Synthetic Human Calcitonin Gene-related Peptide Research Articles

Dimeric Form of Calcitonin Gene-Related Peptide in the Porcine Thyroid Gland

In this study we investigated peptides that increase rat platelet cAMP in porcine thyroid gland. Gel filtration of extracts from porcine thyroid gland showed high and low molecular weight activity. Low molecular weight activity contained peptides, including calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI). We isolated a high molecular weight peptide (M. W. 11,000) showing potent activity able to increase rat platelet cAMP in porcine thyroid gland. The peptide′s N-terminal sequence was determined to be Ser-X-Asn-Thr-Ala-Thr- by gas phase sequencer analysis, a sequence identical to that of porcine CGRP. The peptide had CGRP immunoreactivity as well as platelet cAMP elevating activity. By gel filtration HPLC, synthetic human CGRP (M. W. 3790) was eluted in a position corresponding to M. W. 5,500. These results suggest that judging from its high molecular weight the above peptide is a dimeric form of CGRP.

Effects of calcitonin gene-related peptide on cardiac contractility, coronary hemodynamics and myocardial energetics in idiopathic dilated cardiomyopathy.

This study was performed to examine the effects of calcitonin gene-related peptide on cardiac function and coronary circulation in patients with heart failure. Synthetic human calcitonin gene-related peptide was infused in the left main coronary artery of 9 patients undergoing cardiac catheterization at different doses corresponding to incremental infusion rates of 15, 50, 150 and 600 pmol.min-1. No hemodynamic change was observed in response to administration of the 2 lowest doses. The 2 highest doses induced an increase in cardiac index and a decrease in systemic arterial pressure. The infusion of 600 pmol.min-1 resulted in a decrease of mean systemic arterial pressure (86.8 +/- 6.5 to 71.8 +/- 4.9 mm Hg; p less than 0.01), and an increase in both cardiac index (2.1 +/- 0.1 to 3.1 +/- 0.17 liters.min-1.m-2; p less than 0.01) and heart rate (87 +/- 3.7 to 101 +/- 6.1 beats.min-1; p less than 0.01). These hemodynamic changes were associated with a significant increase in plasma norepinephrine and epinephrine concentrations. Peak positive first derivative of left ventricular pressure did not change at any infusion rate. Left ventricular end-diastolic pressure decreased at the 2 highest doses associated with a decrease in plasma atrial natriuretic factor concentration (730 +/- 140 to 436 +/- 115 pg.ml-1; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular irritative response to YAG laser capsulotomy in rabbits: release of calcitonin gene-related peptide and effects of methysergide.

The Neodymium (Nd):YAG laser is commonly used in ophthalmology mainly for the posterior capsulotomy in patients with secondary cataract after extracapsular cataract extraction. A frequent side-effect following different kinds of YAG laser treatments is an acute increase in the intraocular pressure (IOP). The present study addresses the role of calcitonin gene-related peptide (CGRP) in the ocular irritative response following YAG laser anterior capsulotomy in rabbits. The YAG laser anterior capsulotomy caused an irritative response in the eye, which consisted of an increase in the IOP, miosis and breakdown of the blood-aqueous barrier. Following YAG laser capsulotomy, CGRP-immunoreactivity was found in the aqueous humour in different molecular weight forms as revealed by gel-permeation chromatography. One of the peaks coeluted with synthetic human CGRP. Methysergide attenuated the increase in the IOP and disruption of the blood-aqueous barrier, but not the miosis, following YAG laser anterior capsulotomy. The present study demonstrates the release of CGRP into the aqueous humour following YAG laser capsulotomy, and suggests that CGRP is partly causing the increase in IOP and disruption of the blood-aqueous barrier in this irritative response.

Calcitonin gene-related peptide: effect on contractile activity and luminal cross-sectional area in the isolated, perfused porcine ileum.

Because calcitonin gene-related peptide (CGRP) is an abundant peptide in the enteric nervous system we studied the effect of intra-arterial infusions of synthetic human CGRP I in concentrations from 10(-10) to 10(-8) mol/l on contractile activity and luminal cross-sectional area in the isolated perfused porcine ileum, using manometry and impedance planimetry. The frequency of the basal contractile activity was 0.37 +/- 0.1 contractions per minute. CGRP induced phasic contractions, which at the highest dose were superimposed on tonic contractions, as determined by measurement of luminal cross-sectional area. The frequency of contractions dose-dependently increased to approximately 10/min at 10(-8) mol/l CGRP. The amplitude of contractions increased from a maximum of 35 cm H2O to 51 +/- 3 at 5 x 10(-9) mol/l CGRP and 52 +/- 6 cm H2O at 10(-8) mol/l CGRP. After the termination of CGRP infusion at the highest dose a short phase of up to 5 min with strong tonic contraction was observed. No phasic activity was detected by manometry during this phase. In conclusion, CGRP dose-dependently increased contractile activity in the pig ileum. CGRP may therefore participate in the regulation of small-intestinal motility in the pig.

