Alpha-calcitonin Gene-related Peptide Research Articles

Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene.

Calcitonin (CT) is a known inhibitor of bone resorption. Calcitonin gene-related peptide-alpha (CGRPalpha), produced by alternative RNA processing of the CT/CGRP gene, has no clearly defined role in bone. To better understand the physiologic role of the CT/CGRP gene we created a mouse in which the coding sequences for both CT and CGRPalpha were deleted by homologous recombination. The CT/CGRP(-/-) knockout (KO) mice procreated normally, there were no identifiable developmental defects at birth, and they had normal baseline calcium-related chemistry values. However, KO animals were more responsive to exogenous human parathyroid hormone as evidenced by a greater increase of the serum calcium concentration and urine deoxypyridinoline crosslinks, an effect reversed by CT and mediated by a greater increase in bone resorption than in controls. Surprisingly, KO mice have significantly greater trabecular bone volume and a 1.5- to 2-fold increase in bone formation at 1 and 3 months of age. This effect appears to be mediated by increased bone formation. In addition, KO mice maintain bone mass following ovariectomy, whereas wild-type mice lose approximately one-third of their bone mass over 2 months. These findings argue for dual roles for CT/CGRP gene products: prevention of bone resorption in hypercalcemic states and a regulatory role in bone formation.

The transmembrane domain of receptor-activity-modifying protein 1 is essential for the functional expression of a calcitonin gene-related peptide receptor.

Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [(125)I]h alpha CGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to h alpha CGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.

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.

Involvement of alpha-calcitonin gene-related peptide in heat stress-induced delayed preconditioning in rat hearts.

1. Previous studies have shown that hyperthermia is capable of activating capsaicin-sensitive sensory nerves and stimulating the release of neurotransmitters from their peripheral terminals. Calcitonin gene-related peptide (CGRP) has recently been found to participate in delayed cardioprotection in rat isolated hearts. 2. The purpose of the present study was to explore whether the delayed cardioprotection by heat stress in vivo involves the expression and release of CGRP. 3. Sprague-Dawley rats were pretreated with whole-body hyperthermia (rectal 42 degrees C) for 15 min, 24 h before the experiments and then the left main coronary artery of rat hearts was subjected to a 45 min occlusion followed by 3 h reperfusion. The degree of myocardial injury was evaluated by measurement of infarct size and plasma creatine kinase (CK) activity. The plasma levels of CGRP and expression of CGRP (alpha and beta isoforms) mRNA in lumbar dorsal root ganglia at 4, 8, 16 or 24 h after heat stress treatment were measured. 4. Pretreatment with hyperthermia significantly reduced infarct size and CK release. Heat stress also significantly increased plasma concentrations of CGRP and the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA. The effect of heat stress was completely abolished by pretreatment with capsaicin (50 mg/kg, s.c.), which selectively depletes transmitters in capsaicin-sensitive sensory nerves. 5. In summary, the results suggest that the delayed cardioprotection by heat stress involves the synthesis and release of CGRP and that the protection is mainly mediated by the alpha-CGRP isoform.

Delayed cardioprotection induced by nitroglycerin is mediated by alpha-calcitonin gene-related peptide.

Previous investigations have demonstrated that early preconditioning induced by nitroglycerin is mediated by calcitonin gene-related peptide (CGRP). In the present study, we addressed the question of whether delayed preconditioning induced by nitroglycerin in the rat is related to stimulation of the release and synthesis of CGRP. Sprague-Dawley rats were pretreated with nitroglycerin 24 h before the experiment. The left main coronary artery was occluded for 60 min, followed by 3 h reperfusion. Infarct size, serum creatine kinase activity, serum levels of NO and CGRP and the expression of alpha- and beta-CGRP isoform mRNA in lumbar dorsal root ganglia were measured. Pretreatment with nitroglycerin (60 or 120 microg/kg i.v.) reduced both the infarct size and the release of creatine kinase during reperfusion and caused a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, concomitantly with an increase in concentrations of NO and CGRP. The increase in CGRP expression preceded the increase in CGRP release. The effects of nitroglycerin were abolished completely by pretreatment with methylene blue (30 mg/kg i.p.), an inhibitor of guanylate cyclase, or capsaicin (50 mg/kg s.c.), which selectively depletes transmitters in capsaicin-sensitive sensory nerves. The present results suggest that the delayed cardioprotection induced by nitroglycerin is mediated mainly by the alpha-CGRP isoform via the NO-cGMP pathway.

