Calcitonin Gene-related Peptide Receptor Expression Research Articles

Alleviation of migraine related pain and anxiety by inhibiting calcium-stimulating AC1-dependent CGRP in the insula of adult rats.

Recent animal and clinical findings consistently highlight the critical role of calcitonin gene-related peptide (CGRP) in chronic migraine (CM) and related emotional responses. CGRP antibodies and receptor antagonists have been approved for CM treatment. However, the underlying CGRP-related signaling pathways in the pain-related cortex remain poorly understood. The SD rats were used to establish the CM model by dural infusions of inflammatory soup. Periorbital mechanical thresholds were assessed using von-Frey filaments, and anxiety-like behaviors were observed via open field and elevated plus maze tests. Expression of c-Fos, CGRP and NMDA GluN2B receptors was detected using immunofluorescence and western blotting analyses. The excitatory synaptic transmission was detected by whole-cell patch-clamp recording. A human-used adenylate cyclase 1 (AC1) inhibitor, hNB001, was applied via insula stereotaxic and intraperitoneal injections in CM rats. The insular cortex (IC) was activated in the migraine model rats. Glutamate-mediated excitatory transmission and NMDA GluN2B receptors in the IC were potentiated. CGRP levels in the IC significantly increased during nociceptive and anxiety-like activities. Locally applied hNB001 in the IC or intraperitoneally alleviated periorbital mechanical thresholds and anxiety behaviors in migraine rats. Furthermore, CGRP expression in the IC decreased after the hNB001 application. Our study indicated that AC1-dependent IC plasticity contributes to migraine and AC1 may be a promising target for treating migraine in the future.

Calcitonin gene‑related peptide alleviates hyperoxia‑induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene‑related peptide (CGRP) protects against hyperoxia‑induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia‑induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit‑8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl‑2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non‑selective currents through TRPV1 channels. The CGRP‑induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia‑induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

The Role of Meningeal Immune Cells in the Efficacy of CGRP-based Migraine Therapies

Migraine is a painful, chronic neurological disorder that represents the second most disabling illness worldwide. There are two prominent mechanisms thought to contribute to migraine pain: immune cell activation and calcitonin gene-related peptide (CGRP) signaling. Cortical spreading depression (CSD), thought to underlie the aura that precedes migraine attack in a subpopulation of migraineurs, is associated with increased meningeal macrophage activation. Macrophages can activate meningeal primary afferent neurons and contribute to migraine pain through release of pro-inflammatory cytokines. CSD also increases CGRP synthesis and release, and single-cell RNA sequencing data support the expression of CGRP receptor subunit mRNA in mouse meningeal immune cells. The efficacy of CGRP receptor antagonists as migraine therapies support the critical role of CGRP in the development of migraine pain. However, the mechanism of action of these drugs is incompletely understood. Given the effect of CSD on immune cell activation and CGRP release, the presence of CGRP receptor on immune cells, and the use of CGRP receptor antagonists to treat migraine, the following study uses anatomical, functional, and behavioral approaches to assess the role of meningeal immune cells in the efficacy of CGRP-based migraine therapies. This is done in the context of a minimally invasive model of migraine with aura (optogenetic spreading depression, OSD). I plan to characterize OSD-dependent changes in CGRP receptor expression in meningeal immune cells, assess the ability of CGRP receptor antagonists to prevent OSD-induced changes in macrophage activation, and determine the contribution of macrophage CGRP receptors to OSD-induced pain. The outcomes of these experiments may not only reveal a mechanism underlying the therapeutic efficacy of CGRP antagonists but potentially novel targets and mechanisms to improve existing migraine therapies. Grant support from R01 NS122784.

