Distribution of CGRP and CGRP receptor components in the rat brain.

BACKGROUND: Calcitonin gene-related peptide and its receptor, consisting of receptor activity-modifying protein 1 and calcitonin receptor-like receptor, are of considerable interest because of the role they play in migraine and recently developed migraine therapies. METHODS: To better understand the function of this neuropeptide, we used immunohistochemistry to determine a detailed distribution of calcitonin gene-related peptide, receptor activity-modifying protein 1 and calcitonin receptor-like receptor in the rat brain in a region of 0.5-1.5 mm lateral to the midline. We found calcitonin gene-related peptide immunoreactivity in most of the neurons of the cerebral cortex, hippocampus, cerebellum, thalamic nuclei, hypothalamic nuclei and brainstem nuclei. In contrast, receptor activity-modifying protein 1 and calcitonin receptor-like receptor immunoreactivity were found almost exclusively in the neuronal processes in the investigated regions. CONCLUSION: Overall, the degree of expression of calcitonin gene-related peptide and calcitonin gene-related peptide receptor components in the central nervous system is astonishingly complex and suggestive of many different brain functions, including a possible role in migraine. However, currently, the presence of calcitonin gene-related peptide and the nature of its receptors throughout the brain is an enigma yet to be solved.

Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.

Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.

Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/ß­catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/ß­catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/ß­catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/ß­catenin pathway, including the mRNA of c­myc, cyclin D1, Lef1, Tcf7 and ß­catenin as well as ß­catenin protein. However, the upregulation of these genes and ß­catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled­related protein, an antagonist of the Wnt/ß­catenin pathway. The results of the present study therefore suggested that the Wnt/ß-catenin signaling pathway may be involved in CGRP­ and LiCl-promoted osteoblastic differentiation of BMSCs.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Algo se mueve en la migraña crónica / Something's moving in chronic migraine

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Inervación perivascular de la arteria mesentérica superior: implicaciones fisiopatológicas / Perivascular innervation of the superior mesenteric artery: pathophysiological implications

Introducción. La arteria mesentérica forma parte de la circulación esplácnica y participa, dado el gran volumen de sangre ue circula por ella, en la regulación de la presión arterial, para lo cual resulta fundamental la regulación del tono vascular. Dicho tono está regulado por diversos factores locales, metabólicos, endocrinos y nerviosos, destacando entre estos últimos la inervación perivascular mesentérica. Desarrollo. Se presentan las características más relevantes de la inervación perivascular mesentérica, centrándose en las inervaciones simpática, nitrérgica y sensitiva, y se analiza la implicación de esta inervación en la fisiopatología del envejecimiento, la diabetes, la hipertensión arterial y la vasodilatación esplácnica secundaria a cirrosis/hipertensión portal. Conclusiones. La inervación perivascular mesentérica desempeña un papel importante en la regulación del flujo sanguíneo y la presión arterial. Alteraciones en esta inervación están implicadas en la génesis y mantenimiento de alteracionesvasculares asociadas a la diabetes, la hipertensión arterial, la vasodilatación esplácnica secundaria a cirrosis/hipertensión portal, así como en el proceso de envejecimiento, mediante la alteración del equilibrio entre agentes vasodilatadores y vasoconstrictores. El conocimiento de estas anomalías puede servir para comprender mejor los mecanismos implicados en estos trastornos y ayudar al diseño de nuevos tratamientos (AU)

