The neurotransmitter role of calcitonin gene-related peptide in the rat and guinea-pig ureter: effect of a calcitonin gene-related peptide antagonist and species-related differences in the action of omega conotoxin on calcitonin gene-related peptide release from primary afferents.

In the rat and guinea-pig isolated ureter electrical field stimulation of intrinsic nerves (10 Hz for 10 s) produces transient inhibition of evoked (20 mM KCl or 0.1-1 microM neurokinin A) rhythmic contractions by releasing transmitter(s) from peripheral endings of capsaicin-sensitive primary afferents. The C-terminal fragment of human calcitonin gene-related peptide (8-37) blocked the inhibitory effect of electrical field stimulation as well as that produced by exogenous calcitonin gene-related peptide, while leaving unaffected the inhibitory response to isoprenaline. Human calcitonin gene-related peptide (8-37) was devoid of any inhibitory activity of its own but enhanced the amplitude and frequency of KCl-evoked rhythmic contractions in the rat ureter, probably by antagonizing the inhibitory effect of endogenous calcitonin gene-related peptide released by KCl. Omega conotoxin fraction GVIA, a peptide which possesses a potent blocking activity of N-type voltage-sensitive calcium channels, prevented the inhibitory response to electrical stimulation in the guinea-pig ureter, while leaving the response unaffected in the rat ureter. Conotoxin had no effect toward the inhibition produced by exogenous calcitonin gene-related peptide indicating its prejunctional site of action, demonstrated previously in the guinea-pig ureter [Maggi et al. (1990) Neurosci, Lett. 114, 203-206]. Dermorphin, an amphibian peptide with potent agonist activity on mu-type opioid receptors, inhibited the response to electrical stimulation in the guinea-pig ureter but had no effect in the rat ureter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of calcitonin gene-related peptide type 1 receptor mRNA and their activity-modifying proteins in the rat nucleus accumbens

The calcitonin receptor-like receptor (CRLR) and the orphan receptor RDC-1 have been proposed to be calcitonin gene-related peptide type 1 (CGRP1) receptors, and receptor activity-modifying proteins (RAMPs) determine the ligand specificity of CRLR. Coexpression of RAMP1 and CRLR resulted in functional CGRP1 receptors; the complex of RAMP2 or RAMP3 and CRLR created functional adrenomedullin receptor. Although high levels of CGRP binding sites in the nucleus accumbens have been reported, little is known about the expression of these novel CGRP receptors. In the present study, we used real-time quantitative RT-PCR to detect and quantitate the relative expression of CGRP, CRLR, RAMP1-3 and RDC-1 in the nucleus accumbens of intact rats and rats with inflammation. Our results demonstrate that CGRP, CRLR, RAMP1 and RAMP2 exist in the nucleus accumbens of intact rats, and that they were significantly upregulated in rats with inflammation. In contrast, no expression was detected for RDC-1 and RAMP3. These findings indicated a functional role for CGRP and its receptors in inflammation and pain modulation.     

Expression of calcitonin gene-related peptide receptor components,calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental function during pregnancy. The present study was aimed to investigate mRNA expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP(1)) in the rat placenta. Immunohistochemical staining of rat placentas obtained on day 18 of pregnancy using polyclonal anti-CRLR and RAMP(1) antibodies revealed that labelling was specifically concentrated in the cytotrophoblast and syncytium in labyrinth, trophoblastic giant cells and basophilic cells in trophospongial cell layer, and endothelium and smooth muscle cells in fetal vessels. The intensity of staining was reduced in all these cells except in the syncytium in placentas obtained during labour. RT-PCR analysis showed that mRNA expression of CRLR and RAMP(1) was significantly higher in the rat placenta from gestation day 17 to day 22, than during labour. During pregnancy, 17beta-estradiol inhibits, while progesterone stimulates, placental mRNA and proteins for CRLR and RAMP(1). Antiestrogen, ICI 182780, increased, whereas antiprogesterone, RU 486, inhibited the expression of both CRLR and RAMP(1). In summary, we demonstrate the presence and cellular localization of CRLR and RAMP(1) in the rat placenta. Elevated placental CRLR and RAMP(1) may be involved in CGRP-related increases in blood flow and therefore fetal growth and decreases at term labour may help minimize the blood loss.     

