Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.     

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

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

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses in the renal pelvis and ureter of rats

Summary The distribution of calcitonin gene-related peptide-immunoreactive nerve fibers in the renal pelvis and ureter was examined by immunohistochemistry using whole-mount preparations and cryostat sections. The patterns of innervation were contrasted between the pelvis and ureter; the immunoreactive nerve fibers in the pelvis ran parallel to the long axis of each of the circular and longitudinal muscle layers, causing a lattice-like appearance of the nerve fibers. In the ureter, the immunoreactive fibers were accumulated in the subepithelial region and the longitudinal muscle. In both the pelvis and ureter, a portion of the nerve fibers of smaller caliber showed a swollen or beaded structure; they were located in the musculature and beneath the epithelium extending for considerable distances. Ligation of the ureter caused a marked decrease in the immunoreactive nerves in the pelvis and the proximal portion of the ureter, suggesting that the axonal flow in the calcitonin gene-related peptide-containing neurons of the ureter runs towards the pelvis.     

Calcitonin gene-related peptide (CGRP) receptor antagonists: novel aspartates and succinates

Novel aspartate and succinate CGRP full antagonists were identified through core modification of a potent lead CGRP antagonist, BMS-694153. While aspartates were much less active and had a flat SAR, some of the succinates were very potent CGRP full antagonists and matched the potency of BMS-694153. The most potency resides in the S enantiomer as demonstrated through an asymmetric synthesis.     

Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats

We have previously reported that Chinese traditional medicine rutaecarpine (Rut) produced a sustained hypotensive effect in phenol-induced and two-kidney, one-clip hypertensive rats. The aims of this study are to determine whether Rut could exert antihypertensive and anti-platelet effects in spontaneously hypertensive rats (SHR) and the underlying mechanisms. In vivo, SHR were given Rut and the blood pressure was monitored. Blood was collected for the measurements of calcitonin gene-related peptide (CGRP), tissue factor (TF) concentration and activity, and platelet aggregation, and the dorsal root ganglia were saved for examining CGRP expression. In vitro, the effects of Rut and CGRP on platelet aggregation were measured, and the effect of CGRP on platelet-derived TF release was also determined. Rut exerted a sustained hypotensive effect in SHR concomitantly with the increased synthesis and release of CGRP. The treatment of Rut also showed an inhibitory effect on platelet aggregation concomitantly with the decreased TF activity and TF antigen level in plasma. Study in vitro showed an inhibitory effect of Rut on platelet aggregation in the presence of thoracic aorta, which was abolished by capsazepine or CGRP(8-37), an antagonist of vanilloid receptor or CGRP receptor. Exogenous CGRP was able to inhibit both platelet aggregation and the release of platelet-derived TF, which were abolished by CGRP(8-37). The results suggest that Rut exerts both antihypertensive and anti-platelet effects through stimulating the synthesis and release of CGRP in SHR, and CGRP-mediated anti-platelet effect is related to inhibiting the release of platelet-derived TF.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

Calcitonin gene-related peptide (CGRP) receptor antagonists: investigations of a pyridinone template

In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.     

Calcitonin gene-related peptide (CGRP)-immunoreactive sensory and motor nerves of the mammalian palate

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres in the palate of rat, cat and monkey was studied using immunocytochemistry and radioimmunoassay. CGRP-containing nerve fibres were found, in all species studied, to form a rich plexus in the subepithelial and submucous layers, around excretory ducts and blood vessels. A small number of CGRP-containing nerve fibres penetrated the epithelium of the hard and soft palate, and terminated as free endings. Some CGRP-containing nerve fibres were found in the vicinity of the mucous glands. CGRP-immunoreactive motor end plates were seen in the striated muscle (tensor veli palatini) of the soft palate. Following capsaicin treatment a small depletion in CGRP-immunoreactive nerve fibres in the rat palate epithelium was noted. In contrast, CGRP immunoreactive fibres forming rich plexuses in other layers of the palate, including motor end plates, were not affected. The extractable CGRP showed no significant depletion (normal animals [n = 10] 21.7 +/- 2.4 pmol/g compared with capsaicin-treated animals [n = 10] 17.5 +/- 1.8 pmol CGRP/g wet weight). The reduction in the number of visible immunoreactive nerves following capsaicin application tends to confirm the sensory character of the CGRP-containing nerve fibres terminating in the epithelium of the hard and soft palate. The capsaicin insensitive CGRP-immunoreactive nerve fibres may thus have a predominantly motor function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide (CGRP)-positive neurons and fibers in the cat periaqueductal grey matter

