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.

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.

Characterization and localization of the rabbit ocular calcitonin gene-related peptide (CGRP)-receptor component protein (RCP).

PURPOSE: To determine whether the calcitonin gene-related peptide (CGRP) receptor component protein (RCP), a novel signal transduction molecule, is required for CGRP signaling in the eye and to determine potential ocular sites of CGRP action. METHODS: The cDNA for the rabbit ocular RCP homologue was cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Function of the rabbit ocular RCP was assessed using a sensitive oocyte-based assay, which utilizes the protein kinase A (PKA)-sensitive cystic fibrosis transmembrane conductance regulator (CFTR) as a sensor of cAMP formation. RCP expression in the rabbit eye was localized using immunohistochemistry. RESULTS: A 2063-bp cDNA for the rabbit ocular RCP was cloned and sequenced. Expression of the rabbit RCP cDNA confers CGRP responsiveness in a sensitive oocyte-based assay. Antisense oligonucleotides made to the ocular RCP abolishes CGRP responsiveness of ciliary body and iris mRNA in the oocyte-CFTR assay. Localization of RCP protein in the rabbit eye using immunohistochemistry demonstrated RCP immunoreactivity in the ciliary body and iris blood vessels, as well as in layers of the ciliary epithelium. CONCLUSIONS: The rabbit ocular RCP appears to be required for signal transduction at ocular CGRP receptors and is localized to sites previously reported to bind CGRP, which affect intraocular pressure and neurogenic inflammation.

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.

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.

Neuronal messengers and peptide receptors in the human sphenopalatine and otic ganglia.

A majority of the parasympathetic nerve fibers to cranial structures derive from the sphenopalatine and otic ganglia. In particular, blood vessels are invested with a rich supply of dilator fibers of parasympathetic origin. In the present study, we have examined the occurrence of noncholinergic neuromessengers and neuropeptide receptors in the human sphenopalatine and otic ganglia. Vasoactive intestinal peptide (VIP)-immunoreactive (ir) nerve cell bodies occurred in high numbers in the sphenopalatine and otic ganglia. Likewise, high numbers of NOS- and PACAP-containing nerve cell bodies were seen in both ganglia. Autofluorescent lipofuscin, characteristic of adult human nervous tissue, was present within many nerve cell bodies in both ganglia. Receptor mRNA was studied with reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA from the sphenopalatine and otic ganglia was successfully extracted. By using appropriate sense and antisense primers, oligonucleotides were designed from the human sequences derived from GenBank, corresponding to human NPY Y1, CGRP1 and VIP1 receptors. In the sphenopalatine ganglion, we revealed the presence of mRNA for the human NPY Y1 and VIP1 receptors but not the CGRP1 receptor. The otic ganglion was found to react positively only for primers to mRNA for VIP1 but not for CGRP1 or NPY Y1 receptors.

Adrenomedullin promotes epithelial restitution of rat and human gastric mucosa in vitro.

We have investigated the effect of adrenomedullin (AM) on restitution of mucosal integrity following damage in rat and human gastric mucosa, measuring the potential difference (PD) on a mucosal strip mounted on an Ussing chamber. Mucosal damage was induced by 0.5, 1.0, and 2.0 M NaCl solution, and it caused an immediate and significant decrease in PD. In the rat AM group, PD recovered significantly more than in control group at 120 min after exposure to 0.5 M (p < 0.01) and 1.0 M (p < 0.05) NaCl solution. In the human AM group, PD completely recovered at 120 min after exposure to 0.5 M (p < 0.05) NaCl solution. In rat mucosa damaged by 0.5 M NaCl solution, the effect was inhibited by human (h)-CGRP(8-37) and there was no significant difference between the h-CGRP(8-37) group and control group. On immunohistochemical examination of rat gastric mucosa, AM was detected within the chief cell. AM probably promotes epithelial restitution primarily through the CGRP receptor, but it does not ameliorate more severe damage of gastric mucosa in vitro.

Monoclonal antibodies reveal expression of the CGRP receptor in Purkinje cells, interneurons and astrocytes of rat cerebellar cortex.

The molecular layer of adult rat cerebellum displays high levels of calcitonin gene-related peptide (CGRP) receptors, but the cellular location of the receptor remains unidentified. In an attempt to reveal the expression sites of these receptors, monoclonal antibodies raised against purified CGRP receptors from porcine cerebellar membranes were used in double-immunofluorescence experiments combined with confocal microscopy. PEP-19, a marker that is highly enriched in Purkinje cells (Pc), revealed that CGRP receptors are located in Pc cytoplasm and dendrites, where they label small puncta sometimes arranged in a row along the course of the dendrite itself. CGRP receptors were also located in inhibitory interneurons. Furthermore, as shown by double-labeling experiments with GFAP, CGRP receptor-IR labeled Golgi epithelial cells and their radial fibers (Bergmann fibers), as well as astrocytic processes encircling Pc somata. The simultaneous presence of CGRP receptors in Purkinje cells and in the glial cells that heavily enshroud Purkinje cells allows us to hypothesize that these receptors may be involved in neuron-glia interactions influencing neuronal activity.

