Distinct binding sites for calcitonin gene-related peptide and salmon calcitonin in rat central nervous system

Calcitonin gene-related peptide (CGRP) binding sites have been identified in homogenates from the rat brain and spinal cord. Autoradiography with [¹²⁵I]rat CGRP (rCGRP) revealed high grain density over the lateral hypothalamus, vestibular nuclei, colliculi, medial geniculate body, corpus mamillare and the molecular layer of the cerebellum which lacked binding sites for [¹²⁵I]salmon calcitonin (sCT). In contrast, no rCGRP labeling was seen over the anterior and dorsomedial hypothalamus which showed high sCT binding. The different regional distribution of rCGRP and sCT binding sites indicates that the structurally related peptides interact with separate receptors. The overlap between the localization of CGRP binding sites and endogenous CGRP in many regions of the central nervous system suggests that CGRP exerts unique physiological functions in the central nervous system.     

Localization of calcitonin gene-related peptide and its receptors in a striated muscle

The distribution of calcitonin gene-related peptide (CGRP) and its binding sites in the bulbocavernosus, a striated muscle, are reported. We used immunohistochemistry and [125I]CGRP autoradiography. The pattern of [125I]CGRP binding was restricted to a discrete band that coincides with the distribution of end-plates in this muscle as determined by CGRP immunohistochemistry and acetylcholinesterase staining. CGRP has been shown to increase the level of acetylcholine receptor (AChR) alpha-subunit mRNA. The role of CGRP as the endogenous factor by which motoneurons regulate the expression of junctional AChR is discussed.     

降钙素基因相关肽与内皮素受体A在兔实验性蛛网膜下腔出血后脑组织表达的对照研究

目的探讨降钙素基因相关肽(CGRP)和内皮素受体A(ETRA)在蛛网膜下腔出血(SAH)后脑组织中的表达情况及意义。方法日本大耳白家兔45只,随机分为SAH模型组(20只),盐水对照组(15只),穿刺对照组(5只),空白对照组5只。采用枕大池2次注血法制作SAH的动物模型,并进行CGRP和ETRA的免疫组化染色,观察CGRP和ETRA的免疫表达。结果CGRP和ETRA免疫阳性标记在SAH组模型均有表达,CGRP阳性细胞在1h时少量表达,3～7d时表达明显,其中5d时最显著,10d时阳性细胞表达已经减少,但未达到正常水平。SAH模型组ETRA主要表达在神经元和胶质细胞胞浆内,建模后1h时染色较淡;3d时染色最强烈;5d时染色较3d弱,此后染色逐渐变淡。结论SAH后脑组织CGRP和ETRA的表达增强,并呈现明显的动态改变,但两者的变化趋势并不同步;CGRP和ETRA可能在延迟性神经功能障碍的发生与发展中起重要作用。     

Light and electron microscopic studies of calcitonin gene-related peptide-like immunoreactive neurons and axon terminals of the nucleus of the tractus solitarius of the rat

This study was an examination of the ultrastructural characteristic features of calcitonin gene-related peptide (CGRP)-like immunoreactive neurons and their axon terminals in the nucleus of the tractus solitarius of the rat. Some axon terminals were identified as receiving synaptic inputs from non-immunoreactive axon terminals. This may suggest that part, if not all, CGRP containing afferents are affected presynaptically by other afferents.     

降钙素基因相关肽对缺氧时大鼠海马培养神经元c—fos表达的影响

采用免疫组化（ABC）方法，观察缺氧诱导体外培养大鼠海马神经元c-fos的表达及降钙素基因相关肽（CGRP）的影响。结果显示，缺氧后海马神经元中Fos-免疫反应（Fos—IR）阳性胞核百分率随缺氧时间的延长而逐渐增多，图像分析的结果显示，缺氧后Fos—IR阳性胞核的平均失密度亦随缺氧时间的延长而逐渐增强。经CGRP孵育的海马神经元缺氧后Fos-IR阳性胞核的百分率和Fos-IR阳性胞核的平均光密度均明显低于非CGRP孵育组。本结果表明，缺氧能诱导体外培养海马神经元。c-fos的表达，神经肽CGRP能抑制缺氧海马神经元c-fos的表达。提示CGRP对海马神经元缺氧损伤可能具有一定保护作用。     

Local anesthetics inhibit veratridine-induced secretion of calcitonin gene-related peptide (CGRP) from cultured rat trigeminal ganglion cells

Secretion of calcitonin gene-related peptide (CGRP) was studied with the model system of dispersed adult rat trigeminal ganglion cells. Veratridine stimulated secretion of CGRP immunoreactivity. Tetrodotoxin and local anesthetics inhibited veratridine-stimulated peptide secretion. These observations implicate sodium channels in CGRP secretion and are consistent with a role for the peptide as an extracellular neuromodulator in the sensory nervous system.     

Tissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development

The distribution of calcitonin receptor-like receptor (CRLR) mRNA in developing rats was investigated by in situ hybridization. Signals were found in the piriform cortex, the central and basolateral amygdala and the amygdalostriatal transition area. Among peripheral organs, the CRLR was predominantly expressed in the lung. mRNA expression in blood vessels, liver, midgut, rectum and urethra was restricted to gestational days 16 and/or 20. The CRLR was thought to be a calcitonin gene-related peptide (CGRP) type 1 receptor (Aiyar et al., J. Biol. Chem., 271 (1996) 11325–11329). This contrasts with previously reported evidence that the CRLR is an orphan receptor with no identifiable interactions with CGRP and other related ligands (Flühmann et al., Biochem. Biophys. Res. Commun., 206 (1995) 341–347). In situ hybridization signals have not been detected in the cerebellum and the spleen known to present a high density of CGRP binding sites. The different regional distribution of CGRP receptor binding sites and CRLR mRNA implies the latter encoding a different CGRP receptor subtype.     

Calcitonin Gene-related Peptide Causes Intraspinal Spreading of Substance P Released by Peripheral Stimulation

Experiments were performed in barbiturate-anaesthetized, spinalized cats to investigate the effect of calcitonin gene-related peptide (CGRP) on the spatial distribution of immunoreactive substance P (ir-SP) in the spinal cord released by electrical nerve stimulation and noxious mechanical stimuli. The presence of ir-SP was assessed with microprobes bearing C-terminus-directed antibodies to SP. CGRP was microinjected into the grey matter of the spinal cord near microprobe insertion sites at depths of 2500, 2000, 1500 and 1000 μm using minute amounts (in total 0.2–0.5 μl) of Ringer solution containing CGRP at a concentration of 10⁻⁵ or 10⁻³ M. In the untreated cord electrical stimulation of the tibial nerve (suprathreshold for all C fibres) elicited release of ir-SP which was centred in and around the lamina II. After microinjection of CGRP, stimulation-associated ir-SP was detected in a region extending from the cord surface down to the ventral horn. This pattern was similar to that observed after the microinjection of synthetic peptidase inhibitors (Duggan et al., Brain Res. , 579 , 261–269, 1992). The large expansion of sites accessed by ir-SP was time-dependent, reaching a maximal effect within 10–40 min after microinjection of CGRP, and reversal was observed in subsequent probes. A similar expansion of the regions accessed by ir-SP after microinjection of CGRP was also observed when release of ir-SP was evoked by noxious mechanical stimulation of the toes. These results indicate that one important function of CGRP in the spinal cord may be the control of the intraspinal sites and neuronal circuits accessed by released substance P, possibly by inhibition of endopeptidases responsible for peptide degradation.     

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains

125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide-mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I-CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity.     

降钙素基因相关肽及内皮素A受体与蛛网膜下腔出血

Numerous studies have demonstrated that endothelin-1 combines with endothelin receptor A,resulting in intense vasoconstriction.Although calcitonin gene-related peptide(CGRP) suppresses endothelin-1,CGRP and endothelin receptor A exhibit direct biological effects on brain tissue.The present study analyzed CGRP and endothelin receptor A expression following subarachnoid hemorrhage in rabbits using immunohistochemistry.CGRP expression was significant at 5 days after model establishment,and endothelin receptor A expression was significant at 3 days after model induction.The perimeter of the basilar artery was measured to determine the amount of cerebral vasospasm.Analytical results revealed a significantly shortened basilar artery perimeter following subarachnoid hemorrhage.Changes in the basilar artery perimeter were negatively associated with endothelin receptor A expression,but positively correlated with CGRP expression in vessels.These results suggest that following subarachnoid hemorrhage,CGRP and endothelin receptor A expressions dynamically changed in brain vessels and tissues,although these changes were not synchronous.Changes in endothelin receptor A expression exhibited a significant effect on the occurrence and development of delayed cerebral vasospasm and delayed neuronal death,while CGRP relaxed vessels and protected nerves.     

