Immunohistochemical mapping of neurophysins and calcitonin gene-related peptide in the human brainstem and cervical spinal cord

Our study investigates the distribution of neurophysins (Nph), proteins that are part of the precursors for vasopressin and oxytocin, and calcitonin gene-related peptide (CGRP) in the human brainstem by immunohistochemistry. Both peptides were found in discrete regions of the human hindbrain. Nph could be demonstrated exclusively in fibers and punctate perineural varicosities that were travelling within the mesencephalic central gray, substantia nigra, as well as locus coeruleus, medial longitudinal fascicle, raphe, nucleus of the solitary tract, lateral reticular nucleus and area postrema. A few varicosities were seen in the substantia gelatinosa of the spinal trigeminal tract and its continuation into the dorsal horn of the cervical spinal cord. In contrast to these observations. CGRP-immunoreactive fibers were found to be densest in the spinal tract of the trigeminal nerve and the dorsal horn of the spinal cord. In addition, fibers and varicosities could be demonstrated in numerous distinct brain regions, such as locus coeruleus and subcoeruleus, solitary tract, cuneate nucleus, raphe and periaqueductal gray. CGRP-immunoreactivity was also present in perikarya in the ventral horn of the spinal cord, as well as motor nuclei of cranial nerves, i.e., hypoglossal nucleus, ambiguous nucleus. Our results suggest that Nph-immunoreactivity in the human brainstem may be present predominantly within long fiber projections from hypothalamic neurosecretory nuclei, in analogy to data obtained from rodents, whereas CGRP may play a role in the branchiomotor system as well as in intrinsic or extrinsic projections involved in autonomic regulation and integration of sensory information.     

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.     

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。     

Detection of calcitonin and calcitonin gene-related peptide mRNA in human medullary thyroid carcinoma. A retrospective study

In Situ hybridization finds many applications in modern pathology. In many cases, special attention is paid to the processing of the tissues prior to in situ hybridization. In order to investigate the value of RNA in situ hybridization (RISH) in retrospective studies, we performed RISH for calcitonin and calcitonin gene-related peptide (CGRP)-I and HomRNA in eight medullary thyroid carcinomas processed in 1981–1983. RISH was successful with radioactive calcitonin and CGRP-1 probes. With biotinylated probes, only calcitcnin-specific probes gave adequate results. The concentrations of CGRP mRNA were probably too low to be detected by non-radioactive RISH. The results of RISH were correlated with the immunohistochemical localization of the polypeptides. The results matched in all cases except one, where hybridization for calcitonin mRNA was found, but no immunoreactive calcitonin polypeptide. We con-clude that RISH can be successfully used for retrospective analysis, even after long storage of tissue embedded in poraffin.     

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.     

Immunohistochemical comparison of chromogranins A and B and secretogranin II with calcitonin and calcitonin gene-related peptide expression in normal, hyperplastic and neoplastic C-cells of the human thyroid

Normal and hyperplastic thyroid C-cells and 14 cases of medullary thyroid carcinoma were investigated immunohistochemically with antibodies against chromogranins A and B, secretogranin II, calcitonin and calcitonin gene-related peptide (CGRP). Normal and hyperplastic C-cells showed strong calcitonin and chromogranin A immunoreactivity whereas CGRP, chromogranin B and secretogranin II expression was less intense. Strong calcitonin and chromogranin A immunoreactivity was also found in the majority of tumour cells in medullary thyroid carcinoma. The CGRP, chromogranin B and secretogranin II staining observed was present in variable patterns. In some cases CGRP, chromogranin B and secretogranin II could only be demonstrated in isolated tumour cells with elongated processes suggestive of neuronal differentiation of these cells. The biological function(s) of the chromogranins/secretogranins remain(s) still unclear. There is evidence that these proteins are pro-peptides which give rise to functionally active compounds. Studies on normal C-cells and medullary thyroid carcinoma may elucidate the role of chromogranins/secretogranins in endocrine and neuronal cells.     

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland

The indirect immunofluorescence technique was used to study the distribution of calcitonin gene-related peptide (CGRP) in human submandibular gland. A relatively low number of thin varicose fibers with intense immunofluorescence for CGRP was seen in samples from seven glands. These CGRP-immunoreactive (CGRP-IR) nerve fibers were mainly seen around or in close contact with intra- and interlobular blood vessels. Some CGRP-IR nerve fibers were also found in association with intra- and interlobular salivary ducts and a few around the submandibular acini. By visual estimation there was no difference in the density of CGRP-IR nerve fibers between specimens of recurrent duct obstruction and laryngeal carcinoma. The present results show that the distribution of CGRP-IR nerve fibers in the stroma and in the glandular secretory elements of the human submandibular gland is quite similar to that seen in the rat and the ferret, which have been reported earlier. Furthermore, the regional distribution of CGRP-IR fibers in the human submandibular gland suggests that CGRP has a physiological role in the regulation of salivary gland function in human salivary glands, e.g. blood flow and secretion.     

