Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in rat taste papillae.

Calcitonin gene-related peptide-like and neuron-specific enolase-like immunoreactivity (CGRP-IR and NSE-IR) were surveyed immunohistochemically in the fungi-form, foliate and circumvallate papillae in rats. A dense CGRP-IR network (subgemmal and extragemmal) in the taste papillae is linked to the presence of taste buds, even though CGRP-IR fibers are rarely present in the taste buds. Three typical fiber populations were detected with these two markers. (a) A population of coarse NSE-IR intragemmal fibers characterized by thick neural swellings, never expressing CGRP-immunoreactivity. (b) A population of thin varicose intragemmal NSE/CGRP-IR fibers. (c) A population of subgemmal and extragemmal NSE-/CGRP-IR fibers that partly penetrated the epithelium. The common distribution of CGRP-IR and NSE-IR fibers at the base of taste buds, their differential distribution and morphology within taste buds, added to their restricted nature (gustatory or somatosensory) suggest that a population of CGRP-IR fibers undergoes a target-induced inhibition of its CGRP phenotype while entering the taste buds. The combined use of NSE and CGRP allowed a better characterization of nerve fibers within and between all three types of taste papillae. NSE was also a very good marker for a subtype of taste bud cells in the foliate and in the circumvallate papillae, but no such cells could be observed in the fungiform papillae.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of calcitonin gene-related peptide and substance P in the rat knee cartilage at birth

Substance P (SP) and calcitonin gene-related peptide (CGRP) have been found in the perichondrium and within the cartilage canals. It is still unknown whether they exert a direct effect on chondrocytes during joint development. We processed 28 knees of newborn Wistar rats in 7 different fashions to perform histology and immunohistochemistry studies. Positive immunoreactivity against CGRP and SP was found in the inner aspect of the perichondrium in a close contact with chondrocytes. The presence of CGRP and SP indicates the presence of nerves fibers, and precedes the development of cartilage canals. Nerve fibers may play a role in the development of synovial joints before and during the presence of cartilage canals. The presence of CGRP and SP in the cartilage at birth may be involved in the early postnatal maturation of synovial joints. It remains to be determined whether autonomic innervation is later involved in age-related degenerative joint disease.     

Immunohistochemical localization of a specificileal peptide in the pig

Summary The tissue distribution of a polypeptide purified from pig ileal mucosa tentatively called porcine ileal polypeptide (PIP) and known to have potent acid secretagogue activity has been studied with immunohistochemical methods together with extraction of different tissues followed by radioimmunoassay for PIP content. Histochemically the peptide is found in superficial epithelial cells in the mucosa of the distal 20% of the small intestine and to some extent in the mucosa of the urinary tract. There is no staining of goblet cells or crypt cells. The staining in the urinary tract mucosa is due to antigenic peptides with Mr identical to PIP. While the presence of PIP in the ileum is compatible with a function as an enterooxyntin, it is not possible at present to explain the physiologic role of PIP entirely as a hormone regulating acid secretion in light of the immunohistochemical distribution.Supported in part by a grant from the NIH AM-27077 and the Sinai Hospital General Research Fund     

Potent anti-inflammatory action of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role.     

Immunohistochemical localization of a specific ileal peptide in the pig

The tissue distribution of a polypeptide purified from pig ileal mucosa tentatively called porcine ileal polypeptide (PIP) and known to have potent acid secretagogue activity has been studied with immunohistochemical methods together with extraction of different tissues followed by radioimmunoassay for PIP content. Histochemically the peptide is found in superficial epithelial cells in the mucosa of the distal 20% of the small intestine and to some extent in the mucosa of the urinary tract. There is no staining of goblet cells or crypt cells. The staining in the urinary tract mucosa is due to antigenic peptides with Mr identical to PIP. While the presence of PIP in the ileum is compatible with a function as an enterooxyntin, it is not possible at present to explain the physiologic role of PIP entirely as a hormone regulating acid secretion in light of the immunohistochemical distribution.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland

Summary Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.     

降钙素基因相关肽与偏头痛关系的研究

偏头痛是临床上常见的多发性疾病,主要表现为一侧或双侧头部反复发作的搏动性疼痛,可伴有恶心、呕吐、视物异常等神经症状。关于偏头痛的发病机制研究尚无定论。偏头痛的发病机理一直受到人们的关注,近年来一些研究表明,偏头痛的发作与头颅血管周围组织产生的血管活性物质,特别是与三叉神经血管系统的降钙素基因相关肽(calcitonin gene-relatedpeptide,CGRP)的含量变化关系密切。     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin

The overflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) in the nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz for 3 min or upon capsaicin (0.3 mumol bolus injection) were analysed in the nasal mucosa of sympathectomized pentobarbital anaesthetized pigs. The overflow of CGRP-LI upon antidromic stimulation displayed a slower appearance in the venous effluent than the overflow upon bolus injection of capsaicin. The vascular effects as revealed by the arterial blood flow, the venous blood flow, the blood volume of the nasal mucosa, i.e., the filling of the capacitance vessels and the superficial mucosal blood flow as revealed by the laser-Doppler signal were also studied. Antidromic stimulation of the trigeminal nerve as well as capsaicin bolus injection induced a marked vasodilation which was parallel to the overflow of CGRP. However, capsaicin bolus injection also resulted in a marked increase in the mean arterial blood pressure which may be due to reflex activation of sympathetic fibers. In conclusion, we have demonstrated that chemical stimulation with capsaicin as well as antidromic stimulation of nasal sensory nerves in sympathectomized animals induces both vasodilation and overflow of CGRP-LI in vivo. This indicates that CGRP may contribute to the sensory regulation of the microcirculation in the nasal mucosa.     