Splanchnic and Cerebral Vasodilatory Effects of Calcitonin Gene-related Peptide I in Humans

The ability of synthetic human calcitonin gene-related peptide (CGRP I) to act as an arterial vasodilator was tested in healthy men by measuring arterial blood flow parameters in carotid, superior mesenteric, celiac, and femoral vessels. Calculated volume flow was significantly increased (140 +/- 21% of basal) in the SMA with a 2-ng/kg/min infusion of CGRP. Carotid artery volume flow increased dose dependently (96 +/- 6%, 122 +/- 15%, 135 +/- 15% of basal, respectively, with 2, 4, or 8 ng/kg/min). With steady-state infusion, carotid and superior mesenteric arterial flow parameters remained significantly elevated for 30 minutes after cessation of peptide administration. Blood pressure was unchanged. Pulse increased dose dependently. Arterial diameters were unchanged, implying activity at the arteriolar level.

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 ± 0.09 ng/mg protein) and in intestine (1.06 ± 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 ± 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

Dual effects of calcitonin gene-related peptide on insulin secretion in the perfused dog pancreas

Calcitonin gene-related peptide (CGRP) is an intrapancreatic neuropeptide with potential effects on islet hormone secretion. To investigate its pancreatic actions, we examined the effects of a 10 min perfusion of synthetic human CGRP on islet hormone release from the isolated dog pancreas ( n = 6) at 5.5 mM glucose. At 0.1 nM, CGRP inhibited insulin secretion ( P < 0.01), which was already observed at 2 min after its introduction. After CGRP perfusion was stopped, a stimulatory off-response occurred. In contrast, at higher dose levels, CCRP stimulated insulin secretion. At 1.0 nM, the stimulation was weak and transient ( P < 0.01), occurring only during the first 3 min of CGRP perfusion. At 10 nM, the stimulation continued for 6 min ( P < 0.05), and at 50 nM, the stimulation was marked and sustained throughout the 10 min perfusion period ( P < 0.01). After the CGRP perfusion at 1.0 and 10 nM, but not at 50 nM, a marked stimulatory off-response in insulin secretion was seen. Glucagon and somatostatin secretion were not significantly affected by CGRP at any of the examined concentrations. We conclude that CGRP exerts dual effects on insulin secretion from the perfused dog pancreas: inhibition at low concentrations and stimulation at high concentrations. This pattern of effect might represent a new regulatory concept for neural influences on islet function: the qualitative response being determined by the amount of neurotransmitter released.

Production of calcitonin gene-related peptide from human cancer cells.

Calcitonin (CT) is produced ectopically from a variety of non-thyroidal cancers. The CT genes also encode another peptide, calcitonin gene-related peptide (CGRP) which is a potent vasodilator. In the present study we have used immunochemical and chromatographic methods to demonstrate the presence and characterize the molecular forms of CGRP in cultured human cancer cells. Using two highly sensitive and specific radioimmunoassays, we have detected immunoreactive CGRP (i-CGRP) in cell extracts and cell-exposed media of cultured promyelocytic leukaemia (HL60) and bronchogenic carcinoma (BEN) cells. The mean i-CGRP content of the HL60 and BEN cell extracts was 2 and 45 pmol/g wet weight respectively. On gel filtration and high performance liquid chromatography, the immunoreactive material was found to be heterogeneous, though a major proportion co-eluted with synthetic human CGRP(1-37), suggesting structural identity with the intact CGRP molecule. Finally, we have discussed some interesting features of CT-gene peptide expression in tumour cells.

Calcitonin Gene-Related Peptide in the Human Hypothalamus.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in the human hypothalamus was investigated by radioimmunoassay and by immunocytochemistry. CGRP-LI was detected from two hypothalami obtained at autopsy (2.1 and 7.0 ng/g wet tissue) by radioimmunoassay. Reverse phase high performance liquid chromatography revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP. For immunocytochemistry, human hypothalami obtained at autopsy were fixed and cryostat-sectioned at 40 microns. Free floating sections were immunostained with antibody to CGRP. CGRP-immunoreactive cell bodies were found in the supraoptic nucleus, paraventricular nucleus and infundibular nucleus. These findings indicate that CGRP exists in the cell bodies of the supraoptic nucleus, paraventricular nucleus and infundibular nucleus in the human hypothalamus and CGRP may play some roles in the endocrine and other functions of the human hypothalamus.