Introduction. The mesenteric artery is part of the splanchnic circulation system and, due to the large amount of blood that flows through it, it is involved in the regulation of arterial pressure; to perform this function it is essential to control vascular tone. This tone is regulated by several local, metabolic, endocrine and nervous factors, the most notable of these latter being the mesenteric perivascular innervation.Development. The most significant features of mesenteric perivascular innervation are described, with special attention to the sympathetic, nitrergic and sensory innervations. The article also analyses the involvement of this innervation in the pathophysiology of ageing, diabetes, arterial hypertension and splanchnic vasodilatation secondary to cirrhosis/portal hypertension. Conclusions. Mesenteric perivascular innervation plays an important role in the regulation of blood flow and arterial pressure. Alterations in this innervation are involved in the genesis and continuation of vascular disorders associated with diabetes, splanchnic vasodilatation secondary to cirrhosis/portal hypertension and also in the ageing process, by modifying the balance between vasodilator and vasoconstrictor agents. Greater knowledge of these anomalies can be used to gain a better understanding of the mechanisms involved in these disorders and to help in the design of new forms of treatment (AU)

Emotional stress and orthodontic tooth movement: effects on apical root resorption, tooth movement, and dental tissue expression of interleukin-1 alpha and calcitonin gene-related peptide immunoreactive nerve fibres in rats.

The aim of the study was to investigate the effect of emotional stress on apical root resorption (ARR) and tooth displacement during orthodontic tooth movement in rats. A further area of interest was to evaluate if the expression of interleukin-1 alpha (IL-1alpha) as well as the density and distribution of peptidergic nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) in the periodontal ligament (PDL) are associated with possible stress-induced changes in root resorption and tooth movement. A total of 52 male Wistar rats, aged 6 weeks, were divided in three experimental and one control group (n = 4). Group 1 had orthodontic tooth movement and received foot shocks (OTMS; n = 16), group 2 had orthodontic tooth movement but received no foot shocks (OTMNS; n = 16), and group 3 had no orthodontic tooth movement and received foot shocks (NOTMS; n = 16). Each group was further divided into four subgroups (n = 4), corresponding to the period of the experiment, i.e. 3, 7, 13, and 21 days. At the end of each experimental period, the blood samples were taken, the animals were sacrificed, and the jaws excised, deminerialized, and processed for immunocytochemistry. One-way analysis of variance was used to detect inter-group differences for all investigated variables. CGRP immunopositive nerve fibres were evaluated qualitatively. All the experimental groups demonstrated higher corticosterone levels than the control group, suggesting a stress-induced experience by orthodontic treatment per se. The OTMS group had the least amount of cellular cementum throughout the experimental periods and showed significant reduction in tooth displacement, especially at 3 and 7 days. No obvious changes were observed in the dental tissue expression of IL-1alpha and CGRP immunoreactive nerve fibres between the stressed and non-stressed orthodontically treated groups.

Calcitonin gene-related peptide receptor expression in healthy and inflamed human pulp tissue.

AIM: To use radioreceptor analysis for comparing calcitonin gene-related peptide (CGRP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation. METHODOLOGY: Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another eight pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In four of these premolars, inflammation was induced prior to pulp collection. All the samples were processed and labelled with 125I-CGRP. Binding sites were identified by 125I-CGRP and standard CGRP competition assays. RESULTS: CGRP receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. The Kruskal-Wallis and Mann-Whitney (post-hoc) tests showed statistically significant differences between the groups (P < 0.05). CONCLUSION: CGRP receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.

Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilatory peptide, and its concentration is increased in both maternal and fetal circulation during late pregnancy. The present study was designed to investigate the expression of CGRP receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity modifying protein 1 (RAMP1), and the relaxation response to CGRP in fetoplacental vessels from normotensive pregnant women and women with preeclampsia. Results showed that: 1) mRNA for both CRLR and RAMP1 was expressed in fetoplacental vessels from normal pregnancies; however, these mRNA expressions were substantially reduced in the vessels from preeclamptic women; 2) CRLR and RAMP1 proteins were abundantly expressed in the endothelium and smooth muscle layer of the fetoplacental vessels, as well as the trophoblast cells in normal placentas. In contrast, both vascular tissues and trophoblasts showed decreased expressions for CRLR and RAMP1 proteins and declined CGRP binding sites in preeclamptic placentas; and 3) CGRP produced a dose-dependent relaxation of serotonin-induced contraction of umbilical and chorionic arteries from normal pregnancies, but the response to CGRP was significantly attenuated in the vessels from preeclampsia. We concluded that CGRP may contribute to the low fetoplacental vascular resistance in normal pregnancies; however, CGRP-dependent vascular relaxation appears to be compromised in preeclamptic pregnancies.