Sensory Neurotransmitter CGRP Modulates Oral Cancer Growth and Cancer-associated Immune Response

Head and neck squamous cell carcinoma (HNSCC) induces severe pain due in part to activation of primary afferent neurons by cancer-secreted mediators. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from trigeminal neurons innervating the cancer has been linked to tumorigenesis. We hypothesize that CGRP exerts a dual effect on both cancer-associated pain and tumor progression, suggesting that CGRP may be a promising therapeutic target in HNSCC treatment. We used human tumor tissue and patient-reported outcomes to explore the relationship between CGRP+ sensory nerve innervation and cancer pain in patients. To determine CGRP receptor expression on tumor cells, immunohistochemistry and PCR were performed on human and mouse oral cancer cell lines. We used a syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (i.e. CGRP-KO) to investigate the impact of CGRP signaling on tumor growth and the associate immune response in vivo. We found prominent CGRP-immunoreactive sensory nerve presence innervating human HNSCC tumor tissue, which positively correlated to patient-reported pain (r2=0.357). Furthermore, human HNSCC cell lines expressed 3-fold more CGRP receptor, RAMP1, compared to a non-tumorigenic keratinocyte cell line. In tumor-bearing CGRP-KO mice, we found a significant reduction in tumor size at post-inoculation days 7 and 14 compared to wildtype. We also found a 4-fold increase in tumor infiltrating RAMP1-expressing CD4+ T cells, as well as a 5-fold increase cytotoxic CD8+ T cells and NK1.1+ NK cells in tumor tissue CGRP-KO mice compared to wildtype. This preliminary data suggests that CGRP signaling from sensory neurons may increase cancer associated pain and tumor progression. Further knowledge regarding the relationship between sensory neurons and cancer could allow for the repurposing clinically available nervous system drugs (e.g., anti-CGRP antibodies) for the treatment of cancer and cancer pain. Grant support from the Rita Allen Foundation.

233 Langerhans cells as targets of neuroimmune Communication in the Epidermal Compartment of Human Skin

The immune system is strongly linked to the nervous system, to the point that in many diseases they cannot be discussed as separate entities. Specifically, inflammatory skin diseases such as atopic dermatitis and psoriasis have been reported to be intensified by stress, and, interestingly the skin lesions in these diseases sometimes resolve after nerve injury. The putative cause underlying this empirical observation is that sensory nerves secrete neuropeptides (NPs) and other mediators that are important members of neuro-immune communication. Inside the epidermis, the only dedicated resident immune cells under steady-state are the antigen-presenting cells called Langerhans cells (LCs). LCs have been shown to be anatomically associated with neurons that produce Calcitonin Gene-Related Peptide (CGRP), supporting the hypothesis that NPs possibly play a role in cutaneous inflammation. Previous reports have shown that CGRP reduces LC antigen-presentation to a Th1 clone while simultaneously, enhancing antigen-presentation for Th2 clones in mice, but little is known in humans. As a first, exploratory step we utilized RNA sequencing from monocyte-derived LCs samples from five donors and we detected the expression of neuropeptide receptor genes. We found high expression of CGRP receptor and its coreceptor RAMP1 and also one of the neurotensin receptors (SORT1, also known as NTR3) and the Brain Natriuretic peptide receptor NPR1, and validated these with RT-qPCR. We also determined the effect of all three neuropeptides on the differentiation of monocyte-derived LCs with flow cytometry and found that NPR1 activation significantly increased the differentiation of LCs, while the others had no effect. Interestingly, BNP had only minimal effect on the T cell stimulatory capability of LCs, or on their cytokine (IL-6, IL-8, IL-10 and IL-12) production. These data suggest that neuroimmune communication might be an important prerequisite of LC differentiation.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8–37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8–37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Role of Calcitonin Gene-Related Peptide in Nociceptive Modulationin Anterior Cingulate Cortex of Naïve Rats and Rats With Inflammatory Pain

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC.

Novel calcitonin gene-related peptide/chitosan-strontium-calcium phosphate cement: Enhanced proliferation of human umbilical vein endothelial cells in vitro.