Calcitonin gene-related peptide-like immunoreactivities in pheochromocytomas.

Calcitonin gene-related peptide (CGRP) is reported to exist in high concentrations in plasma and tumor tissues of medullary thyroid carcinomas. CGRP-like immunoreactivity (LI) in tumor tissues of pheochromocytomas was investigated by radioimmunoassay. CGRP-LI in 9 pheochromocytomas ranged from 0.50 to 1240 ng/g wet tissue. Sephadex G-50 column chromatography revealed that most of CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP. Reverse-phase high pressure liquid chromatography revealed that CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP and in a more hydrophobic position. These results indicate that high concentrations of CGRP-LI also exist in tumor tissues of pheochromocytomas.

Calcitonin gene-related peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn

Effects of synthetic human calcitonin gene-related peptide (hCGRP) on Ca 2+ mobilization in crude synaptosomes prepared from rat spinal dorsal horn were studied using 45Ca 2+ as a tracer. hCGRP promoted the influx of 45Ca 2+ into synaptosomes of the spinal dorsal horn, in a dose dependent manner (at dose ranges from 10 −9 to 10 −6 M). The maximum effect of hCGRP on 45Ca 2+ influx was achieved 5 min after addition of the peptide and was significantly inhibited by nifedipine. hCGRP did not alter the influx of 45Ca 2+ into synaptosomes of the rat cerebral cortex, a brain area with reportedly few CGRP binding sites. In measurements of the 45Ca 2+ efflux from synaptosomes of the spinal dorsal horn, 10 −7 M of hCGRP had no effect on the efflux of 45Ca 2+ from the resting synaptosomes, but there was significantly inhibitory effect on synaptosomes depolarized by 40 mM K +. The inhibitory effect of hCGRP on the efflux of 45Ca 2+ was completely abolished by lowering the incubation temperature of the medium to 0 °C. The inhibitory effect of hCGRP on the efflux of 45Ca 2+ was not observed in synaptosomes of the cerebral cortex. These findings suggest that hCGRP increases the levels of Ca 2+ level in synaptosomes of the spinal dorsal horn, through interactions with its own receptors and the release of neurotransmitters from the nerve terminals is thus enhanced.

Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect

The occurrence and distribution of calcitonin gene-related peptide (CGRP) in the lower airways was studied by means of immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-like immunoreactivity (-LI) was observed in nerves from the epiglottis down to peripheral bronchi in rat, cat and guinea pig and also in human bronchi. Double staining revealed colocalization of CGRP-LI and substance P (SP)-LI in cell bodies of nodose and jugular ganglia as well as in axons and nerve terminals of the airways. Systemic capsaicin pretreatment induced a marked loss of the CGRP- and SP-immunoreactive (-IR) nerves in the lower airways. CGRP-IR was also present in epithelial endocrine cells and neuroepithelial bodies. The content of CGRP-LI as measured with RIA in guinea pig bronchi was significantly lower after capsaicin pretreatment. Analysis of human bronchial extracts revealed that CGRP-LI coeluted with synthetic human CGRP on HPLC. In the isolated perfused guinea pig lung capsaicin exposure caused overflow of CGRP-LI suggesting release from peripheral branches of sensory nerves. Both in vivo experiments in the guinea pig measuring insufflation pressure as well as in vitro studies on isolated guinea pig and human bronchi showed that whereas tachykinins contracted bronchial smooth muscle no contractile or relaxing effect was elicited by human or rat CGRP. However, CGRP caused relaxation of serotonin precontracted guinea pig and human pulmonary arteries. In conclusion, the presence and release of CGRP-LI from capsaicin sensitive nerves in the lower airways adds another possible mediator, in addition to tachykinins, of vascular reactions upon sensory nerve irritation.