Elevated sympathetic nervous activity in mice deficient in alphaCGRP.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic neuropeptide implicated in a variety of physiological processes. To better understand the biological functions of alphaCGRP, we developed an alphaCGRP-null mouse model using a gene targeting approach. Recordings of mean arterial pressure (MAP) and heart rate (HR) showed that basal MAP and HR were significantly higher in both anesthetized and conscious, unrestrained alphaCGRP-null mice than in corresponding wild-type mice. The elevated MAP in alphaCGRP-null mice was shown to be the result of elevated peripheral vascular resistance by alpha-adrenergic blockade with prazosin and by transthoracic echocardiogram, which revealed no significant differences between alphaCGRP-null and wild-type mice in the stroke volume, fractional shortening, and ejection fraction. Moreover, evaluation of autonomic nervous activity by measuring HR after pretreatment of atropine and/or atenolol and by analyzing arterial baroreceptor reflexes showed sympathetic nervous activity to be significantly elevated in alphaCGRP-null mice; elevated levels of urinary catecholamine metabolites and decreased HR variability in mutant mice were also consistent with that finding. These findings suggest that alphaCGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity.

Induction of alpha-calcitonin gene-related peptide mRNA expression in rat dorsal root ganglia by heat stress involves the heme oxygenase-1/carbon monoxide pathway

SD rats were pretreated with whole body hyperthermia (rectal 42°C) for 15min. The level of calcitonin gene-related peptide (CGRP) in plasma, and α- and β-CGRP mRNA as well as heme oxygenease-1 and heme oxygenase-2 mRNA in dorsal root ganglia were determined by radioimmunoassay and semi-quantitative reverse-transcription polymerase chain reaction, respectively. Heat stress induced only the expression of α-CGRP or heme oxygenease-1 but not β-CGRP or heme oxygenase-2 mRNA, and the release of CGRP and induction of α-CGRP mRNA expression were abolished by pretreatment with Zinc protoporphyrin IX, the heme oxygenase inhibitor, or methylene blue, the inhibitor of soluble guanylate cyclase. These results indicate that induction of α-CGRP mRNA expression in rat DRG by heat stress involves the heme oxygenase-1/carbon monoxide pathway.

Altered neuroadaptation in opiate dependence and neurogenic inflammatory nociception in alpha CGRP-deficient mice.

The neuropeptide alpha CGRP (calcitonin gene-related peptide) is involved in the complex process of pain signaling, but the precise contribution of alpha CGRP remains unclear. Here we show that mice lacking alpha CGRP display an attenuated response to chemical pain and inflammation. Furthermore, alpha CGRP(-/-) mice do not show changes in heroin self-administration or morphine tolerance, but display a marked decrease in morphine withdrawal signs, suggesting an important contribution of alpha CGRP to opiate withdrawal.

Proceedings of the 4th International Meeting on Calcitonin Gene-Related Peptide and its Receptor.

Calcitonin Gene-Related Peptide (CGRP), a 37 amino acid peptide identified as the alternately spliced gene product of calcitonin gene, is a sensory neuropeptide with potent cardiovascular effects. CGRP is distributed throughout the central and peripheral nervous systems and possesses diverse biological actions. CGRP has been suggested to play a role in diseases such as migraine, diabetes, pain, and inflammation. Two forms of CGRP (alpha and beta) that differ in three amino acids have been identified and are encoded by different genes. Based on the differential biological activities of various CGRP analogs, the CGRP receptors have been classified into CGRP1 and CGRP2. Structure-activity studies of CGRP analogs showed that the C- and N-terminal regions of the peptide interact independently with their receptors. While C-terminal peptide, CGRP (8-37) behaves as a CGRP1 receptor antagonist, N-terminal peptide CGRP (1-12) behaves as a weak agonist. Structural modifications of CGRP(28-37) have yielded micromolar to nanomolar affinity ligands. CGRP receptor belongs to the calcitonin receptor like receptor (CRLR) family of G-protein-coupled receptors and has been shown to require a single transmembrane domain protein called receptor activity modifying protein-1 (RAMP1) for its functional expression as well as activity. Human, rat, and porcine CRLRs have been cloned and characterized. Currently, the major focus is on the identification of potent and specific nonpeptide antagonists for this receptor in order to understand the physiological and pathophysiological role of this peptide.

Cellular localization of neprilysin in mouse bone tissue and putative role in hydrolysis of osteogenic peptides.

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell-surface zinc-metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half-life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone-forming cells including presumptive osteoblast precursors, preosteoblasts, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone-related peptide 1-43 (PTHrP1-34), osteostatin (PTHrP107-139), osteogenic growth peptide (OGP), calcitonin, alpha-calcitonin gene-related peptide (alpha-CGRP), and PTH1-34. Except for PTH1-34, all peptides were found to be NEP substrates.

Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (α-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect was partially antagonized by the CGRP 1 receptor antagonist α-CGRP-(8-37) (10 −6 M). Amylin and the CGRP 2 receptor agonist [Cys(acetylmethoxy) 2,7]CGRP had a positive inotropic effect in some trabeculae, whereas adrenomedullin had no inotropic effect. Using reverse transcriptase–polymerase chain reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP 1 and CGRP 2 receptors may mediate a positive inotropic effect at both the atrial and ventricular level of the human heart. mRNAs for calcitonin receptor-like receptor and specific RAMPs further support the presence of CGRP receptors.

Positive inotropy of calcitonin gene-related peptide and amylin on porcine isolated myocardium

Isolated porcine myocardial trabeculae from right atria and left ventricles were paced at 1.5 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Alpha calcitonin gene-related peptide (α-CGRP) increased contractile force in nearly half of the trabeculae, whereas the selective CGRP 2 receptor agonist [Cys(acetylmethoxy) 2,7]-CGRP had effect in only a few. Preincubation with the CGRP 1 receptor antagonist α-CGRP-(8-37) (10 −6 M) almost completely blocked positive inotropic responses to α-CGRP. Amylin had weak positive inotropic effects in some atrial, but not in ventricular trabeculae. Adrenomedullin did not affect contractility in either atrial or ventricular trabeculae. In conclusion, these results suggest that α-CGRP has a positive inotropic effect that can be mediated by both CGRP 1 and CGRP 2 receptors. Amylin seems to have a potential positive inotropic effect on atrial tissue, whereas no direct effect of adrenomedullin could be measured.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides.

Structure-activity relationships for the binding of human alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[Tyr0]-CGRP28-37 (apparent pKBs of 7.49+/-0.25, 5.89+/-0.20, 6.18+/-0.19, 5.85+/-0.19 and 5.25+/-0.07). The SK-N-MC receptor appeared CGRP1-like. On Col 29 cells, only halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (M432) had apparent pKBs of 6.64+/-0.16 and 6.42+/-0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. M433 was almost as potent as halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery.

1. Effects of adrenomedullin and alpha-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and alpha-CGRP were also investigated. 2. Adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and alpha-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMMA, 100 microM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 microM). 3. Adrenomedullin and alpha-CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle. 4. An antagonist of the CGRP1 receptor, CGRP (8-37), antagonized the relaxant effects of alpha-CGRP and the beta-isoform of CGRP (beta-CGRP) but not those of adrenomedullin in the rat aorta. 5. In the porcine coronary artery contracted with U46619, adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or alpha-CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation. 6. Adrenomedullin and alpha-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery. 7. CGRP (8-37) significantly antagonized the relaxant effects of adrenomedullin and alpha-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the beta-isoform of CGRP (beta-CGRP). 8. These results suggest that in the rat aorta, adrenomedullin and alpha-CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and alpha-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to alpha-CGRP, beta-CGRP and CGRP (8-37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to alpha-CGRP, CGRP (8-37) and adrenomedullin and the other is sensitive only to beta-CGRP.

Changes in the mRNA expression pattern, with special reference to calcitonin gene-related peptide, after axonal injuries in rat motoneurons depends on age and type of injury.