Bone cement materials have some disadvantages, including slow degradation and no biological activity, which greatly weakens their clinical application. Therefore, the search for a multifunctional bioactive bone cement has become urgent. In this study, a novel bone cement sample of calcitonin gene-related peptide (CGRP)/chitosan-strontium (Sr)-calcium phosphate cement (CPC) was developed. The structure and morphology were observed by scanning electron microscope (SEM). The cytotoxicity and proliferation of CGRP/chitosan-Sr-CPC were also measured. The expression of CGRP receptor 1 was measured using an immunofluorescence assay. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were employed to quantify the VEGF mRNA and protein levels, respectively. Finally, the ability of the material to improve angiogenesis was assessed by using human umbilical vein endothelial cells (HUVECs) tube formation assay. The results showed that CGRP/Chitosan-Sr-CPC had the characteristics of a good orthopedic material without showing cell cytotoxicity to HUVECs. Meanwhile, CGRP/chitosan-Sr-CPC could release CGRP and enhance the proliferation of HUVECs via CGRP receptors. Moreover, CGRP/chitosan-Sr-CPC significantly upregulated the expression of the VEGF gene and protein in HUVECs, which might help improve the angiogenesis microenvironment. Besides, CGRP/chitosan-Sr-CPC could significantly improve angiogenesis of HUVECs. These findings provide new therapeutic material for the treatment of osteoporotic bone injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 19-28, 2019.

Increased receptor activity-modifying protein 1 in the nervous system is sufficient to protect against autonomic dysregulation and hypertension

Calcitonin gene-related peptide (CGRP) can cause migraines, yet it is also a potent vasodilator that protects against hypertension. Given the emerging role of CGRP-targeted antibodies for migraine prevention, an important question is whether the protective actions of CGRP are mediated by vascular or neural CGRP receptors. To address this, we have characterized the cardiovascular phenotype of transgenic nestin/hRAMP1 mice that have selective elevation of a CGRP receptor subunit in the nervous system, human receptor activity-modifying protein 1 (hRAMP1). Nestin/hRAMP1 mice had relatively little hRAMP1 RNA in blood vessels and intravenous injection of CGRP caused a similar blood pressure decrease in transgenic and control mice. At baseline, nestin/hRAMP1 mice exhibited similar mean arterial pressure, heart rate, baroreflex sensitivity, and sympathetic vasomotor tone as control mice. We previously reported that expression of hRAMP1 in all tissues favorably improved autonomic regulation and attenuated hypertension induced by angiotensin II (Ang II). Similarly, in nestin/hRAMP1 mice, hypertension caused by Ang II or phenylephrine was greatly attenuated, and associated autonomic dysregulation and increased sympathetic vasomotor tone were diminished or abolished. We conclude that increased expression of neuronal CGRP receptors is sufficient to induce a protective change in cardiovascular autonomic regulation with implications for migraine therapy.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients.

BackgroundCalcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood.MethodsSynovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3–4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist.ResultsCGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists.ConclusionsPGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

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/β-catenin 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/β-catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β-catenin 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/β-catenin pathway, including the mRNA of c-myc, cyclin D1, Lef1, Tcf7 and β-catenin as well as β-catenin protein. However, the upregulation of these genes and β-catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled-related protein, an antagonist of the Wnt/β-catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP- and LiCl-promoted osteoblastic differentiation of BMSCs.

Expression and function of calcitonin gene-related peptide (CGRP) receptors in trigeminal ganglia of R192Q Cacna1a knock-in mice

Migraine is a neurovascular brain disorder suggested to be due to dysfunction of the trigeminovascular system with sensitization of trigeminal ganglion (TG) nociceptors. Since the neuropeptide calcitonin gene-related peptide (CGRP) has been established as a key player in the pathogenesis of migraine, CGRP receptor antagonists have been considered useful compounds to block headache originating from hyperactivation of such TG neurons. Whereas there is some information on the expression of CGRP receptors in postmortem human tissue, data are lacking for migraineurs suffering from common or genetic migraine. To help to clarify these issues it is very useful to study a transgenic knock-in (KI) mouse model of hemiplegic migraine expressing a R192Q missense mutation in the α1 subunit of CaV2.1 calcium channels previously found in patients with familial hemiplegic migraine type-1 (FHM-1). The aim of the present study, therefore, was to compare CGRP receptor expression and function in wildtype (WT) versus KI mouse TG. The principal components of the CGRP receptor, namely the CLR and RAMP-1 proteins, were similarly expressed in WT and KI TG neurons (in situ or in culture) and responded to exogenous CGRP with a strong rise in cAMP concentration. Hence, the previously reported phenotype of sensitization of KI TG neurons is not due to up-regulation of CGRP receptors but is likely caused by a constitutively larger release of CGRP. This observation implies that, in FHM-1 TG, normal TG sensory neuron signaling can be restored once the extracellular concentration of CGRP returns to control level with targeted treatment.