Effects of the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS on alpha-CGRP-induced regional haemodynamic changes in anaesthetised rats.

Several studies suggest that a calcitonin gene-related peptide (CGRP) receptor antagonist may have antimigraine properties, most probably via the inhibition of CGRP-induced cranial vasodilatation. We recently showed that the novel selective CGRP receptor antagonist, BIBN4096BS (1-piperidinecarboxamide, -N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [[R-(R,(R*,S*)]), attenuated the CGRP-induced porcine carotid vasodilatation in a model predictive of antimigraine activity. In order to evaluate the potential safety of BIBN4096BS in migraine therapy, this study was designed to investigate the effects of intravenous BIBN4096BS on alpha-CGRP-induced systemic and regional haemodynamic changes in anaesthetised rats, using radioactive microspheres. In vehicle-pretreated animals, consecutive intravenous infusions of alpha-CGRP (0.25, 0.5 and 1 microg kg(-1) min.(-1)) dose-dependently decreased mean arterial blood pressure with an accompanying increase in heart rate and systemic vascular conductance whereas cardiac output remained unchanged. Alpha-CGRP also increased the vascular conductance to the heart, brain, gastrointestinal tract, adrenals, skeletal muscles and skin, whilst that to the kidneys, spleen, mesentery/pancreas and liver remained unaltered. The above systemic and regional haemodynamic responses to alpha-CGRP were clearly attenuated in BIBN4096BS (3 mg kg(-1) intravenously)-pretreated animals. These results indicate that exogenously administered alpha-CGRP dilates regional vascular beds via CGRP receptors on the basis of the antagonism produced by BIBN4096BS. Moreover, the fact that BIBN4096BS did not alter baseline haemodynamics suggests that endogenously produced CGRP does not play an important role in regulating the systemic and regional haemodynamics under resting conditions.

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery.

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.

In-depth characterization of CGRP receptors in human intracranial arteries.

The purpose of the present study was to characterize the effects of human (h) alpha- and beta-calcitonin gene-related peptide (CGRP) on intracranial arteries from man and to investigate the presence of mRNA for the calcitonin receptor like receptor (CRLR) and the receptor activity modifying proteins (RAMPs) 1, 2 and 3, in cerebral and middle meningeal arteries with and without endothelium, in microvessels and in the endothelial cells isolated from the human basilar artery. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of CRLR, RAMP 1, RAMP 2 and RAMP 3 in cerebral and middle meningeal arteries with and without endothelium as well as in microvessels and in the endothelial cells. Human and rat alpha- and beta-CGRP, amylin, adrenomedullin and [acetamidomethyl-Cys(2,7)]human CGRP induced strong concentration-dependent relaxation of human cerebral and middle meningeal arteries. Removal of the endothelium neither changed the maximum relaxant response nor the pIC(50) values for alpha- and beta-CGRP as compared to the responses in arteries with an intact endothelium. Human alpha-CGRP-(8-37) caused a shift of h alpha- and h beta-CGRP-induced relaxations in cerebral and middle meningeal arteries. Calculation of pK(B) values revealed that h alpha-CGRP-(8-37) could not significantly discriminate between relaxations induced by h alpha-CGRP (pK(B) around 6.8) and h beta-CGRP (pK(B) around 5.4). There was no significant difference in pK(B) value of h alpha-CGRP-(8-37) on h beta-CGRP-induced relaxation of human cerebral and middle meningeal arteries with and without endothelium. In conclusion, our molecular and pharmacological data support the existence of a single type of CGRP(1) receptors in the human intracranial circulation.

The somatostatin sst2A receptor in the rat trigeminal ganglion.