The axotomy reaction in motoneurons after a peripheral nerve transection in the adult animal is characterized by a robust upregulation of alpha-calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) together with mRNAs encoding cytoskeletal and growth-related proteins. Here we have examined whether the nature of the lesion and the age of the animal have any impact on the mRNA regulation in severed cells. Thus, the effect of a sciatic nerve transection in the adult rat was compared with, on the one hand, ventral root avulsions in the adult animal and, on the other hand, sciatic nerve transection in the immature animal. In the two latter cases, a proportion of the lesioned cells die and overall chances of regeneration are small. In the adult animal a sciatic nerve transection induced an upregulation of alpha-CGRP mRNA from the 3rd day after surgery and throughout the first 3 weeks (the time span of the study). Also low-affinity nerve growth factor receptor (p75) and growth-associated protein-43 (GAP-43) mRNAs were upregulated during the entire 3-week period. In contrast, after ventral root avulsion, the expression of alpha-CGRP, c-jun, and p75 mRNAs were normalized within the 1st postoperative week, while GAP-43 mRNA was still upregulated at 3 weeks. Galanin message-associated peptide (GMAP) mRNA became upregulated preferentially in motoneurons subjected to ventral root avulsion, while nitric oxide synthase (NOS) mRNA was expressed exclusively after the latter type of injury. In the immature animal, alpha-CGRP mRNA was downregulated after sciatic nerve transection in rats aged 3 days or 7 days at the time of surgery; while, in contrast, an upregulation was seen in 12- or 21-day-old animals. GAP-43 and c-jun mRNAs were upregulated in lesioned motoneurons of all ages, while GMAP mRNA was upregulated preferentially in lesioned motoneurons of early postnatal animals. p75 mRNA was expressed in unlesioned immature motoneurons until the age of 7-10 days. The downregulation of p75 mRNA in intact cells at this age coincided with a developmental switch in the ability of axotomized cells to express increased levels of p75 mRNA. No expression of NOS mRNA was detectable in lesioned cells of any of the age groups. These results show that the age of the animal and the type of axonal injury are indeed to a high degree influencing the changes seen in the protein expression pattern in axotomized rat motoneurons. The different responses in these paradigms suggest differences in the trophic response from surrounding glia or the trophic responsiveness of lesioned motoneurons. Also, the results may indicate different roles for the studied substances during the regenerative response of lesioned neurons. Of the substances studied here, upregulation of alpha-CGRP and p75 mRNAs best correlated with a possibility of axon regeneration.

VAMP-1 and VAMP-2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury.

Vesicle-associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP-1 and VAMP-2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP-1 and VAMP-2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion-induced changes in the expression of choline acetyl transferase (ChAT) and alpha-calcitonin gene-related peptide (alpha-CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP-1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP-1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP-1 mRNA and a 48% decrease in ChAT mRNA. VAMP-1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP-2 and alpha-CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP-2 and alpha-CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for alpha-CGRP mRNA (163%) and after 14 days for VAMP-2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP-1- or VAMP-2-like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP-1- and VAMP-2-LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP-1 mRNA is downregulated and VAMP-2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.

Central action of adrenomedullin to inhibit gastric emptying in rats.

The central action of human adrenomedullin (AM) to influence gastric emptying and the peripheral mechanisms involved were studied in conscious rats. The 20-min rate of gastric emptying of a methylcellulose solution was assessed after intracisternal (i.c.) injection of AM or rat alpha-calcitonin gene-related peptide (alphaCGRP). AM and alphaCGRP dose-dependently inhibited gastric emptying with i.c. ED50 values of 120 and 100 pmol, respectively. Human proadrenomedullin N-terminal 20 peptide (150-600 pmol, i.c.) and AM (150 pmol, i.v.) had no effect. The inhibitory actions of AM and alphaCGRP (150 pmol, i.c.) were completely blocked by the CGRP antagonist, human CGRP-(8-37) injected i.c. at 30 microg, but not at 15 microg. The CRF antagonist, [D-Phe12,Nle(21,38),C(alpha)MeLeu37]CRF-(12-41) (10 microg/rat) injected i.c. prevented i.c. rat/human CRF (150 pmol)-induced 53% inhibition of gastric emptying while not modifying the effect of AM. The action of AM (150 pmol, i.c.) was abolished by bilateral adrenalectomy or the beta-adrenergic blocker, propranolol (1 mg/kg, i.p.), but was not altered by indomethacin (5 mg/kg, i.p.) or subdiaphragmatic vagotomy. These results indicate that i.c. AM and alphaCGRP equipotently inhibit gastric emptying through mechanisms similarly antagonized by a high dose of CGRP-(8-37). The central AM action is mediated through adrenal-dependent, beta-adrenergic pathways independently from activation of central CRF receptors.

Characterization of Amylin and Calcitonin Receptor Binding in the Mouse α-Thyroid-Stimulating Hormone Thyrotroph Cell Line*