Synovial macrophage-derived IL-1β regulates the calcitonin receptor in osteoarthritic mice.

Recent studies have reported that calcitonin gene-related peptide (CGRP) contributes to joint pain. However, regulation of the CGRP/CGRP receptor signalling in osteoarthritis (OA) is not fully understood. To investigate the regulation of CGRP/CGRP receptor signalling by macrophages in the synovial tissue (ST) of OA joints, we characterized the gene expression profiles of CGRP and CGRP receptors in the ST of OA mice (STR/Ort). In addition, we examined whether macrophage depletion by the systemic injection of clodronate-laden liposomes affected the expression of CGRP and CGRP receptors in ST. CD11c(+) macrophages in the ST of STR/Ort and C57BL/6J mice were analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to evaluate the expression of interleukin (IL)-1β, CGRP, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in F4/80(+) and F4/80(-) cells. The effects of IL-1β on the expression of CGRP and CLR by cultured synovial cells were also examined. The percentage of CD11c(+) macrophages in the ST of STR/Ort was higher than that in C57/BL6J mice. Notably, the F4/80(+) cell fraction expressed IL-1β highly, whereas the F4/80(-) cell fraction expressed CGRP, CLR, and RAMP1 highly. In addition, expression of the IL-1β and CLR genes was increased in ST, but was decreased upon macrophage depletion, and the IL-1β treatment of cultured synovial cells up-regulated CLR. Taken together, the present findings suggest that synovial macrophages are the major producers of IL-1β and regulators of CLR in OA mice. Therefore, macrophages and IL-1β may be suitable therapeutic targets for treating OA pain.

The novel CGRP receptor antagonist BIBN4096BS alleviates a postoperative intestinal inflammation and prevents postoperative ileus.

Abdominal surgery results in neuronal mediator release and subsequent acute intestinal hypomotility. This phase is followed by a longer lasting inflammatory phase resulting in postoperative ileus (POI). Calcitonin gene-related peptide (CGRP) has been shown to induce motility disturbances and in addition may be a candidate mediator to elicit neurogenic inflammation. We hypothesized that CGRP contributes to intestinal inflammation and POI. The effect of CGRP in POI was tested in mice treated with the highly specific CGRP receptor antagonist BIBN4096BS and in CGRP receptor-deficient (RAMP-1(-/-) ) mice. POI severity was analyzed by cytokine expression, muscular inflammation and gastrointestinal (GI) transit. Peritoneal and muscularis macrophages and mast cells were analyzed for CGRP receptor expression and functional response to CGRP stimulation. Intestinal manipulation (IM) resulted in CGRP release from myenteric nerves, and a concurrent increased interleukin (IL)-6 and IL-1β transcription and leukocyte infiltration in the muscularis externa and increased GI transit time. CGRP potentiates IM-induced cytokine transcription within the muscularis externa and peritoneal macrophages. BIBN4096BS reduced cytokine levels and leukocyte infiltration and normalized GI transit. RAMP1(-/-) mice showed a significantly reduced leukocyte influx. CGRP receptor was expressed in muscularis and peritoneal macrophages but not mast cells. CGRP mediated macrophage activation but failed to induce mast cell degranulation and cytokine expression. CGRP is immediately released during abdominal surgery and induces a neurogenic inflammation via activation of abdominal macrophages. BIBN4096BS prevented IM-induced inflammation and restored GI motility. These findings suggest that CGRP receptor antagonism could be instrumental in the prevention of POI.

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 [3H]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.

CGRP in the trigeminovascular system: a role for CGRP, adrenomedullin and amylin receptors?