Immunohistochemistry for the somatostatin sst2A receptor was performed on the rat trigeminal ganglion to know its function in the trigeminal nervous system. The immunoreactivity was detected in 9.4% of primary sensory neurons in the ganglion. These neurons were small to medium-sized (range=106.5-1123.2 microm(2); mean+/-S.D.=506.3+/-213.2 microm(2)) and predominantly located in the rostromedial part of the ophthalmo-maxillary division. They were also immunoreactive for calcitonin gene-related peptide and the vanilloid receptor subtype 1. In addition, 13.7% of trigeminal neurons which were retrogradely traced with fluorogold from the nasal mucosa exhibited sst2A receptor-immmunoreactivity. Trigeminal neurons which innervated the facial skin and tooth pulp were devoid of the immunoreactivity. In the brainstem trigeminal sensory nuclear complex, both the neuronal cell body and the neuropil exhibited sst2A receptor-immunoreactivity in the superficial medullary dorsal horn.The present study indicates that sst2A receptor-immunoreactive trigeminal nociceptors innervate the nasal mucosa. They may project to the superficial laminae of the medullary dorsal horn.

Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas.

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.

Localization of calcitonin gene-related peptide receptors in rat gastric mucosa.

The location of calcitonin gene-related peptide (CGRP) receptors in the rat stomach has not been elucidated. It was recently reported that the CGRP receptor is formed when a calcitonin-receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1 are co-expressed on the cell membrane. The aim of this study was to determine the location and the role of CGRP receptors in the rat gastric mucosa. Gene expressions of CRLR and RAMP1 were investigated by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization. Immunohistochemical stainings for CGRP, somatostatin, gastrin, and chromogranin A were performed. Gastric endocrine cells were collected by counterflow-elutriation and their responses to CGRP were studied. CRLR and RAMP1 mRNA was expressed mainly in small gastric epithelial cells in the pyloric glands. The mRNA expression had a similar distribution to that of D cells. In cultured gastric endocrine cells, CGRP enhanced somatostatin production, while it inhibited the secretion of histamine and gastrin. Our results suggest that CGRP receptors are expressed in D cells in the rat gastric mucosa and control production and secretion of somatostatin.

Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature.

Calcitonin gene-related peptide and adrenomedullin belong to a structurally related neuropeptide family and are potent vasodilators expressed in the trigeminovascular system. The molecular identity of receptors for these proteins has only recently been elucidated. Central to functional binding of these neuropeptides is the G-protein-coupled receptor, the calcitonin receptor-like receptor (CRLR), whose cell surface expression and pharmacology is determined by coexpression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP binds calcitonin gene-related peptide with high affinity, whereas CRLR coexpression with RAMP2 or -3 confers high-affinity binding of adrenomedullin. The authors investigated the expression of these receptor components in human cerebral vasculature to further characterize neuropeptide receptor content and the potential functions of these receptors. Localization has been carried out using specific antisera raised against immunogenic peptide sequences that were subsequently applied using modern immunohistochemical techniques and confocal microscopy. The results are the first to show the presence of these receptor component proteins in human middle meningeal, middle cerebral, pial, and superficial temporal vessels, and confirm that both calcitonin gene-related peptide and adrenomedullin receptors may arise from the coassembly of RAMPs with CRLR in these vessel types. These novel data advance the understanding of the molecular function of the trigeminovascular system, its potential role in vascular headache disorders such as migraine, and may lead to possible ways in which future synthetic ligands may be applied to manage these disorders.

Immunohistochemical detection of calcitonin gene-related peptide receptor (CGRPR)-1 in the endothelium of human coronary artery and bronchial blood vessels.