Co-localization of tachykinins and calcitonin gene-related peptide in capsaicin-sensitive afferents in relation to motility effects on the human ureter in vitro

Tachykinin- and calcitonin gene-related peptide (CGRP) immunoreactivities were localized by immunohistochemistry in the same nerves of the kidney, renal pelvis and ureter as well as in spinal ganglion cells of both the guinea-pig and man. The tachykinin and CGRP-immunoreactive nerves in the ureter were present within the smooth muscle layers, around blood vessels, close to and within the lining epithelium. The levels of neurokinin A-, substance P- and CGRP-like immunoreactivity per tissue weight, as determined by radioimmunoassay, were about 30–100-fold higher in the guinea-pig than in the human ureter, which was in good agreement with the relative density of immunoreactive nerve fibres, as seen by immunohistochemistry. Capsaicin treatment caused an almost total disappearance of both neurokinin A-, substance P- and CGRP-immunoreactive nerve fibres in the guinea-pig ureter and a 90% depletion of neurokinin A, substance P- and CGRP-like immunoreactivity, further supporting a sensory origin of these nerves. Reversed-phase high performance liquid chromatography of water extracts of the human ureter revealed the presence of neurokinin A- and eledoisin-like material using antiserum K12, which does not cross-react with substance P. Most of the CGRP-like immunoreactivity in human ureter extracts co-eluted with synthetic human CGRP. Capsaicin both caused inhibition of spontaneous motility of the human ureter in vitro and initiated contractions in some preparations. Neurokinin A and neuropeptide K potently initiated phasic contractions of the ureter, while substance P had only minor contractile effects. CGRP inhibited both spontaneous and neurokinin A-induced ureteric contractions. In conclusion, peptides with potent opposite motility effects are present in the same, presumably sensory nerves of the ureter in both the guinea-pig and man. It will be of importance to determine whether local release of neuropeptides can account for ureteric motility changes accompanying sensory nerve activation upon ureteral obstruction, by e.g. renal calculi.

Basal and stimulated release of calcitonin gene-related peptide (CGRP) in patients with medullary thyroid carcinoma.

Calcitonin gene-related peptide (CGRP) is a recently discovered peptide whose existence was first predicted following sequence analysis of the rat calcitonin gene. In the present study, plasma levels of CGRP were measured in patients with medullary thyroid carcinoma, both basally and following calcium or pentagastrin stimulation. Using a sensitive radioimmunoassay for CGRP, 19 of 21 patients with medullary thyroid carcinoma had elevated plasma levels of immunoreactive CGRP (median value 114 pmol/l) while 23 normal subjects had plasma CGRP levels below the detection limit of the assay (less than 10 pmol/l). Calcium or pentagastrin infusion in medullary thyroid carcinoma patients were potent stimuli to CGRP release, increasing plasma levels two- to five-fold. Chromatographic characterization using high pressure liquid chromatography of the CGRP immunoreactivity in plasma from two patients with medullary carcinoma of the thyroid demonstrated the presence of two molecular forms, neither of which co-eluted with synthetic human CGRP. The role of CGRP in the pathophysiology of medullary thyroid carcinoma is not clear but it may contribute to some of the clinical features associated with the disease. Furthermore, measurement of CGRP in conjunction with calcitonin could help in determining the prognosis of these patients.

Human calcitonin gene-related peptide possesses weak inhibitory potency of bone resorption in vitro.

Synthetic human calcitonin gene-related peptide (CGRP) was examined for the action on the bone in vitro. Human CGRP inhibited bone resorption stimulated by both human parathyroid hormone and basal. The mode of inhibitory action of human CGRP seemed to be similar to that of calcitonin and the relative potency of human CGRP to inhibit bone resorption is one five-hundredth of that of human calcitonin. Thus, a novel pharmacological action of CGRP was demonstrated.

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3×10 −9−3×10 −7 M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10 −5 M) and adenosine (10 −4 M) reduced the positive inotropic effect of hCGRP at concentrations of 3×10 −9 and 3×10 −8 M, but not at that of 3×10 −7 M. Ouabain (5×10 −5 M) inhibited the effect of hCGRP in concentrations of 3×10 −7 and 3×10 −8 M, but not in that of 3×10 −9 M. Simultaneous pretreatment with verapamil (10 −5 M) and ouabain (5×10 −5 M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10 −6 M) potentiated only the positive inotropic effect of 3×10 −7 M hCGRP. Metoprolol (10 −7 M) and theophilline (10 −3 M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by β-adrenoceptors but by two distinct mechanisms of action, (a) which was inhibited by verapamil but not by ouabain (facilitation of Ca ++ influx in lower concentrations of hCGRP) and (b) which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na +/Ca ++ exchange mechanism at higher concentrations of hCGRP).

Calcitonin gene-related peptide: Detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.

Chemicals as tools in the study of excitable membranes.

Chemicals as tools in the study of excitable membranes.