The neuropeptide calcitonin gene-related peptide (CGRP) is reported to play an important role in migraine. It is expressed throughout the trigeminovascular system. Antagonists targeting the CGRP receptor have been developed and have shown efficacy in clinical trials for migraine. However, no CGRP antagonist is yet approved for treating this condition. The molecular composition of the CGRP receptor is unusual because it comprises two subunits; one is a GPCR, the calcitonin receptor-like receptor (CLR). This associates with receptor activity-modifying protein (RAMP) 1 to yield a functional receptor for CGRP. However, RAMP1 also associates with the calcitonin receptor, creating a receptor for the related peptide amylin but this also has high affinity for CGRP. Other combinations of CLR or the calcitonin receptor with RAMPs can also generate receptors that are responsive to CGRP. CGRP potentially modulates an array of signal transduction pathways downstream of activation of these receptors, in a cell type-dependent manner. The physiological significance of these signalling processes remains unclear but may be a potential avenue for refining drug design. This complexity has prompted us to review the signalling and expression of CGRP and related receptors in the trigeminovascular system. This reveals that more than one CGRP responsive receptor may be expressed in key parts of this system and that further work is required to determine their contribution to CGRP physiology and pathophysiology. This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7.

Localization of CGRP Receptor Components, CGRP, and Receptor Binding Sites in Human and Rhesus Cerebellar Cortex

The cerebellum is classically considered to be mainly involved in motor processing, but studies have suggested several other functions, including pain processing. Calcitonin-gene-related peptide (CGRP) is a neuropeptide involved in migraine pathology, where there is elevated release of CGRP during migraine attacks and CGRP receptor antagonists have antimigraine efficacy. In the present study, we examined CGRP and CGRP receptor binding sites and protein expression in primate cerebellar cortex. Additionally, mRNA expression of the CGRP receptor components, calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), was examined. In addition, expression of procalcitonin was studied. We observed high [(3)H]MK-3207 (CGRP receptor antagonist) binding densities in the molecular layer of rhesus cerebellar cortex; however, due to the limit of resolution of the autoradiographic image the exact cellular localization could not be determined. Similarly, [(125)I]CGRP binding was observed in the molecular layer and Purkinje cell layer of human cerebellum. CLR and RAMP1 mRNA was expressed within the Purkinje cell layer and some expression was found in the molecular layer. Immunofluorescence revealed expression of CGRP, CLR, and RAMP1 in the Purkinje cells and in cells in the molecular layer. Procalcitonin was found in the same localization. Recent research in the biology of cerebellum indicates that it may have a role in nociception. For the first time we have identified CGRP and CGRP receptor binding sites together with CGRP receptor expression through protein and mRNA localization in primate cerebellar cortex. These results point toward a functional role of CGRP in cerebellum. Further efforts are needed to evaluate this.

The neuropeptide calcitonin gene‐related peptide affects allergic airway inflammation by modulating dendritic cell function

The neuropeptide calcitonin gene-related peptide (CGRP) is released in the lung by sensory nerves during allergic airway responses. Pulmonary dendritic cells (DC) orchestrating the allergic inflammation could be affected by CGRP. To determine the immunomodulatory effects of CGRP on DC function and its impact on the induction of allergic airway inflammation. CGRP receptor expression on lung DC was determined by RT-PCR and immunofluorescence staining. The functional consequences of CGRP receptor triggering were evaluated in vitro using bone marrow-derived DC. DC maturation and the induction of ovalbumin (OVA)-specific T cell responses were analysed by flow cytometry. The in vivo relevance of the observed DC modulation was assessed in a DC-transfer model of experimental asthma. Mice were sensitized by an intrapharyngeal transfer of OVA-pulsed DC and challenged with OVA aerosol. The impact of CGRP pretreatment of DC on airway inflammation was characterized by analysing differential cell counts and cytokines in bronchoalveolar lavage fluid (BALF), lung histology and cytokine responses in mediastinal lymph nodes. RT-PCR, immunofluorescence and cAMP assay demonstrated the expression of functionally active CGRP receptors in lung DC. RT-PCR revealed a transcriptional CGRP receptor down-regulation during airway inflammation. CGRP specifically inhibited the maturation of in vitro generated DC. Maturation was restored by blocking with the specific antagonist CGRP(8-37) . Consequently, CGRP-pretreated DC reduced the activation and proliferation of antigen-specific T cells and induced increased the numbers of T regulatory cells. The transfer of CGRP-pretreated DC diminished allergic airway inflammation in vivo, shown by reduced eosinophil numbers and increased levels of IL-10 in BALF. CGRP inhibits DC maturation and allergen-specific T cell responses, which affects the outcome of the allergic airway inflammation in vivo. This suggests an additional mechanism by which nerve-derived mediators interfere with local immune responses. Thus, CGRP as an anti-inflammatory mediator could represent a new therapeutic tool in asthma therapy.