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.     

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.     

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.     

Substance P and calcitonin gene-related peptide: effects on mast cells and in human skin

Mast cells are found in close association with blood vessels, and histamine is known to be a potent vasodilator in humans. It is now clear that mast cells form neuroeffector junctions and that one of the types of nerve involved is the peptide-containing primary afferent neurone (C fibre). Nerve stimulation produces vasodilation which is blocked by antihistamines or by depletion of mast cell histamine with compound 48/80. Nerve stimulation also releases histamine and degranulates mast cells. Substance P and other neuropeptides release histamine from isolated rat and human skin mast cells. The actions of substance P and calcitonin gene-related peptide in human skin are compatible with a role for these two peptides in neurogenic inflammation. The inflammatory effects of substance P in human skin are inhibited by antihistamines. The possible role of the mast cell in neurogenic inflammation is discussed.     

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)     

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.     

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.     

Immunohistochemical analysis of the ontogeny of calcitonin gene-related peptide-like immunoreactivity in the rat central nervous system

The ontogeny of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the rat central nervous system was examined using the indirect immunofluorescence technique. Caudally located neurons displayed CGRP-LI relatively early in development. For instance, CGRP-immunoreactive (CGRP-IR) cells were first visualized in the hypoglossal nucleus and dorsal nucleus of the lateral lemniscus at day 19 of gestation, while by day 1 postnatal the facial and ambiguus nuclei possessed their full complement of immunoreactive neurons relative to that observed in the adult. By contrast, CGRP-IR somata in more rostral regions such as the lateral septal nucleus were not visualized until much later in development, i.e. day 12 postnatal. By day 15 postnatal, however, CGRP-LI was at a maximum in almost all areas scrutinized. This pattern of immunostaining persisted up to the oldest age studied (day 28 postnatal), except in the lateral septal and parabrachial nuclei, along the dorsal border of the medial lemniscus, and in the inferior olive, where many of the previously immunopositive cells could no longer be detected. In summary, CGRP-IR somata in the rat central nervous system appear in a principally caudal to rostal direction, beginning in late gestation. In some regions, CGRP is present only ephemerally. The ontogeny of CGRP during maturation of the rat brain is consistent with its suggested neurotrophic and neurotransmitter roles.     

Immunohistochemical localization of calcitonin gene-related peptide and cotransmitters in a subpopulation of post-ganglionic neurons in the porcine inferior mesenteric ganglion.

Applying double-fluorescence immunohistochemistry, adrenergic and non-adrenergic postganglionic sympathetic neurons, in the porcine inferior mesenteric ganglion (IMG) are subdivided according to size and cotransmitter content. Calcitonin gene related peptide (CGRP)-immunoreactive (IR) neurons are demonstrated to belong to the non-adrenergic, i.e. tyrosine hydroxylase- and DOPAmine-beta-hydroxylase-(D beta H)-negative subpopulation of postganglionic perikarya. Virtually all of the CGRP-IR postganglionic neurons exhibit colocalization with somatostatin (SOM), and, some of them with neuropeptide tyrosine (NPY). Additionally, NPY-, SOM-, and NPY/SOM-IR subpopulations of adrenergic and non-adrenergic neurons are observed. CGRP-immunoreactivity is seen in dense networks of intraganglionic varicose nerve fibres, adjacent to the TH- and SOM-IR neurons. NPY-IR perikarya are sparsely supplied by CGRP-IR fibres. SOM- and NPY-IR nerve fibres also exist in the inferior mesenteric ganglion. The functional relevance of CGRP-IR postganglionic neurons, as well as target organs of these neurons remain to be elucidated.     

The effect of calcitonin gene-related peptide (CGRP) on human forearm blood flow

The effect of intravenous and intra-arterially administered calcitonin gene-related peptide (CGRP) on the human forearm blood flow and cutaneous blood flow were investigated by means of venous occlusion plethysmography and laser-Doppler flowmetry, respectively. Infusion of CGRP (11-216 pmol min-1) into the brachial artery resulted in a dose-dependent increase in forearm blood flow and cutaneous blood flow which persisted for up to 90 min after the infusion was stopped. Repeated infusions resulted in an identical response. Systemic intravenous infusion of CGRP (104-520 pmol min-1) resulted in a dose-dependent flush in the face, neck, upper trunck and upper arms, and an increase in the forearm blood flow. The cutaneous blood flow was dramatically increased on the forehead, whereas on the hand only a slight increase was noted. By intravenous infusions a significant drop in blood pressure and increase in heart rate were seen at 520 pmol min-1. Thus, it is possible to give CGRP in doses that increase the blood flow in muscle and skin without resulting in a fall in systemic arterial blood pressure and tachycardia, suggesting that CGRP may be used as a tool for the treatment of various conditions in man with compromised blood flow.