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.     

Origin and course of nerves immunoreactive for calcitonin gene-related peptide surrounding the femoral artery in rat

Appreciation of anatomic relationships between perivascular nerve fibers and blood vessels is essential in reconstructive surgery. We examined the origin and neural connections of perivascular nerve fibers containing calcitonin gene-related peptide surrounding the femoral artery that regulate vascular tone. We used immunohistochemistry, denervation, and retrograde labeling methods. Peptide-immunoreactive fibers surrounding the femoral artery formed a complex network, with numerous small fibers extending from nerve fiber bundles located in the perivascular connective tissue. In middle and distal arterial segments, these fibers originated from the femoral nerve, the arterys main accompanying nerve. More proximally, fibers arose from the genitofemoral nerve and sympathetic nerves. Nerve branches terminating in various arterial segments had origins corresponding to those of somatic sensory nerve fibers, although pathways innervating the femoral artery took different courses.     

Dietary calcium modulates spinal cord content of calcitonin gene-related peptide in the rat

This study was undertaken to determine if calcium status modulates calcitonin gene-related peptide (CGRP) neuronal content. In two separate experiments, young, growing rats and mature rats were placed on low, normal, and high calcium diets for four weeks. CGRP immunostaining was localized immunocytochemically in laminae I and II of the upper thoracic spinal cord in young rats and in the upper thoracic and lumbar spinal cord in mature rats. Low calcium intake decreased dorsal horn CGRP content in young, growing rats, while high calcium diet significantly increased CGRP content, as determined by computer-assisted image processing, in both young and adult rats. A significant positive correlation was found between the serum ionized calcium and CGRP content in laminae I and II. Thus, calcium balance appears to modulate neuronal CGRP content.     

The presence of a calcitonin gene-related peptide in the olivocochlear bundle in rat

Summary The origins of calcitonin gene-related peptide immunoreactive (CGRPI) fibers in the cochlea were examined in rats. Parasagittal transection of the brain just medial to the principal sensory trigeminal nucleus resulted in the ipsilateral disappearance of CGRPI fibers in the cochlea, indicating that the origins of these fibers lie in the central nervous system. Next, we used a highly sensitive method combining retrograde tracing and immunohistochemistry to identify the origins of the CGRPI fibers in the cochlea. After injection of biotin-wheat germ agglutinin (b-WGA) into the cochlea, CGRPI neurons in the ipsilateral lateral superior olivary nucleus also contained b-WGA granules. These findings indicated tht CGRPI efferent fibers are major components of the olivocochlear bundle.     

降钙素基因相关肽合成与释放调节

降钙素基因相关肽（calcitonin gene-related peptide, CGRP）是迄今发现的最强的舒血管物质,具有多种重要的生物学作用。作为感觉神经主要递质,CGRP主要由背根神经节合成,某些非神经细胞(如淋巴细胞和内皮细胞)也有表达。CGRP的合成与释放受多种因素调节,除辣椒素受体外,肾上腺素受体、血管紧张素受体、神经生长因子受体和一氧化氮等也参与了CGRP合成与释放的调节。     

Effects of cerebral lesions on binding sites for calcitonin and calcitonin gene-related peptide in the rat nucleus accumbens and ventral tegmental area.

