A second trigeminal CGRP receptor: function and expression of the AMY1 receptor.
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ABSTRACT: Objective
The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system.Methods
Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons.Results
mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons.Interpretation
The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine.
SUBMITTER: Walker CS
PROVIDER: S-EPMC4479521 | biostudies-literature | 2015 Jun
REPOSITORIES: biostudies-literature
Publications
A second trigeminal CGRP receptor: function and expression of the AMY1 receptor.
Walker Christopher S CS Eftekhari Sajedeh S Bower Rebekah L RL Wilderman Andrea A Insel Paul A PA Edvinsson Lars L Waldvogel Henry J HJ Jamaluddin Muhammad A MA Russo Andrew F AF Hay Debbie L DL
Annals of clinical and translational neurology 20150401 6
<h4>Objective</h4>The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system.<h4>Methods</h4>Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohis ...[more]
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Project description:BackgroundStimulation of trigeminovascular pathway is widely used to establish the headache animal model. Headache is a common neurological disorder, in which symptomatic attacks are mediated by calcitonin-gene-related peptide (CGRP). CGRP is synthesized and released from the trigeminal ganglion to transmit pain signals under stimulation. On the other hand, Neuropeptide FF (NPFF) is a candidate transmitter/modulator for migraine, and stimulation of its receptor, NPFFR2, increases the expression and release of CGRP in mice sensory neurons. Here, we investigate the impact of NPFFR2 on trigeminal CGRP level in a capsaicin-induced headache mouse model.MethodsMice were intracisternally injected with capsaicin into the cisterna magna to activate the trigeminovascular pathway and induce headache symptoms. Mice pretreated with Npffr2-shRNA or NPFFR2 knockouts were adopted to test the impact of NPFFR2 on capsaicin-induced CGRP upregulation in trigeminal ganglion. The gene silencing effect of Npffr2-shRNA in trigeminal ganglion was confirmed by real-time PCR. Trigeminal CGRP level was determined by immunofluorescence staining, and the percentage of CGRP-positive cell was calculated after setting the signal intensity threshold by Image J software. Amount of trigeminal CGRP in NPFFR2 overexpressed mice was also measured by CGRP ELISA.FindingsInfusion of capsaicin into the cisterna magna upregulated the CGRP in trigeminal ganglion and induced spontaneous pain behaviors including the reduction of locomotor activity and the increase of freezing behavior. Intracisternal injection of Npffr2-shRNA reduced the mRNA of Npffr2 in trigeminal ganglion. Mice pretreatment with Npffr2-shRNA prevented capsaicin-induced CGRP upregulation in trigeminal ganglion. Similarly, CGRP upregulation was also reduced in NPFFR2 knockout mice. On the contrary, trigeminal CGRP was increased in NPFFR2 overexpressed mice.ConclusionsReducing the level of NPFFR2 leads to the downregulation of capsaicin-induced CGRP in trigeminal ganglion, which would consequently attenuate the activation of trigeminovascular pathway. Thus, NPFFR2 could serve as a potential target for neuromodulation of cephalic pain.