Age-related increase of calcitonin gene-related peptide in rat thyroid and circulation.

Elevated calcitonin levels in thyroid gland extracts and in plasma accompanied by C-cell hyperplasia are frequently found in old rats, in particular those raised in laboratory conditions. In parallel with calcitonin, we demonstrate here that the thyroidal content and plasma levels of immunoreactive calcitonin gene-related peptide (i-CGRP) significantly increase with age in rats (p less than 0.0001). C18 Sep-Pak-extractable i-CGRP level in plasma was 35% of the total i-CGRP. Gel permeation chromatography and rp-HPLC studies revealed a number of immunoreactive molecular forms of CGRP and only 40-50% of the acid-extracted immunoreactivity was coeluted with the synthetic CGRP(1-37). The i-CGRP level measured in plasma was highly correlated with the thyroidal content of CGRP (p less than 0.001) and also with the age of the rat (p less than 0.001), suggesting an age-related increase of contribution of CGRP from thyroid gland into the circulation.     

Immunohistochemical distribution of calcitonin gene-related peptide in nerve fibres of the anterior buccal gland of the rat

Summary Calcitonin gene-related peptide immunoreactive (CGRP-IR) nerves in rat were studied as to their distribution and origin in anterior buccal glands, which are unique minor mucous salivary glands in the rat. A moderate number of CGRP-IR nerve fibres were located, mostly perivascularly and around intralobular ducts, but they were also found around acini. The latter fibres were mainly of sensory origin, as suggested by their disappearance after denervation of the ophthalmic and maxillary branches of the trigeminal nerve. On the contrary, CGRP-IR nerves around interlobular ducts and some of those in a perivascular location remained both after sensory denervation and after extirpation of the sympathetic superior cervical ganglion. Whether these fibres originate in dorsal root ganglia C₃−C₄ or represent parasympathetic fibres is not known. Based on the present data and the previous findings showing a regulatory role of CGRP both on blood and salivary flow, it is possible that CGRP in sensory, and possibly also in parasympathetic nerves, participates in the regulation of reflex blood flow and salivary secretion in the anterior buccal gland of the rat.     

Light- and electron-microscopical localization of calcitonin, calcitonin gene-related peptide, somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light- and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.     

Genomic and expression analysis of canine calcitonin receptor-stimulating peptides and calcitonin/calcitonin gene-related peptide

Calcitonin receptor-stimulating peptides (CRSPs) are new members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified in pigs, dogs and other domestic animals, and CRSP-1 is an active ligand for the CT receptor (CT-R). We recently sequenced porcine CRSP genes (Crsps) and found similarity with the CT/CGRP gene (Ct/Cgrp) in sequence and genomic organization. In this study, we identified five Crsps, Crsp-1 to Crsp-5, in dogs. Crsp-1 has five exons with an exon-intron organization identical to that of porcine Crsp-1 or Crsp-2, while Crsp-2 and Crsp-3 have additional CT-2- and CT-3-coding exons like Ct/Cgrp. Crsp-2 was renamed as Ct-2/Crsp-2 because both CRSP-2 and CT-2 mRNAs were tissue-specifically expressed. Crsp-4 and Crsp-5 are presumably generated by retrotransposition. We postulate that Crsps were generated from the gene duplication of Ct/Cgrp, and gained their diversity during mammalian evolution. Among the canine CTs and CRSPs, CRSP-1, CT-1 and CT-2 are active ligands for the CT-R, but CRSP-2 and others are inactive. Canine CRSP-1 and CT-2 are expressed in the central and peripheral systems, while CT-1 is localized in the thyroid gland. These findings indicate that dogs can be used for an experimental model as analysing the physiological roles of the CT/CGRP/CRSP family.     

Presence and release of calcitonin gene-related peptide in rat stomach

Immunoreactive (IR)-calcitonin gene-related peptide (CGRP) was identified throughout the entire stomach of rats, being most highly concentrated in the pyloric region, and the concentrations in muscular layers being higher than those in mucosal layers. In addition, IR-CGRP was also present in the venous effluent from isolated perfused rat stomach, and its release was stimulated by dibutyryl cyclic AMP or theophylline but not by glucagon. Gel chromatography as well as HPLC of both tissue extracts and gastric perfusate showed three identical major peaks of IR-CGRP, one of which coeluted with synthetic CGRP. These results suggest that CGRP in the stomach plays a role in the regulation of gastric function.