Binding site densities of [125I]-labelled salmon calcitonin and human calcitonin gene-related peptide were investigated in the rat nucleus accumbens and ventral tegmental area by means of quantitative autoradiography following selective brain lesions. [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites were highly concentrated in the accumbens, whereas the ventral tegmental area only contained [125I]salmon calcitonin binding sites. Unilateral injection of 6-hydroxydopamine into the ventral tegmental area did not alter [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding site densities in the ipsilateral accumbens, while it produced a significant decrease in [125I]salmon calcitonin binding sites in the lesioned ventral tegmental area (-50%). In contrast, following unilateral injection of quinolinic acid into the accumbens, the densities of [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites were significantly decreased in the lesioned accumbens (-57% and -56%, respectively), while [125I]salmon calcitonin binding site densities were not modified in the ipsilateral ventral tegmental area. The present study clearly suggests that [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites are located on intrinsic neurons but not on the dopaminergic nerve terminals in the accumbens. Moreover, a certain proportion of [125I]salmon calcitonin binding sites could be present on dopaminergic cell bodies in the ventral tegmental area.     

Immunohistochemical analysis of calcitonin and calcitonin gene-related peptide in human lung

Calcitonin and calcitonin gene-related peptide (CGRP) have been localized immunohistochemically in neuroendocrine cells of normal and diseased human lungs. Cells immunoreactive for calcitonin and CGRP first appeared in immature bronchi in the 27th gestational week. Thereafter, both peptides were found in the same bronchial neuroendocrine cells throughout fetal and neonatal life. In adult lungs with or without neuroendocrine cell hyperplasia, only calcitonin was present. In 17 of 18 (94%) pulmonary tumorlets, variable numbers of calcitonin-positive cells were identified. A few CGRP immunoreactive cells, as a subset of calcitonin-containing cells, were found in only three (17%) lesions. Of 37 bronchial carcinoids, calcitonin was detected in 14 and CGRP was detected in 16 (38% and 43%, respectively), and both peptides were predominantly localized in the same cells. Ten of 45 (22%) small cell lung carcinomas were calcitonin-immunoreactive. CGRP was noted in only one (2%) of these tumors, and both peptides coexisted in single cells. These findings indicate that the patterns of calcitonin/CGRP expression in hyperplastic bronchial neuroendocrine cells, pulmonary tumorlets, and, to some extent, small cell lung carcinomas are similar to those of normal adult lungs. On the other hand, calcitonin/CGRP expression in bronchial carcinoids is similar to that of late fetal and neonatal lungs.     

The role of calcitonin gene-related peptide in gastric mucosal protection in the rat

The presence of circulating antibodies to calcitonin gene-related peptide (CGRP) enhanced the damaging effect of ethanol on the rat gastric mucosa. Taken together with previous experimental and morphological data the results suggest that CGRP released from the peripheral terminals of visceral afferent fibres plays a role in mediating gastric mucosal defence mechanisms.     

Projections of calcitonin gene-related peptide immunoreactive neurons in the vagal ganglia of the rat

We have studied the connections of calcitonin gene-related peptide immunoreactive (CGRP-ir) sensory neurons in the ganglia of the vagus nerve. Many CGRP-ir neurons were identified in the jugular ganglion located in the cranial cavity, while fewer CGRP-ir neurons were found in the nodose ganglion located at the level of the jugular foramen. Application of Fluorogold to the cut end of the cervical vagus nerve resulted in many Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Application of Fluorogold to the cut end of the subdiaphragmatic vagus nerve resulted in Fluorogold-labeled neurons mostly in the nodose ganglion with only a few labeled neurons in the jugular ganglion. Injection of Fluorogold into the heart resulted in Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Double labeling combining CGRP immunohistochemistry and Fluorogold retrograde tracing showed that in cases of both the application of Fluorogold to the cut end of the cervical vagus nerve and the injection of Fluorogold into the heart, about 40% of the Fluorogold-labeled neurons in the jugular ganglion expressed CGRP-like immunoreactivity. These results indicate that many CGRP-ir neurons in the jugular ganglion innervate the cervical and thoracic visceral organs, including the heart, but only a few CGRP-ir neurons project to the abdominal visceral organs.     