| S-EPMC7346335 | biostudies-literature
Project description:ObjectiveThe goal of this narrative review is to provide an overview of migraine pathophysiology, with an emphasis on the role of calcitonin gene-related peptide (CGRP) within the context of the trigeminovascular system.BackgroundMigraine is a prevalent and disabling neurological disease that is characterized in part by intense, throbbing, and unilateral headaches. Despite recent advances in understanding its pathophysiology, migraine still represents an unmet medical need, as it is often underrecognized and undertreated. Although CGRP has been known to play a pivotal role in migraine for the last 2 decades, this has now received more interest spurred by the early clinical successes of drugs that block CGRP signaling in the trigeminovascular system.DesignThis narrative review presents an update on the role of CGRP within the trigeminovascular system. PubMed searches were used to find recent (ie, 2016 to November 2018) published articles presenting new study results. Review articles are also included not as primary references but to bring these to the attention of the reader. Original research is referenced in describing the core of the narrative, and review articles are used to support ancillary points.ResultsThe trigeminal ganglion neurons provide the connection between the periphery, stemming from the interface between the primary afferent fibers of the trigeminal ganglion and the meningeal vasculature and the central terminals in the trigeminal nucleus caudalis. The neuropeptide CGRP is abundant in trigeminal ganglion neurons, and is released from the peripheral nerve and central nerve terminals as well as being secreted within the trigeminal ganglion. Release of CGRP from the peripheral terminals initiates a cascade of events that include increased synthesis of nitric oxide and sensitization of the trigeminal nerves. Secreted CGRP in the trigeminal ganglion interacts with adjacent neurons and satellite glial cells to perpetuate peripheral sensitization, and can drive central sensitization of the second-order neurons. A shift in central sensitization from activity-dependent to activity-independent central sensitization may indicate a mechanism driving the progression of episodic migraine to chronic migraine. The pathophysiology of cluster headache is much more obscure than that of migraine, but emerging evidence suggests that it may also involve hypersensitivity of the trigeminovascular system. Ongoing clinical studies with therapies targeted at CGRP will provide additional, valuable insights into the pathophysiology of this disorder.ConclusionsCGRP plays an essential role in the pathophysiology of migraine. Treatments that interfere with the functioning of CGRP in the peripheral trigeminal system are effective against migraine. Blocking sensitization of the trigeminal nerve by attenuating CGRP activity in the periphery may be sufficient to block a migraine attack. Additionally, the potential exists that this therapeutic strategy may also alleviate cluster headache as well.
| S-EPMC6593989 | biostudies-literature
Project description:The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization.
| S-EPMC9655983 | biostudies-literature
Project description:The neuropeptide calcitonin gene-related peptide (CGRP) is expressed in the trigeminal ganglia, a key site in craniofacial pain and migraine. CGRP potently activates two receptors: the CGRP receptor and the AMY1 receptor. These receptors are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with either the calcitonin receptor-like receptor (CLR) to form the CGRP receptor or the calcitonin receptor (CTR) to form the AMY1 receptor. The expression of the CGRP receptor in trigeminal ganglia has been described in several studies; however, there is comparatively limited data available describing AMY1 receptor expression and in which cellular subtypes it is found. This research aimed to determine the relative distributions of the AMY1 receptor subunit, CTR, and CGRP in neurons or glia in rat, mouse and human trigeminal ganglia. Antibodies against CTR, CGRP and neuronal/glial cell markers were applied to trigeminal ganglia sections to investigate their distribution. CTR-like and CGRP-like immunoreactivity were observed in both discrete and overlapping populations of neurons. In rats and mice, 30-40% of trigeminal ganglia neurons displayed CTR-like immunoreactivity in their cell bodies, with approximately 78-80% of these also containing CGRP-like immunoreactivity. Although human cases were more variable, a similar overall pattern of CTR-like immunoreactivity to rodents was observed in the human trigeminal ganglia. CTR and CGRP appeared to be primarily colocalized in small to medium sized neurons, suggesting that colocalization of CTR and CGRP may occur in C-fiber neurons. CGRP-like or CTR-like immunoreactivity were not typically observed in glial cells. Western blotting confirmed that CTR was expressed in the trigeminal ganglia of all three species. These results confirm that CTR is expressed in trigeminal ganglia neurons. The identification of populations of neurons that express both CGRP and CTR suggests that CGRP could act in an autocrine manner through a CTR-based receptor, such as the AMY1 receptor. Overall, this suggests that a trigeminal ganglia CTR-based receptor may be activated during migraine and could therefore represent a potential target to develop treatments for craniofacial pain and migraine.