The potent vasodilatory peptide, calcitonin gene-related peptide (CGRP) is present in the innervation of vascular tissue. The actions of CGRP occur via a receptor, CGRP receptor(R)-1, which is also a target for the cardioprotective peptide adrenomedullin. The human version of the pharmacologically-defined CGRPR-1 has been cloned but its distribution and cellular location is unknown. A rabbit antibody was generated to a synthetic peptide that corresponds to the C-terminus of human CGRPR-1 Immunochemical analysis of the human cell-line, SK-N-MC, which exhibits functional expression of the CGRPR-1 confirmed the antibody's specificity. The antiserum revealed specific staining in the endothelium of human coronary arteries. The vascular smooth muscle and ventricular myocardium were not immunoreactive. In bronchial blood vessels CGRPR-1-immunoreactivity was detected in the endothelium of the venules and not in the arterioles, which is particularly relevant for elucidating the putative role of CGRP in inflammation in this tissue.

Regional localization and abundance of calcitonin gene-related peptide receptors in guinea pig heart.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released within the heart during myocardial ischemia. The present study was done to determine the regional localization and abundance of CGRP receptors in the guinea pig heart. CGRP binding sites in 20 microm frozen sections of heart were labeled using [125I]CGRP. Non-specific binding was determined in the presence of 1 microm unlabeled CGRP or CGRP(8-37). Significant amounts of specific CGRP binding were identified in atrial and ventricular myocardium, all portions of the conducting system, coronary arteries, the aorta and pulmonary trunk and intracardiac ganglia. Specific binding of CGRP to the left atrium was two-fold higher than binding to the right atrium (0.667+/-0.052 v 0.340+/-0.029 fmol/mg tissue, n=5, CGRP(8-37)group). In contrast to the atria, a lower and uniform density of CGRP receptors occurred in contractile tissue of the ventricular myocardium (e.g. 0.239+/-0.013 fmol/mg left ventricle, n=5). The highest concentration of CGRP receptors in guinea pig cardiac tissue occurred at the bundle of His and the bundle branches (0.752+/-0.087 and 0.713+/-0.138 fmol/mg tissue, respectively, n=5). CGRP receptors were localized to coronary vessels throughout the heart and to the ascending aorta and pulmonary trunk. Lastly, intracardiac ganglia exhibited moderate levels of specific [125I]CGRP binding (0.475+/-0.043 fmol/mg, n=5). These findings support the concept that CGRP can have direct effects on atrial and ventricular function as well as coronary flow. The high density of CGRP receptors in the distal conducting system and the presence of CGRP receptors in intracardiac ganglia further suggest that CGRP could have important effects on cardiac conduction velocity and parasympathetic regulation of the heart.

Autoradiographic localization of tachykinin and calcitonin gene-related peptide receptors in adult urinary bladder.

PURPOSE: In bladder, sensory afferent nerve fibers contain the "sensory neuropeptides" substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), which interact with tachykinin NK-1 and NK-2 receptors and CGRP receptors, respectively. The purpose of this study was to examine the autoradiographic distribution of these three receptor types in the human bladder, to determine whether the anatomic location of the receptors was consistent with their known functional roles. MATERIALS AND METHODS: Specimens of urinary bladder from 9 patients (58-74 years) were obtained at cystectomy. Frozen sections of dome were labeled with [125I]-Bolton-Hunter [Sar9,Met(O2)11]-SP (NK-1 receptors), [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) (NK-2 receptors) and [125I]-rat CGRP-I. Binding sites were visualized using emulsion autoradiography. RESULTS: NK-1 receptors were found over the endothelium of arterial blood vessels within the detrusor muscle and lamina propria, and over small vessels in the subepithelium. NK-2 receptors were seen over the detrusor muscle and very sparsely over blood vessels, whereas CGRP receptors were expressed densely over the smooth muscle layer of arteries and arterioles, and weakly over collecting venules. NK-1 and CGRP receptors were not observed over the detrusor muscle. CONCLUSIONS: Although the afferent nerves contain all three peptides, not all cell types express receptors for each peptide. The general distribution of receptors is in good agreement with the location of nerves, and with the known actions of SP and CGRP as vasodilator agents, and of NKA (but not SP or CGRP) in contracting the detrusor muscle.