降钙素基因相关肽的血管生物学功能多样性

降钙素基因相关肽是由辣椒素敏感的感觉神经末梢释放的一种生物活性多肽,广泛分布于神经及心血管系统。在心血管系统,降钙素基因相关肽生物活性具有多样性,如强大的舒血管作用、抑制平滑肌细胞增殖和内皮细胞凋亡,也能促进细胞分化增殖,参与血管新生。这种功能的多样性与维持机体的心血管系统功能有密切关系。     

Vascular actions of calcitonin gene-related peptide and adrenomedullin

This review summarizes the receptor-mediated vascular activities of calcitonin gene-related peptide (CGRP) and the structurally related peptide adrenomedullin (AM). CGRP is a 37-amino acid neuropeptide, primarily released from sensory nerves, whilst AM is produced by stimulated vascular cells, and amylin is secreted from the pancreas. They share vasodilator activity, albeit to varying extents depending on species and tissue. In particular, CGRP has potent activity in the cerebral circulation, which is possibly relevant to the pathology of migraine, whilst vascular sources of AM contribute to dysfunction in cardiovascular disease. Both peptides exhibit potent activity in microvascular beds. All three peptides can act on a family of CGRP receptors that consist of calcitonin receptor-like receptor (CL) linked to one of three receptor activity-modifying proteins (RAMPs) that are essential for functional activity. The association of CL with RAMP1 produces a CGRP receptor, with RAMP2 an AM receptor and with RAMP3 a CGRP/AM receptor. Evidence for the selective activity of the first nonpeptide CGRP antagonist BIBN4096BS for the CGRP receptor is presented. The cardiovascular activity of these peptides in a range of species and in human clinical conditions is detailed, and potential therapeutic applications based on use of antagonists and gene targeting of agonists are discussed.     

Leg uptake of calcitonin gene-related peptide during exercise in spinal cord injured humans.

Exercise-induced increases in cardiac output (CO) and oxygen uptake (VO2) are tightly coupled, as also in absence of central motor activity and neural feedback from skeletal muscle. Neuromodulators of vascular tone and cardiac function - such as calcitonin gene related peptide (CGRP) - may be of importance. Spinal cord injured individuals (six tetraplegic and four paraplegic) performed electrically induced cycling (FES) with their paralyzed lower limbs for 29 +/- 2 min to fatigue. Voluntary cycling performed both at VO2 similar to FES and at maximal exercise in six healthy subjects served as control. In healthy subjects, CGRP in plasma increased only during maximal exercise (33.8 +/- 3.1 pmol l(-1) (rest) to 39.5 +/- 4.3 (14%, P<0.05)) with a mean extraction over the working leg of 10% (P<0.05). Spinal cord injured individuals had more pronounced increase in plasma CGRP (33.2 +/- 3.8 to 46.9 +/- 3.6 pmol l-1, P<0.05), and paraplegic and tetraplegic individuals increased in average by 23% and 52%, respectively, with a 10% leg extraction in both groups (P<0.05). The exercise induced increase in leg blood flow was 10-12 fold in both spinal cord injured and controls at similar VO2 (P<0.05), whereas CO increased more in the controls than in spinal man. Heart rate (HR) increased more in paraplegic subjects (67 +/- 7 to 132 +/- 15 bpm) compared with controls and tetraplegics (P<0.05). Mean arterial pressure (MAP) was unchanged during submaximal exercise and increased during maximal exercise in healthy subjects, but decreased during the last 15 min of exercise in the tetraplegics. It is concluded that plasma CGRP increases during exercise, and that it is taken up by contracting skeletal muscle. The study did not allow for a demonstration of the origin of the CGRP, but its release does not require activation of motor centres. Finally, the more marked increase in plasma CGRP and the decrease in blood pressure during exercise in tetraplegic humans may indicate a role of CGRP in regulation of vascular tone during exercise.     