| S-EPMC9128745 | biostudies-literature
Project description:BackgroundCalcitonin gene-related peptide (CGRP) is part of the calcitonin peptide family, which includes calcitonin (CT), amylin (AMY), and adrenomedullin (ADM). CGRP and its receptor are highly present in the trigeminovascular system (TVS). Recent research suggests that other members of the calcitonin family could be feasible therapeutic targets in the treatment of migraine. The present study aims to elucidate the distribution of ADM, AMY, CT, and their receptors in the rat TVS, and to explore potential sex differences in their expression.MethodsTrigeminal ganglia (TG) were dissected from male and female adult rats. Protein and gene expression were assessed through immunohistochemistry and RT-qPCR. Additionally, the dura mater was isolated for further investigation of protein expression and fiber localization using immunohistochemistry.ResultsQuantitative gene expression analysis revealed the presence of all genes in male and female TGs, except for calcitonin receptor (CTR). Notably, CGRP mRNA levels in TG were several folds higher than those of other genes. The receptor activity-modifying protein-1 (RAMP1) mRNA levels were significantly higher in female compared to male. No AMY or CT immunoreactivity was observed in the TVS. In contrast, immunoreactivity for ADM, CGRP, RAMP1, CTR, and calcitonin-like receptor (CLR) were observed in the cytoplasm of TG neurons. Immunoreactive Aδ-fibers storing RAMP1, ADM and CLR were also identified. RAMP2 and RAMP3 were expressed in nucleus of TG neurons and in satellite glial cells. Furthermore, RAMP1 and CLR were co-localized with CASPR in the nodes of Ranvier located in Aδ-fibers.ConclusionsThis study provides valuable insights into the distribution of the CGRP family of peptides and their receptors in the TVS. CGRP mRNA levels in the TG were markedly higher than those of other genes, demonstrating the key role of CGRP. The co-localization of CLR and RAMP1 on Aδ-fibers with CASPR suggests a potential role for this receptor in modulating trigeminal nerve function and neuronal excitability, with implications for migraine pathophysiology. Additionally, RAMP1 mRNA levels were significantly higher in female TG compared to males, indicating sex-specific differences in gene expression. These findings underscore the need for further research into the functional significance of gender-related variations.
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Project description:There are several endogenous molecules that can trigger migraine attacks when administered to humans. Notably, calcitonin gene-related peptide (CGRP) has been identified as a key player in a signalling cascade involved in migraine attacks, acting through the second messenger cyclic adenosine monophosphate (cAMP) in various cells, including intracranial vascular smooth muscle cells. However, it remains unclear whether intracellular cAMP signalling requires CGRP receptor activation during a migraine attack in humans. To address this question, we conducted a randomized, double-blind, placebo-controlled, parallel trial using a human provocation model involving the administration of CGRP and cilostazol in individuals with migraine pretreated with erenumab or placebo. Our study revealed that migraine attacks can be provoked in patients by cAMP-mediated mechanisms using cilostazol, even when the CGRP receptor is blocked by erenumab. Furthermore, the dilation of cranial arteries induced by cilostazol was not influenced by the CGRP receptor blockade. These findings provide clinical evidence that cAMP-evoked migraine attacks do not require CGRP receptor activation. This discovery opens up new possibilities for the development of mechanism-based drugs for the treatment of migraine.