The morphology and topographical distribution of neurons and terminals containing calcitonin gene-related peptide (CGRP) immunoreactivity in the cat periaqueductal grey (PAG) were studied using a rabbit antiserum raised against the C-terminal region of rat alpha-CGRP. In normal cats, numerous fibers, but rarely immunoreactive neurons, were observed in the PAG. CGRP-containing fibers showed bouton-like swellings along their length and expanded in terminal clusters of boutons. In many cases, CGRP-positive fibers were also observed in close association with small blood vessels. Immunoreactive fibers were particularly numerous at caudal PAG levels, mostly in its ventrolateral portion. In colchicine-treated cats, the pattern of CGRP-containing fibers was basically unchanged, despite a reduction of both the number of fibers and the intensity of fiber staining; in addition, numerous CGRP-positive neurons were found, mostly in the ventrolateral portion of the caudal PAG. These neurons were fusiform, spheroidal, and triangular in shape. The selective distribution of CGRP-positive elements in the PAG suggests a functional specialization of these neurons in the activation of pain-modulating mechanisms.     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Calcitonin gene-related peptide(CGRP) stimulates the release of atrial natriuretic peptide(ANP) from isolated rat atria

The effect of calcitonin gene-related peptide(CGRP) on the release of atrial natriuretic peptide(ANP) was studied in spontaneously beating, isolated rat atria. CGRP stimulated the ANP release in a dose-dependent manner. When the atria were incubated with a combination of phentolamine, propranolol, and atropine, these antagonists blocked neither the rise in ANP release nor the positive chronotropic and inotropic effects of CGRP. Therefore, we conclude that CGRP stimulates ANP release as well as cardiac contractility independently of adrenergic and cholinergic receptors.     

降钙素基因相关肽的研究

降钙素基因相关肽(cGRP)是近年来发现的一种新肽,广泛分布于中枢和外周神经系统以及某些非神经组织,在心血管和消化道等部位尤其丰富;它参与机体多种调节扼制,特别是感觉和胃肠功能的调节,对血压、血流、心率等也有较强的调节作用。     

Prolactin and LH release in response to vasoactive intestinal peptide (VIP) administration in spontaneously hypertensive and normotensive rats.

Vasoactive intestinal peptide (VIP) was injected intravenously at a dose of 10 micrograms in spontaneously hypertensive and normotensive Wistar-Kyoto rats. In order to evaluate the hemodynamic and hormonal effects of this peptide, the mean arterial pressure, heart rate as well as a serum rLH and rPRL levels, the contents of LH-RH in hypothalamus and the content of LH in pituitary tissue were determined. The same procedure was applied in rats receiving placebo. Serum rPRL concentration was measured additionally after combined administration of VIP+dopamine. VIP injection produced a decrease in mean arterial pressure and an increase in heart rate in both spontaneously hypertensive and normotensive rats. Serum rPRL concentration was significantly increased at 10 minutes after injection. The combined therapy (VIP+dopamine) partially inhibited this response. Serum rLH concentration, the content of LH-RH in hypothalamic tissue as well as the content of pituitary LH after VIP injection in spontaneously hypertensive and normotensive rats did not differ from the values obtained for the control group. Conclusions: 1. VIP injection produced the dramatic hypotensive effects in hypertensive rats; 2. A marked increase in PRL concentration in response to VIP was partially inhibited by dopamine in hypertensive and normotensive rats; 3. VIP injection did not change LH-RH and LH release in both hypertensive and normotensive rats.     