The intramural pelvic nerves immunoreactive for calcitonin gene-related peptide in the rectum of normal and aganglionosis rat

Summary The distribution of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was investigated immunohistochemically in the rectum of normal, capsaicin-treated and congenital aganglionosis rats. The rectum of the normal rat was densely supplied with both extrinsic and intrinsic nerves exhibiting CGRP-like immunoreactivity. Numerous CGRP-LI nerve fibres were seen in both the myenteric and submucous plexuses. Intrinsic CGRP-LI nerve cell bodies were sparsely found in both the ganglionated plexuses, while a large inflow of extrinsic CGRP-LI nerves was characteristically observed in the rat rectum. CGRP-like immunoreactive fibres were abundant in the intramural pelvic nerves which ascend proximally in the intermuscular zone and connect with the myenteric plexus of the rat distal bowel. As compared with CGRP-positive fibres, SP- or SK-positive fibres in the intramural pelvic nerves were far less frequent. The treatment with capsaicin in the neonatal period led to a marked depletion of CGRP-immunoreactivity in these extrinsic nerves as well as in the most terminal varicose fibres seen in the whole layers of the rectal wall. These findings suggest that the vast majority of CGRP-LI fibres in the intramural pelvic nerves are sensory in nature, and that the positive nerve fibres of extrinsic origin directly innervate each layer of the rat rectum. These CGRP-LI sensory fibres associated with the intramural pelvic nerves, may be of importance in the regulation of rectal and colonic function in normal rats. A dense innervation of CGRP-LI nerve fibres, some of which showed the varicose appearance, was also found in the rectum of congenital aganglionosis rats. Thus, it is suggested that there is a large inflow of extrinsic CGRP-LI fibres from the pelvic plexus in the affected rectum. The extrinsic CGRP-LI nerves in the aganglionic segment of the mutant rat might also be related to the regulation of rectal function, providing afferent pathways.     

Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat

The interactions between the inflammatory mediators bradykinin, serotonin, prostaglandin E(2) and acid pH were studied in rat dorsal root ganglion neurons in culture. For this purpose, the cultures were stimulated by inflammatory mediators (bradykinin, serotonin, prostaglandin E(2), 10(-5)M each) or acid solution (pH 6.1) for 5 min and the content of calcitonin gene-related peptide was determined in the supernatant before, during and after stimulation, using an enzyme immunoassay. Acid solution resulted in a threefold increase of the basal calcitonin gene-related peptide release which was entirely dependent on the presence of extracellular calcium. The release could not be blocked by the addition of the capsaicin antagonist capsazepine (10(-5)M). Bradykinin (10(-5)M) caused a 50% increase of the basal calcitonin gene-related peptide release which was again dependent on the presence of extracellular calcium, whereas serotonin and prostaglandin E(2) were each ineffective at 10(-5)M concentration. The combination of bradykinin, serotonin and prostaglandin E(2) led to a fivefold increase of the calcitonin gene-related peptide release which could not be further enhanced by acidification. The competitive capsaicin receptor antagonist capsazepine (10(-5)M) significantly reduced the release induced by the combination of bradykinin, serotonin and prostaglandin E(2).It is suggested that the inflammatory mediators co-operate and together may act as endogenous agonists at the capsaicin receptor to cause calcium influx and consecutive neuropeptide release.     

Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis

The distribution of immunoreactive calcitonin gene-related peptide in the rat brain was investigated by means of an indirect immunofluorescence method. In addition to previously reported calcitonin gene-related peptide-like immunoreactive structure-containing sites such as the nucleus ambiguus, nucleus originis nervi facialis, nucleus originis nervi hypoglossi, nucleus peripeduncularis and nucleus parabrachialis, the present study demonstrated a far wider distribution of calcitonin gene-related peptide-like immunoreactive structure-containing cells in the rat brain, i.e. the nucleus hypothalamicus lateralis, nucleus ventromedialis thalami, colliculus superior, lemniscus lateralis, gyrus dentatus, nucleus olivaris superior, nucleus tractus solitarii, nucleus cuneiformis, nucleus parabigeminalis and a proportion of the Purkinje cells. We have also demonstrated a more extensive network of calcitonin gene-related peptide-like immunoreactive fibers distributed in various areas throughout the rat brain than has been reported previously such as the colliculus inferior, nucleus olivaris superior, nucleus vestibularis lateralis and inferioris, and nucleus cochlearis dorsalis and ventralis, etc.