| S-EPMC10690017 | biostudies-literature
Project description:The involvement of testosterone in pain, inflammation, and analgesia has been reported, but the role of androgen receptor (AR), a steroid receptor for testosterone, is not well understood. We have previously shown that peripheral inflammation upregulates μ-opioid receptor (MOR) in rat trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we hypothesized that testosterone regulates MOR expression via transcriptional activities of AR in TG. We first examined whether AR is co-expressed with MOR in TG neurons. Our immunohistochemical experiment revealed that AR staining is detected in neurons of all sizes in TG and that a subset of AR is expressed in MOR as well as in TRPV1-positive neurons. We identified the promoter region of the rat MOR gene contains putative AR binding sites. Using chromatin immunoprecipitation assay, we demonstrated that AR directly binds to these sites in TG extracts. We confirmed with luciferase reporter assay that AR activated the MOR promoter in response to androgens in a human neuroblastoma cell line (5H-5YSY). These data demonstrated that AR functions as a transcriptional regulator of the MOR gene activity. Finally, we showed that flutamide, a specific AR antagonist, prevents complete Freund's adjuvant (CFA)-induced upregulation of MOR mRNA in TG, and that flutamide dose-dependently blocks the efficacy of DAMGO, a specific MOR agonist, on CFA-induced mechanical hypersensitivity. Our results expand the knowledge regarding the role of androgens and their receptor in pain and analgesia and have important clinical implications, particularly for inflammatory pain patients with low or compromised plasma testosterone levels.
| S-EPMC5498154 | biostudies-literature
Project description:BackgroundCalcitonin gene-related peptide (CGRP) has a key role in migraine pathophysiology and is associated with activation of the trigeminovascular system. The trigeminal ganglion, storing CGRP and its receptor components, projects peripheral to the intracranial vasculature and central to regions in the brainstem with Aδ- and C-fibers; this constitutes an essential part of the pain pathways activated in migraine attacks. Therefore it is of importance to identify the regions within the brainstem that processes nociceptive information from the trigeminovascular system, such as the spinal trigeminal nucleus (STN) and the C1-level of the spinal cord. Immunohistochemistry was used to study the distribution and relation between CGRP and its receptor components - calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) - in human and rat STN and at the C1-level, using a set of newly well characterized antibodies. In addition, double-stainings with CGRP and myelin basic protein (MBP, myelin), synaptophysin (synaptic vesicles) or IB4 (C-fibers in general) were performed.ResultsIn the STN, the highest density of CGRP immunoreactive fibers were found in a network around fiber bundles in the superficial laminae. CLR and RAMP1 expression were predominately found in fibers in the spinal trigeminal tract region, with some fibers spanning into the superficial laminae. Co-localization between CGRP and its receptor components was not noted. In C1, CGRP was expressed in fibers of laminae I and II. The CGRP staining was similar in rat, except for CGRP positive neurons that were found close to the central canal. In C1, the receptor components were detected in laminae I and II, however these fibers were distinct from fibers expressing CGRP as verified by confocal microscopy.ConclusionsThis study demonstrates the detailed expression of CGRP and its receptor components within STN in the brainstem and in the spinal cord at C1-level, and shows the possibility of CGRP acting postjunctionally in these areas putatively involved in primary headaches.
| S-EPMC3282678 | biostudies-literature
Project description:Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.
| S-EPMC3320266 | biostudies-literature
Project description:In the cell walls of the pathogenic yeast phases of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum, the outer ?-(1,3)-glucan layer behaves as a virulence factor. In H. capsulatum, an ?-(1,4)-amylase gene (AMY1) is essential for the synthesis of this polysaccharide, hence related to virulence. An orthologous gene to H. capsulatum AMY1 was identified in P. brasiliensis and also labeled AMY1. P. brasiliensis AMY1 transcriptional levels were increased during the yeast phase, which correlates with the presence of ?-(1,3)-glucan as the major yeast cell wall polysaccharide. Complementation of a H. capsulatum amy1 mutant strain with P. brasiliensis AMY1, suggests that P. brasiliensis Amy1p may play a role in the synthesis of cell wall ?-(1,3)-glucan. To study some biochemical properties of P. brasiliensis Amy1p, the enzyme was overexpressed, purified and studied its activity profile with starch and amylopeptin. It showed a relatively higher hydrolyzing activity on amylopeptin than starch, producing oligosaccharides from 4 to 5 glucose residues. Our findings show that P. brasiliensis Amy1p produces maltooligosaccharides which may act as a primer molecule for the fungal cell wall ?-(1,3)-glucan biosynthesis by Ags1p.
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