Calcitonin gene-related peptide and hypertension

Capsaicin-sensitive sensory nerves participate in the regulation of cardiovascular functions both in the normal state and the pathophysiology of hypertension through the actions of potent vasodilator neuropeptides, including calcitonin gene-related peptide (CGRP). CGRP, a very potent vasodilator, is the predominant neurotransmitter in capsaicin-sensitive sensory nerves, and plays an important role in the initiation, progression and maintenance of hypertension via: (1) the alterations in its synthesis and release and/or in vascular sensitivity response to it; (2) interactions with pro-hypertensive systems, including renin-angiotensin-aldosterone system, sympathetic nervous system and endothelin system; and (3) anti-hypertrophy and anti-proliferation of vascular smooth muscle cells. The decrease in CGRP synthesis and release contributes to the elevated blood pressure, as shown in the spontaneously hypertensive rats, alpha-CGRP knockout mice, Dahl-salt or phenol-induced hypertensive rats. In contrast, the increase in CGRP levels or the enhancement of vascular sensitivity response to CGRP plays a beneficial compensatory depressor role in the development of hypertension, as shown in deoxycorticosterone-salt, sub-total nephrectomy-salt, N(omega)-nitro-L-arginine methyl ester or two-kidney, one-clip models of hypertension in rats. We found that rutaecarpine causes a sustained depressor action by stimulation of CGRP synthesis and release via activation of vanilloid receptor subtype 1 (VR1) in hypertensive rats, which reveals the therapeutic implications of VR1 agonists for treatment of hypertension.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Calcitonin gene-related peptide (CGRP) modulates cholinergic neurotransmission in the small intestine of man, pig and guinea-pig via presynaptic CGRP receptors.

The effect of calcitonin gene-related peptide (CGRP) on the cholinergically mediated twitch contraction in longitudinal muscle strips of the small intestine (duodenum, jejunum, ileum) of guinea-pig, pig and man was investigated. Independently of the anatomical region, CGRP inhibited the twitch response in the different specimens of all three species by about 40% with similar IC50 values (1.5-2.4 nmol/l). Only in the guinea-pig small intestine CGRP induced a contraction of the smooth muscle which was sensitive to scopolamine and tetrodotoxin. The electrically evoked [3H]acetylcholine release from jejunal longitudinal muscle strips with myenteric plexus attached of the guinea-pig, which were incubated with [3H]choline, was concentration-dependently inhibited by CGRP. A direct relaxant effect of CGRP on smooth muscle tone of carbachol precontracted preparations was only observed in specimens of the guinea-pig. In conclusion, presynaptic inhibitory CGRP receptors on cholinergic neurones modulate the release of acetylcholine in different parts of the small intestine.     

Calcitonin gene-related peptide (CGRP) receptor antagonists: pyridine as a replacement for a core amide group

In our continuing efforts to identify CGRP receptor antagonists that can be dosed orally for the treatment of migraine headache, we have investigated a pyridine bioisosteric replacement of a polar amide portion of a previous lead compound, BMS-694153. Pyridine derivatives were discovered and their SAR was studied. Some of them showed excellent binding potency. However, oral bioavailability was low, even for compounds with good Caco-2 cell permeability.     

Cold-restraint induced gastric lesions in normotensive and spontaneously hypertensive rats

Spontaneously hypertensive (SHR) rats and normotensive Wistar-Kyoto (WKY) rats were subjected to 2 hr of cold-restraint stress at 2–6°C following a 24 hr fast. WKY rats had a significantly greater incidence and degree of ulceration of the gastric glandular mucosa than did SHR rats. Mean arterial pressure, obtained from a chronic arterial cannula, fell during 2 hr of cold-restraint stress in both SHR and WKY rats. Heart rate was unchanged in WKY but fell significantly in SHR. Plasma norepinephrine (NE) and epinephrine (E), determined by radioenzymatic assay, increased significantly following stress. Increased levels of NE remained similar for both SHR and WKY rats, while post-stress levels of E for the SHR rats greatly exceeded E levels for WKY rats. A greater degree of hypothermia was also noted in SHR rats. Decreased stress induced ulcerogenesis in the SHR may be due to the well-known altered hemodynamic and autonomic nervous system reactivity in this strain or other factors not yet discovered.