Kidney: a target organ for calcitonin gene-related peptide

In the present study we have evaluated the effect of calcitonin gene-related peptide (CGRP) on cyclic AMP levels in intact mouse kidneys. We have used an in vivo bioassay based on microwave irradiation as a means of rapid tissue fixation, followed by the determination of cyclic AMP concentration using a protein binding assay. CGRP was found to cause a dose-dependent elevation of renal cyclic AMP levels, and despite being slightly less potent than calcitonin (CT), the action of CGRP was significantly more prolonged. The extended time course of action of CGRP is in sharp contrast to its known effect on bone cyclic AMP levels and could be of physiological relevance. CGRP may act on a receptor different from that of CT to produce distinct functional effects.     

A highly specific and sensitive enzyme-immunometric assay for calcitonin gene-related peptide based on enzyme amplification

Calcitonin gene-related peptide (CGRP) is an important member of the peptide family encoded by the calcitonin gene. It has been found to be a potent vasodilator in man and a major circulating gene product (Girgis et al., 1985). The present study reports the development of a sensitive and rapid two-site immunoassay for CGRP based on enzyme amplification (Self, 1985). The assay has been easy to construct, taking advantage of available antisera raised for other purposes. Nevertheless it has been found to be clearly superior to our previous radioimmunoassay in terms of sensitivity, specificity, speed and convenience.     

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)     

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。     

降钙素基因相关肽（CGRP）在败血症休克中的作用

为探讨调节肽CGRP在休克中的发病学意义,本工作在结扎大鼠盲肠加穿孔的败血症休克模型上,观察了CGRP的变化及外源性CGRP对败血症休克过程的影响。结果发现,败血症休克时,血浆中CGRP显著增加(14.3±4.0 vs 5.5±1.8 Pg/ml,P<0.01)。在休克早期、晚期运用CGRP治疗(5μg/kg CGRP ⅳ)都能明显改善动物的低血压状态(分别是14.8±0.8,15.5±0.9 vs休克组10.3±1.6kPa,P<0.05),减轻心肌组织病理损伤,减少血浆中酶(LDH,CD)的漏出。同时,也可抑制休克动物血浆中AGT-Ⅱ含量的增加,提高血浆6-kcto-PGF_1α/TXB_2的比值(早期:69.1±5.3,晚期:65.8±4.1 vs休克组51.0±4.7%,P<0.05)。结果提示,CGRP可能参与败血症休克的代偿调节,适当应用CGRP治疗败血症休克是有益的。     

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.     

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.     

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。     

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.     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8–37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8–37) [0.01–10 μmol L^?1] dose-dependently decreased (29 ± 4–86 ± 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 μmol L^?1). Renal pelvic perfusion with 900 m M NaCl also increased ipsilateral ARNA (23 ± 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 ± 4% increase, P < 0.05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8–37). Renal pelvic perfusion with 1 or 10 μM h-CGRP (8–37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 m M ). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

The action of a calcitonin gene-related peptide antagonist in human skin

The CGRP antagonist, CGRP_8-37, antagonized the ability of CGRP to increase blood flow in human skin and cause erythema. The mechanism of the antagonist effect of CGRP_8-37 on the CGRP-induced erythema was an increase by the antagonist of the rate of decay of the CGRP-induced erythema. Since CGRP_8-37 activates rat peritoneal mast cells to release their granular contents, we conclude that the degradation of CGRP by protease released from skin mast cells by CGRP_8-37. It was not possible to demonstrate any antagonistic effect of CGRP_8-37 on the CGRP receptor mediating increased blood flow in human skin.     

Inhibition of natural killer activity by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied. CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10(-7) M CGRP was about 60%. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells. Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Evidence for calcitonin gene-related peptide contacts on a population of lamina I projection neurons

Using double-labeling techniques, we evaluated small diameter primary afferent input, as indicated by calcitonin gene-related peptide-immunoreactive varicosities, to a population of lamina I projection neurons in the rat lumbar spinal cord. About one third of the lamina I neurons labeled after injections of a retrograde tracer into the region surrounding the brachium conjunctivum received contacts from immunoreactive varicosities. Significantly fewer immunoreactive varicosities were in apposition to fusiform neurons than pyramidal or flattened neurons. A positive correlation was found between the size of the retrogradely labeled neuron and the number of contacts received. This study demonstrates that a known population of nociceptive lamina I neurons received direct input from presumed nociceptive primary afferents.     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors.