The effect of neuropeptides on proliferation of rat bone marrow mesenchymal stem cells

Objective To investigate the effects and mechanism of calcitonin gene-related peptide (CGRP) and substance P (SP) on proliferation of rat bone marrow mesenchymal stem cells. Methods The rBMSCs were isolated using whole bone marrow adherence method. In the different periods of culturing (1, 2,and 3 weeks), expressions of the neuropeptide receptors were detected by Western Blot and reserve transcriptase-polymerase chain reaction (RT-PCR). The BMSCs were treated with CGRP and SP at concentration 10-8 mol/L at different time (1,3,5,7,9 days), cell proliferation was detected with MTT assay, the protein expressions of cyclin D1 ,cyclin E and p53 were examined using Western Blot. Results The CGRP receptor and SP receptor were expressed in BMSCs. The expression of CGRP receptor was statistically higher than that of SP receptorat the same time point. The growth curves of BMSCs cultured by both neuropeptides had similar appearance. CGRP and SP stimulated the proliferation of BMSCs significantly at 9 days and 7 and 9 days. In this process, the expressions of cyclinDl and cyclinE were up-regulated by CGRP, SP only enhanced the expression of cyclinE; these effects all reached a peak at 5 days. The expression of p53 was down-regulated by both neuropeptides. Conclusion CGRP and SP had direct effects on the proliferation of BMSCs, the regulation of cell cycle proteins is one of the mechanisms. Key words: Calcitonin gene-related peptide; Substance P; Myeloid progenitor cells; Cell cycle

Calcitonin gene-related peptide modulates interleukin-13 in circulating cutaneous lymphocyte-associated antigen-positive T cells in patients with atopic dermatitis

Neuropeptides (NPs) may play an important role in the pathogenesis of atopic dermatitis (AD) by regulating immune responses and contributing to the cross-talk between the immune and nervous systems. To assess the ability of NPs to influence interleukin (IL)-13 and interferon (IFN)-gamma production and the expression of the activation marker HLA-DR in skin memory T cells [cutaneous lymphocyte-associated antigen (CLA)+ T cells] from patients with AD with severe, chronic lesions and intense pruritus, and from nonatopic controls. Cells were cultured in the presence and absence of different NPs, calcitonin gene-related peptide (CGRP), somatostatin (SOM) and substance P (SP). IL-13 and IFN-gamma production and HLA-DR expression were measured in both CLA+ and CLA- T-cell subsets by flow cytometry. CGRP increased IL-13 production in peripheral blood mononuclear cells from patients with AD (P < 0.05), with no changes detected in the presence of SOM or SP. These patients with AD had a lower expression of CGRP receptor compared with controls (P < 0.05). Memory T cells incubated with CGRP also showed an increase in IL-13 (P < 0.05) and HLA-DR (P < 0.05) in CLA+ T cells from patients with AD compared with controls, but not in CLA- T cells. Patients with a higher production of IL-13 were those with higher total IgE and percentage of skin area involved. Furthermore, the IL-13/IFN-gamma ratio was increased in patients with AD after cells were cultured with CGRP (P < 0.05). Our results suggest an immunomodulatory role of CGRP towards a Th2 pattern in CLA+ T cells, which may contribute to exacerbating clinical symptoms in patients with AD.