侧脑室注射肾上腺髓质素增加大鼠心血管相关核团中c-Fos的表达

目的 研究侧脑室注射肾上腺髓质素(1nmol／kg，3．nmoL／kg)，对大鼠脑内心血管相关核团中c-fos表达的影响。方法 应用Fos蛋白免疫组化技术。结果 (1)侧脑室注射肾上腺髓质素诱发脑干的孤束核、最后区、蓝斑核、臂旁核和外侧巨细胞旁核，下丘脑的室旁核、视上核和腹内侧核以及前脑的中央杏仁核和外侧缰核等多个部位的心血管中枢出现大量Fos样免疫反应神经元。(2)降钙素基因相关肽受体拮抗剂CGRP8-37(30nmol／kg)可明显减弱肾上腺髓质素(3nmol／kg)的效应。结论 肾上腺髓质素可兴奋脑内多个心血管相关核团的神经元，降钙素基因相关肽受体可能介导这一效应。     

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations. The lack of any rise in the brainstem CGRP content raises the possibility that this process spares central terminations. In contrast, the level of neuropeptide Y, a peptide mainly contained in sympathetic terminals, fell to 35% of control values in the iris and pial arteries whilst the trigeminal ganglion and brainstem concentrations remained unchanged. The possible relevance of these observations to the clinical syndrome of postsympathectomy pain (sympathalgia) is discussed. There are similarities between the delayed onset of the human pain state and the delayed rise in sensory peptides after sympathectomy.     

Calcitonin and calcitonin gene-related peptide enhance calcium-dependent potentials

Recent data suggests that calcitonin (CT) and/or calcitonin gene-related peptide (CGRP) may be potential transmitters or modulators in the nervous system. The present study analyzed the effect of CT and CGRP on the neuronal membranes of cat parasympathetic ganglia of the urinary bladder. The related peptides prolonged the duration of the afterhyperpolarization of the action potential but had no effect on resting potential or input resistance. CT and CGRP enhanced the duration of a calcium spike recorded in the presence of agents blocking Na and K channels while under similar conditions forskolin, an activator of adenylate cyclase, did not affect the calcium spike. These data suggest that the neural mechanism of action of CT and CGRP is to prolong a calcium conductance and that these effects are not mediated through cyclic AMP.     

天舒胶囊对偏头痛动物模型血浆一氧化氮、一氧化氮合酶、降钙素基因相关肽含量及血流动力学的影响

目的 研究天舒胶囊对偏头痛动物模型血浆及脑组织血管活性物质及血流动力学的影响.方法 皮下注射硝酸甘油分别制作大鼠和兔偏头痛模型.给予天舒胶囊后用放免法和分光光度法测大鼠血浆一氧化氮(NO)和一氧化氮合酶(NOS)、降钙素基因相关肽(CGRP)含量;通过免疫组织化学染色观察三叉神经脊束核神经元型NOS(NOS1)和CGRP表达;用经颅多普勒检测兔颈内动脉血流速度改变.结果 模型组大鼠血浆NO、NOS和CGRP较对照组明显升高;经不同剂量天舒胶囊灌胃后大鼠血浆NO、NOS和CGRP的增加受到抑制,尤以中、高剂量组明显(P<0.05～0.01).模型组兔颈内动脉收缩期峰值流速明显下降,经中剂量天舒胶囊干预后流速下降也受到抑制 (P<0.05).免疫组织化学染色发现灌胃天舒胶囊后,偏头痛大鼠三叉神经脊束核NOS1和CGRP表达增加的程度减小(均P<0.05).结论 天舒胶囊可改善偏头痛发作时血管活性物质和神经递质水平失常,从而缓解偏头痛症状.     

Rat facial motoneurons express increased levels of calcitonin gene-related peptide mRNA in response to axotomy

The expression and localization of the mRNA encoding the calcitonin gene-related peptide (CGRP) were analyzed in the rat facial nucleus after axotomy by Northern blot analysis and by in situ hybridization histochemistry (ISH) using a synthetic 32P-labeled oligonucleotide probe. Northern blot analysis revealed the presence of the 1.2 kb CGRP mRNA in RNA extracted from the facial nucleus. This mRNA species was strongly increased after axotomy of the facial nerve. By ISH increased levels of CGRP mRNA were observed as soon as 16 hr after axotomy compared with the unoperated nucleus. CGRP mRNA could be localized in more than 50% of the motoneurons. Three populations of motoneurons with no, moderate, or strong labeling for CGRP mRNA could be distinguished. Peak expression of CGRP mRNA during the first 48 hr was followed by a decline to moderate levels at day 4 after lesion, and to almost basal levels at days 7 and 9. These data demonstrate that axotomy of the facial nerve leads to an early and strong induction of CGRP gene expression in motoneurons of the facial nucleus.     