Recently, a high affinity amylin binding site was identified in the mouse alpha-TSH thyrotroph cell line. In this study, we have characterized binding sites for 125I-salmon calcitonin (125I-sCT), 125I-rat alpha-calcitonin gene-related peptide (125I-CGRP), and 125I-rat amylin in alpha-TSH cells. Using 125I-CGRP or 125I-rat amylin, equilibrium was rapidly reached, and binding was fully reversible. Competition binding revealed the relative potency of peptides was sCT>amylin, CGRP>>rCT, which is similar to the specificity profile of amylin receptors characterized in rat brain. Furthermore, specific binding of 125I-rat amylin and 125I-CGRP to membrane preparations was reduced by 52% and 39%, respectively, in the presence of 20 microM GTP-gamma-s, indicating a requirement of G protein coupling for high affinity binding. In contrast, 125I-sCT binding reached equilibrium more slowly, was essentially irreversible, and was unaltered by GTP-gamma-s. Competition binding studies using 125I-sCT as radioligand demonstrated only weak interaction by CGRP or amylin, consistent with other described CT receptors. Assessment of ligand-induced cAMP accumulation and intracellular calcium signaling revealed a relative specificity profile of sCT>rCT with little or no second messenger signaling stimulated by amylin or CGRP, consistent with a C1-CT receptor phenotype. RT-PCR amplification of messenger RNA indicated that the predominant isoform was the C1a CT receptor. In cross-linking studies, 125I-rat amylin and 125I-CGRP specifically labeled a major band of relative molecular mass (Mr) approximately 80K, being approximately 10 kDa higher than the major 125I-sCT binding protein. Full deglycosylation of N-linked carbohydrates with endoglycosidase F reduced the Mr of each of the labeled proteins to approximately 50K. Cross-linked amylin or CT receptors were immunoprecipitated with C-terminally directed antimouse or antirat CT receptor antibodies but were not immunoprecipitated with nonimmune sera or antihuman CT receptor antibodies. The current data demonstrate expression of two biochemically distinct receptor phenotypes in mouse alpha-TSH cells, a CT receptor phenotype and an amylin receptor phenotype that have highly similar protein backbones.

Calcitonin Gene Related Peptide Enhances Bone Colony Development In Vitro

Recent evidence suggests that sensory nerve fibers of the dental pulp secrete calcitonin gene related peptide alpha exactly where secondary or tertiary dentin is mineralized. In addition, calcitonin gene related peptide raises the level of cyclic adenosine monophosphate in osteoblasts, indicating a potential effect on secretory activity in bone. Because calcitonin and calcitonin gene related peptide are formed from the same gene, the authors tested whether calcitonin gene related peptide or calcitonin has an osteogenic potential in vitro. To this end, 0.01, 0.1, and 1 microg/ml of salmon calcitonin or rat calcitonin gene related peptide in Bigger, Gwatkin, Jackson b medium was added daily to 3 x 10(6) rat light density bone marrow leukocytes that were separated with the Ficoll-Paque density gradient separation method, then seeded on a previously prepared fibroblast layer in Petri dishes. After 7 days, the number and size of bone colonies formed in the calcitonin gene related peptide (0.1 or 1 microg/ml) added group was significantly greater than that of the control group. There was no statistically significant difference between the calcitonin added and control groups. Calcitonin gene related peptide has an osteogenic stimulating effect, either by stimulating stem cell mitosis or osteoprogenitor cell differentiation (or both), whereas salmon calcitonin has no effect.

Development of calcitonin gene-related peptide slow-release tablet implanted in CSF space for prevention of cerebral vasospasm after experimental subarachnoid haemorrhage.

The calcitonin gene-related peptide (CGRP), a known potent intrinsic cerebral vasodilator, is contained in the sensory nerves from trigeminal ganglia that inervate the cerebral arteries. We previously reported that human alpha CGRP (hCGRP) dilates spastic cerebral arteries after experimental subarachnoid haemorrhage (SAH) in rabbits. In the present study, we investigated the prophylactic potential of a sustained higher cerebrospinal fluid level of hCGRP against experimental cerebral vasospasm. An hCGRP slow-release tablet (hCGRP s-r tablet) was developed for cisternal implantation. Experimental SAH was induced by percutaneous cisternal injection of autologous arterial blood. Angiography was initiated on day 1 (before SAH) and performed everyday. The hCGRP s-r tablet was implanted into the cisterna magna on day 2 in the treated groups. The spastic response of the basilar artery was maximized on day 4 in the non-treated (80.7% of day 1) and the placebo-treated (79.3%) groups. In contrast, the arterial diameters on day 4 were 96.1% and 90.5% of day 1 in the groups implanted with hCGRP 24 micrograms and 153 micrograms s-r tablets, respectively. We also measured the concentration of hCGRP in the cerebrospinal fluid (CSF) following implantation of the hCGRP 24 micrograms s-r tablet in the cisterna magna. The hCGRP concentration before implantation was below the dectable level. Following implantation, the hCGRP level in the CSF was 23.12 nmol/L on the second day and remained at elevated levels until the fifth day. These experiments suggest that the intrathecal single implantation of the hCGRP s-r tablet could produce an elevated concentration of hCGRP in the CSF over five days and have prevented the cerebral vasospasm after SAH in the rabbit. The hCGRP s-r tablet may be clinically applicable in the treatment of patients with SAH against cerebral vasospasm.