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8-37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8-37) [0.01-10 micromol L-1] dose-dependently decreased (29 +/- 4-86 +/- 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 micromol L-1). Renal pelvic perfusion with 900 mM NaCl also increased ipsilateral ARNA (23 +/- 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 +/- 4% increase, P < 0. 05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8-37). Renal pelvic perfusion with 1 or 10 microM h-CGRP (8-37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 mM). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

Postnatal calcitonin gene-related peptide in the superior olivary complex

The purpose of this study was to investigate quantitatively the distribution of calcitonin gene-related peptide (CGRP)-positive neurons within the superior olivary complex (SOC) at various postnatal ages. In the lateral superior olive (LSO), most if not all, CGRP-positive cells correspond to the cholingeric portion of the lateral olivocochlear system which innervates the cochlea. Brains from 1-day old (P1) through juvenile (P30) hamsters were used. At all ages, CGRP-positive neurons were seen throughout the various nuclei of the SOC. There was a dramatic shift in the distribution of CGRP-positive neurons from being predominantly periolivary to being predominantly confined within the LSO. The number of CGRP-positive neurons clearly increased as a function of increasing age. At the earliest postnatal ages, the LSO contained few if any immunostained cells, whereas at later ages the LSO contained the majority (greater than 70%) of the immunostained cells. Assuming that these CGRP-positive cells within the LSO correspond to olivocochlear neurons, these data suggest that in hamsters the lateral olivocochlear system may be immature at birth up until the second postnatal week.     

Pulmonary calcitonin gene-related peptide immunoreactivity: nerve-endocrine cell interrelationships

Respiratory epithelium has been reported to be supplied with sensory nerves and to contain irritant and other receptors. In this immunohistochemical study, we examined the incidence, morphology, and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in epithelial cells the rat respiratory tract, using peroxidase anti-peroxidase (PAP) techniques. CGRP immunoreactivity was localized in capsaicin-sensitive nerve fibers and in capsaicin-nonsensitive endocrine cells occurring singly or in groups. These CGRP-immunoreactive structures reached close to or actually touched the airway lumen, were widely and abundantly present in the respiratory epithelium, and were arranged in distinct and characteristic patterns. CGRP-immunoreactive nerves innervated not only grouped cells but also single cells, and the innervation of these cells differed depending on whether they were in extrapulmonary or intrapulmonary epithelium. The specificity of the immunoreactivity was confirmed by absorption tests that excluded cross-reactivity with other peptides. The results suggest that epithelial nerve fibers and endocrine-like cells exhibiting CGRP immunoreactivity form a morphologic, and probably also a functional, complex throughout the respiratory epithelium. CGRP innervation may be related to receptor functions of respiratory epithelium.     

Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons

Summary By use of fluorescence immunohistochemistry it is shown that sciatic nerve section in cat and rat induces increased levels of immunoreactive calcitonin gene-related peptide (CGRP) in axotomized motoneurons. In the rat, this effect was clearly seen at 2–5 days postoperatively, but could not be demonstrated after 11–21 days. These findings are discussed in relation to previously proposed roles for CGRP in motoneurons.     

降钙素基因相关肽表达载体的构建

背景：为研究降钙素基因相关肽基因治疗在骨质疏松中的应用,首先要建立高效的降钙素基因相关肽基因载体。 目的：构建降钙素基因相关肽真核表达载体pBaBb-puro-CGRP。 方法：设计引物并通过PCR扩增出降钙素基因相关肽,酶切后用T4 DNA连接酶将其与pBaBb-puro进行连接,经过转化、阳性克隆筛选后,进行酶切和测序鉴定。 结果与结论：经PCR扩增获得了含430 bp的降钙素基因相关肽基因编码序列,经过酶切、连接、转化后,挑选10个菌落进行PCR检测,筛查到8个菌落含有重组质粒,进一步行酶切和测序鉴定,结果与理论预期完全相符。提示实验成功构建了pBaBb-puro-CGRP真核表达载体。     

降钙素基因相关肽对内皮素释放的影响

本实验在大鼠的整体及离体血管条上观察了降钙素基因相关肽（ＣＧＲＰ）对内皮素（ＥＴ）释放的影响。结果表明：ＣＧＲＰ静脉注射（５μｇ／ｋｇ）能明显降低大鼠内毒素血症时血浆ＥＴ含量，ＣＧＲＰ孵育（１０＾－８ｍｏｌ／Ｌ）离体的大鼠主动脉血管条能有效地抑制凝血酶（ｔｈｒｏｍｂｉｎ）引起的ＥＴ释放，但是ＣＧＲＰ静注及孵育不影响血浆ＥＴ的基础含量及离体血管条ＥＴ的基础释放。结果提示ＣＧＲＰ能抑制病理条件下ＥＴ的     

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.