Alteration of capsaicin and endotoxin-induced calcitonin gene-related peptide release from mesenteric arterial bed and spinal cord slice in 18-month-old rats

In the present study, we investigated the changes in capsaicin- and endotoxin-induced calcitonin gene-related peptide (CGRP) release from mesenteric arterial bed (MAB) and spinal cord slices (SCS) in 2-month-old and 18-month-old Wistar rats. The isolated MAB or SCS was perfused or incubated in vitro. The CGRP-like immunoreactivity in perfusate or supernatant was measured by radioimmunoassay. The results showed that endotoxin triggered CGRP release from isolated rat MAB and SCS, which represent the peripheral and central terminals of CGRP-containing sensory nerves, respectively. Either basal or stimulated CGRP release induced by capsaicin and endotoxin was significantly decreased as the rats aged from 2 to 18 months. The basal CGRP release was 14.9 ± 1.8 and 5.8 ± 1.0 pg/ml from MAB and 3.50 ± 0.54 and 1.78 ± 0.16 pg/ml/mg from SCS in 2-month-old and 18-month-old rats, respectively. The release of CGRP evoked by capsaicin (10⁻⁷ mol/L) and endotoxin (1 ≈ 5 μg/ml) from MAB and SCS was significantly decreased by more than 50% in 18-month-old rats. These data suggest that both the basal and capsaicin- or endotoxin-stimulated CGRP release from MAB and SCS display a significant decrement in aged rats that may have some physiological, pathological, and behavioural relevance in age-related diseases. J. Neurosci. Res. 53:385–392, 1998. © 1998 Wiley-Liss, Inc.     

Parabrachial Origin of Calcitonin Gene-Related Peptide-Immunoreactive Axons Innervating Meynert''s Basal Nucleus

Meynert's basal nucleus is innervated by calcitonin gene-related peptide (CGRP)-immunoreactive axons synapsing with cholinergic principal cells. Origin of CGRP-immunopositive axons was studied in the albino rat. Since beaded axons containing the nicotinic acetylcholine receptor (nAChR) are also present in the basal nucleus, the microstructural arrangement raises the question whether or not an interaction between CGRP and nAChR exists like in the neuromuscular junction. We found that electrolytic lesion of the parabrachial nucleus results in degeneration of CGRP-immunoreactive axons in the ipsilateral nucleus basalis and induces shrinkage of principal cholinergic neurons while the contralateral nucleus basalis remains intact. Electrolytic lesions in the thalamus, caudate-putamen, and hippocampus did not induce alterations in Meynert's basal nucleus. Disappearance of CGRP after lesions of the parabrachial nucleus does not impair presynaptic nAChR in the basal nucleus, suggesting that, unlike in the neuromuscular junction, CGRP is not involved in the maintenance of nAChR in the basal forebrain. It is concluded that the parabrachial nucleus is involved in the activation of the nucleus basalis-prefrontal cortex system, essential in gnostic and mnemonic functions.     

Plasticity of the serotonergic innervation of the dorsal horn of the rat spinal cord following neonatal capsaicin treatment.

Neonatal capsaicin treatments (25 or 50 mg/kg, 12, 24, or 48 hr after birth given subcutaneously) were applied in order to follow by immunocytochemical techniques the postnatal development and plasticity of the serotonergic system in the dorsal horn of the rat spinal cord. Two markers of the lesions of C primary afferents induced by capsaicin were tested by immunocytochemical detection: substance P and calcitonin gene-related peptide (CGRP). We show that the internal part of substantia gelatinosa (lamina Ili) which does not contain serotonergic fibers in intact or vehicle-treated rats is invaded within a few days after capsaicin treatment by serotonergic fibers apparently sprouting from the deepest laminae. Moreover, these fibers often establish axodendritic synapses while synapses are rare in intact animals in the whole dorsal horn. This reorganization is stable whatever the dose of capsaicin used or the moment chosen for its injection. On the other hand, while lesions of substance P-ergic fibers appeared quite stable, partial recovery of CGRP innervation was found after 3 to 6 months, especially with the low dose of capsaicin. We discuss the ability of the serotonergic system innervating the dorsal cord either to find new targets or to fill vacated sites when one of its putative targets is removed.