Chronotropic, inotropic and lusitropic effects of capsaicin on isolated rat Atria

This work includes results on chronotropic, inotropic and lusitropic changes induced by capsaicin on isolated rat atria. As regards spontaneous frequency, it was stimulated from 10(-9) M up to 7 x 10(-7) M of capsaicin. A simultaneous depression in developed force (F) showed a signigicant correlation with this positive chronotropic effect up to 7 X 10(-8) M of capsaicin, which is the result of the negative staircase phenomenon in the rat heart. The correlation was lost at 2 and 7 x 10(-7) M of capsaicin since in spite of the sustained increase in atrial rate the decrease in F was reversed and then depressed again at 2 and 7x 10(-6) M of capsaicin without changes in frequency. A concentration of capsaicin that overcome the negative staircase phenomenon, 5 x 10(-7) M, was tested as unique dose resulting in stimulation of the chronotropic, inotropic and lusitropic states of the atria. Percentual differences with respect to control values were maximal after 1-3 minutes for frequency (10+3 per cent), F (29+4 per cent), maximal velocity of force development (+F=50+12 per cent) (in all cases +F and -F,bold indicates +F and -F, respectively) and maximal velocity of relaxation (-F=64+13 per cent); a positive lusitropic effect was significant after 8-10 minutes (+F/-F=-17+7 per cent). Capsaicin did not affect the rat atria in the presence of 10(-6) M of ruthenium red, a blocker of capsaicin activation of sensory nerves, indicating that the stimulatory effects were entirely mediated by the release of neurotransmitters and that this concentration of capsaicin was not deleterous "per se". Capsaicin elicited similar inotropic responses in electrically driven isolated atria (+F=41+9 per cent) but the positive lusitropic effect was lost suggesting that capsaicin-induced increases in -F are limited at a frequency higher than the spontaneous frequency (11+6 vs. 32+4 per cent, respectively). 10(-6) M of CGRP8(-37), an antagonist of CGRP1 receptors, suppress the stimulatory effects of capsaicin on atrial contraction. In summary, atrial rate as compared to atrial contraction is more sensitive to the neurotransmitter released by capsaicin, which results in mechanical effects expressing the negative staircase phenomenon in the rat at low concentrations of capsaicin. The positive chronotropic, inotropic and lusitropic responses elicited by capsaicin are mediated by the reelease of neurotransmitters from sensory fibbers and no deletereous effects...

ATP-sensitive potassium channels mediate vasodilatation produced by calcitonin gene-related peptide in human internal mammary but not gastroepiploic arteries.

The purpose of this study was to elucidate the mechanism of action of calcitonin gene-related peptide-induced vasodilatation of human gastroepiploic and internal mammary arteries. Calcitonin gene-related peptide (0.1-100 nmol L-1) elicited relaxations of preconstricted vessels, with a significantly greater effect in the gastroepiploic artery (P < 0.05). This effect was independent of endothelium-derived vasodilating substances. The response of the internal mammary artery but not the gastroepiploic artery to calcitonin gene-related peptide was attenuated by glybenclamide (1.0 mumol L-1) (P < 0.05). In vitro autoradiography indicated that [125I]-calcitonin gene-related peptide bound to the tunica media but not the endothelial cells in both types of artery, with a significantly higher degree of binding in the gastroepiploic artery. It is concluded that calcitonin gene-related peptide acts directly on vascular smooth muscle via specific binding sites to induce vasodilatation. In addition, KATP channels are involved in the action of calcitonin gene-related peptide in the internal mammary artery but not in the gastroepiploic artery.

Differential effects of guanine nucleotides on [125I]-hCGRP(8-37) binding to porcine lung and human neuroblastoma cell membranes.

Calcitonin gene-related peptide (CGRP) mediates its effects by binding to specific receptors which are positively coupled to adenylyl cyclase. CGRP(8-37), a CGRP fragment devoid of the N-terminal region, was shown to be a competitive CGRP receptor antagonist. Only a limited amount of data exists on the usefulness of this ligand in studying CGRP receptors. In the present study, we used [125I]-hCGRP(8-37) to characterize CGRP receptors in porcine lung and human neuroblastoma cell (SK-N-MC) membranes. [125I]-hCGRP(8-37) displayed specific and high affinity binding in both membrane preparations. Displacement studies using [125I]-hCGRP(8-37) and the agonist CGRP revealed the presence of high and low affinity CGRP binding sites in SK-N-MC cell and porcine lung membranes. Addition of guanylimidodiphosphate [Gpp(NH)p] shifted the competition curve to the right and changed the two affinity states of the receptor to a single affinity in SK-N-MC cell membranes. On the other hand, in porcine lung membranes, the whole competition curve was shifted to the right while maintaining the two affinity states. Thus, our data indicate that the new radioligand [125I]-hCGRP(8-37) is a useful tool for characterizing CGRP receptors and their coupling to guanine nucleotide binding proteins.

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions.

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin are structurally related peptides with N-terminal 6-7 amino acid ring structures linked by a disulfide bridge and with amidated C-termini. Among the related bioactive peptides, the structures of the calcitonin receptor and subtypes thereof have been identified so far through molecular cloning. Cross-reaction between receptors of calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin, as well as overlapping biological actions, anticipate that the respective receptors belong to a family of G-protein-coupled receptors that include those of parathyroid hormone, secretin and vasointestinal peptide.

