (3 microM) did not significantly affect the inhibitory action of the three agonists tested.4. In the rat isolated vas deferens, a or PCGRP or amylin produced a concentration-related inhibition of twitch contractions evoked by electrical field stimulation (0.2 Hz frequency, 0.5 ms duration at 60 volts). Amylin was about 100 times less potent than a or PCGRP. Human aCGRP(8-37) at 3 microM did not significantly affect the inhibitory action of amylin and at 3 microM antagonized the responses to rat a and PCGRP with apparent pKB values of 5.86 +/- 0.15 and 6.11 +/- 0.13, respectively.5. These findings indicate that multiple receptors mediate the actions of peptides of the CGRP/amylin family in the preparations investigated. In the guinea-pig atrium both a and P forms of rat CGRP as well as amylin act by stimulating a single class of receptors which are sensitive to the inhibitory action of human aCGRP(8-37). In rat isolated vas deferens, at least two receptors could be present, one activated by a and PCGRP and partially sensitive to human aCGRP(8-37) and another which is sensitive to amylin but not recognised by human aCGRP(8-37). This latter type of receptor could be entirely responsible for the action of the agonists in the guinea-pig urinary bladder.

Peptide Contents of Neuropeptide Y, Vasoactive Intestinal Polypeptide, andβ-Calcitonin Gene-Related Peptide and Their Messenger Ribonucleic Acids after Dexamethasone Treatment in the Isolated Rat Islets of Langerhans

A number of neuropeptides including neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and beta-calcitonin gene related peptide (beta-CGRP) are known to influence insulin secretion. In order to investigate whether they might have a local autocrine/paracrine effect within the islets of Langerhans we screened isolated islets by Northern blot analysis and RIA for a number of peptides and found evidence for the presence of messenger RNA (mRNA) encoding NPY, VIP, and beta-CGRP. Dexamethasone treatment for 12 days increased the content of NPY, VIP, and beta-CGRP significantly from 1.3 +/- 0.3 to 19.8 +/- 1.6; 0.25 +/- 0.03 to 0.91 +/- 0.1; 2.2 +/- 0.2 to 4.8 +/- 0.1 fmol/islet respectively, mean +/- SEM (n = 4, P less than 0.05) and remained elevated 24 h after recovery. However when the results were normalized and expressed as a ratio of insulin content only NPY and VIP were significantly raised. Five days post treatment VIP was still significantly elevated compared to controls. mRNA for NPY increased 10-fold and for VIP increased 2 1/2 times after dexamethasone whereas mRNA for beta-CGRP was not significantly different from controls. Neither capsaicin nor 6-hydroxydopamine affected islet content or message of NPY, VIP, and beta-CGRP. Immunoreactive NPY and its mRNA were detected in two cultured beta-cell lines, HIT T-15 and RIN m5F cells whereas VIP and beta-CGRP were undetectable. The local islet synthesis of neuropeptides, which are known to influence islet hormone release pharmacologically, suggests the possibility that they may play a role in intraislet paracrine regulation.

A calcitonin gene‐related peptide (CGRP) antagonist (CGRP8–37) inhibits microvascular responses induced by CGRP and capsaicin in skin

1. The effect of the calcitonin gene-related peptide (CGRP) antagonist CGRP8-37 on responses to CGRP and other mediators was investigated in rabbit dorsal skin. 2. Blood flow changes at intradermally-injected sites were measured by a multiple site 133xenon clearance technique. CGRP8-37 had little effect on blood flow at doses up to 0.3 nmol/site, when injected alone, although a significant increase in blood flow was observed at the highest dose tested (1 nmol/site). 3. CGRP8-37 dose-dependently inhibited the increased blood flow induced by human alpha CGRP and human beta CGRP, but had no effect on equivalent vasodilator responses induced by vasoactive intestinal peptide (VIP) and prostaglandin E1 (PGE1). CGRP8-37 showed a preferential ability to inhibit alpha CGRP (IC50 0.04 nmol), when compared with beta CGRP (IC50 greater than or equal to 0.3 nmol). 4. Capsaicin, which selectively activates sensory nerves, caused a dose-dependent increase in blood flow when injected intradermally into rabbit skin. The effects of capsaicin (0.01-0.1 mumol/site) were inhibited by CGRP8-37 (0.3 nmol/site), with a partial but significant attenuation of blood flow induced by the highest dose of capsaicin. 5. Oedema formation, induced by intradermal histamine injection (3 nmol/site), was measured in rabbit skin by the local accumulation of intravenously-injected 125I-labelled albumin. Vasodilator doses of CGRP, PGE1 and capsaicin potentiated, in a synergistic manner, oedema formation induced by histamine. GRP8-37 totally inhibited the potentiating effect of CGRP, partially inhibited the synergistic effect of capsaicin, but did not affect PGE1-induced responses.6. The results suggest that capsaicin acts to release a rabbit form of CGRP in skin and that CGRP8 37 is a useful antagonist for investigating the potential of CGRP as a neurogenic mediator of inflammation.

Regional haemodynamic effects of human alpha- and beta-calcitonin gene-related peptide in conscious Wistar rats.

1. A comparative study was carried out to assess the regional haemodynamic effects of intravenous infusion of human alpha- and beta-calcitonin gene-related peptide (CGRP; 0.006, 0.06 and 0.6 nmol h-1) in conscious, unrestrained Wistar rats. 2. With human alpha-CGRP, tachycardia was always accompanied by a fall in mean arterial blood pressure (MBP), but human beta-CGRP at the middle dose caused tachycardia with no significant hypotension. 3. Human alpha- and beta-CGRP both caused dose-dependent falls in MBP accompanied by common carotid and hindquarters vasodilatations and increased flows. The highest dose of alpha- and beta-CGRP caused mesenteric vasoconstriction and renal vasodilatation, but the latter was not associated with an increase in flow. 4. With the intermediate dose, human alpha-CGRP caused more significant haemodynamic changes than did human beta-CGRP. In addition, comparison with previous experiments involving infusion of rat alpha-CGRP indicated that human alpha-CGRP had more potent haemodynamic effects than the former.

Both alpha- and beta-calcitonin gene-related peptides are present in plasma, cerebrospinal fluid and spinal cord in man.

The presence of both alpha- and beta-calcitonin gene-related peptide (CGRP) was demonstrated by oxidation and by CNBr cleavage in extracts of plasma, cerebrospinal fluid and spinal cord in man. This was achieved by the use of both CNBr cleavage and oxidation of the methionine residue present in the human beta-CGRP molecule. This study demonstrates that around 50% of CGRP immunoreactivity in plasma, cerebrospinal fluid and spinal cord is not alpha-CGRP, but corresponds to beta-CGRP-like activity. Furthermore, experiments with CNBr also suggest the presence of another methionine-containing CGRP-like peptide in all three extracts.

Human α- and β-calcitonin gene-related peptides are vasodilators in human chorionic plate vasculature

Calcitonin gene-related peptide receptors are known to be present in the placenta. We studied the effect of alpha- or beta-calcitonin gene-related peptide or carrier on human chorionic plate arteries preconstricted with angiotensin II. Both forms of calcitonin gene-related peptides evoked dose-dependent relaxation, the magnitude of which was proportional to the initial response to angiotensin II.