Comparative distribution of receptors for amylin and the related peptides calcitonin gene related peptide and calcitonin in rat and monkey brain.

The distribution of amylin receptors (125I-labelled rat amylin) in brains of rat and monkey were mapped and compared with the distribution of receptors for calcitonin (CT) (125I-labelled salmon CT) and calcitonin gene related peptide (CGRP) (rat, 125I-labelled rat CGRP alpha; monkey, 125I-labelled human CGRP alpha. In rat, amylin receptors were discretely distributed with the highest receptor densities found in mid-caudal accumbens nucleus, parts of the bed nucleus of the stria terminalis, amygdala, and hypothalamus. Moderate to high densities of binding also occurred in the area postrema, subfornical organ, vascular organ of the lamina terminalis, locus ceruleus, dorsal raphe, and caudal solitary tract nucleus. In monkey, the distribution of amylin binding sites was similar, although the highest densities of receptors were in the hypothalamus, with relatively fewer sites present in the accumbens nucleus. In rat, the distribution of amylin receptors formed a subset of the receptor distributions for 125I-labelled salmon CT and 125I-labelled rat CGRP alpha. In contrast, in monkey, although the amylin receptors again formed a subset of the binding sites identified with 125I-labelled salmon CT, there was very little overlap with the pattern of CGRP receptor distribution. This suggests that the specificity profile of amylin receptors in primates differs from that of amylin receptors in the rat, with CGRP alpha having relatively lower affinity for the primate amylin receptors.

Receptors for calcitonin, calcitonin gene related peptide, amylin, and adrenomedullin.

Calcitonin, calcitonin gene related peptide, amylin, and adrenomedullin are structurally related polypeptides characterized by a six or seven amino acid ring structure linked by a disulfide bridge and an amidated C-terminus. They exhibit overlapping biological actions as a result of cross-reactivity between the different receptors. In this article, the respective receptors and G-protein-coupled postreceptor events are reviewed in relation to some of the biological actions of the peptides.

Receptors for calcitonin gene related peptide (CGRP) in gastrointestinal epithelia.

A pharmacological comparison of calcitonin gene related peptide (CGRP) receptors expressed in normal rat colon mucosa and two human adenocarcinoma cell lines has been undertaken. Using voltage-clamp techniques electrogenic ion transport was continuously monitored across either mucosal preparations or confluent epithelial monolayers grown on permeable supports. The data presented at this meeting show that CGRP receptors are preferentially located on the basolateral epithelial surface and that their stimulation by a variety of CGRP analogues results in enhanced CI- secretion mediated via a cyclic AMP dependent mechanism. Responses to rat alpha CGRP in rat descending colon mucosa and in the adenocarcinoma cell line Colony-29 are insensitive to the inhibitory effects of the C-terminal fragment human CGRP(8-37); however, significant inhibition of rat alpha CGRP responses was observed in the parent epithelial cell line HCA-7. This together with the subtle differences seen in agonist orders of potency in the three preparations indicates that different CGRP receptor subtypes exist in the basolateral domains of HCA-7 compared with rat colon and Colony-29 epithelia.

[A study of CGRP receptor and its effect on the growth of human pancreatic carcinoma cells].

In the present studies the calcitonin gene-related peptide (CGRP) receptors were characterized by radioligand analysis and the effect of CGRP and CGRP receptor antagonist on the growth of human pancreatic carcinoma cells (PU-PAN-1) was determined by 3H-TdR incorporation assay. The results indicated that human pancreatic carcinoma cells possess distinct CGRP receptors. CGRP stimulated the growth of PU-PAN-1 tumor cells, and CGRP receptor antagonist CGRP8-37 inhibited these effects. These results suggests that CGRP receptors may play an important role in the growth of human pancreatic carcinoma cells (PU-PAN-1).

Characterization of a calcitonin gene-related peptide (CGRP) receptor on mouse bone marrow cells.

Calcitonin gene-related peptide, (CGRP), a vasoactive neuropeptide, is found throughout the peripheral nervous system, and CGRP receptors are present on mature lymphocytes. The current studies describe a CGRP receptor on isolated mouse bone marrow cells. The affinity, distribution and specificity of CGRP receptors were analyzed using radioligand binding assays. [125I]CGRP binding in mouse bone marrow cells was dependent on cell concentration and was stable from 5 to 60 min at room temperature. The average Kd is 3.29 +/- 1.24 nM and the average receptor density is 2796 +/- 365 sites/cell. Competition binding analysis found rat alpha and beta CGRP to be the most inhibitory, (Ki values 0.899 and 0.711 nM, respectively), followed by human alpha CGRP and the antagonist human CGRP8-37. The neuropeptides human and salmon calcitonin did not inhibit [125I]CGRP binding to bone marrow cells. The presence of CGRP receptors on mouse bone marrow cells provides further evidence for a direct role for CGRP in modulating the function and differentiation of cellular components of the immune and inflammatory systems.