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BACKGROUND: The upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY1) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG. METHODS: CTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG. RESULTS: Amylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences. CONCLUSIONS: Amylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets.
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Péptido Relacionado con Gen de Calcitonina , Receptores de Calcitonina , Ratas , Femenino , Masculino , Humanos , Ratones , Animales , Péptido Relacionado con Gen de Calcitonina/genética , Ganglios Espinales , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Hibridación Fluorescente in Situ , Dolor , ARN MensajeroRESUMEN
We read with great interest the recent article by Yoo and colleagues [...].
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Neurología , Hormonas Peptídicas , Animales , Ratas , Polipéptido Amiloide de los Islotes Pancreáticos , EncéfaloRESUMEN
OBJECTIVE: Migraine is a prevalent and disabling neurological disease. Its genesis is poorly understood, and there remains unmet clinical need. We aimed to identify mechanisms and thus novel therapeutic targets for migraine using human models of migraine and translational models in animals, with emphasis on amylin, a close relative of calcitonin gene-related peptide (CGRP). METHODS: Thirty-six migraine without aura patients were enrolled in a randomized, double-blind, 2-way, crossover, positive-controlled clinical trial study to receive infusion of an amylin analogue pramlintide or human αCGRP on 2 different experimental days. Furthermore, translational studies in cells and mouse models, and rat, mouse and human tissue samples were conducted. RESULTS: Thirty patients (88%) developed headache after pramlintide infusion, compared to 33 (97%) after CGRP (p = 0.375). Fourteen patients (41%) developed migraine-like attacks after pramlintide infusion, compared to 19 patients (56%) after CGRP (p = 0.180). The pramlintide-induced migraine-like attacks had similar clinical characteristics to those induced by CGRP. There were differences between treatments in vascular parameters. Human receptor pharmacology studies showed that an amylin receptor likely mediates these pramlintide-provoked effects, rather than the canonical CGRP receptor. Supporting this, preclinical experiments investigating symptoms associated with migraine showed that amylin treatment, like CGRP, caused cutaneous hypersensitivity and light aversion in mice. INTERPRETATION: Our findings propose amylin receptor agonism as a novel contributor to migraine pathogenesis. Greater therapeutic gains could therefore be made for migraine patients through dual amylin and CGRP receptor antagonism, rather than selectively targeting the canonical CGRP receptor. ANN NEUROL 2021;89:1157-1171.
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Agonistas de los Receptores de Amilina/efectos adversos , Polipéptido Amiloide de los Islotes Pancreáticos/efectos adversos , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/efectos adversos , Estudios Cruzados , Método Doble Ciego , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Ganglio del Trigémino/metabolismoRESUMEN
BACKGROUND AND AIM: Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. METHODS: We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. RESULTS: All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. CONCLUSIONS: Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.
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Péptido Relacionado con Gen de Calcitonina , Trastornos Migrañosos , Animales , Encéfalo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ratones , Ratas , Receptores de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Receptores de Polipéptido Amiloide de Islotes PancreáticosRESUMEN
Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.
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Péptido Relacionado con Gen de Calcitonina , Trastornos Migrañosos , Ratones , Animales , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Inmunohistoquímica , Trastornos Migrañosos/metabolismoRESUMEN
Amylin is a 37-amino acid endocrine hormone secreted from the pancreas in response to nutrient intake, acting centrally to promote meal-ending satiation. With many studies linking amylin action to the nervous system, determining the distribution or expression of amylin in the nervous system is critical. However, amylin shares sequence identity and structural homology to the related neuropeptide calcitonin gene-related peptide (CGRP). This creates challenges in identifying selective amylin antibodies that do not cross-react with CGRP, especially in neural tissues, where CGRP is densely packed into secretory vesicles. Here, we characterized eight amylin antibodies to determine their ability to detect amylin and cross-react with rat or human αCGRP, using immunoblots and preabsorption controls in rat pancreas. We observed that amylin antibodies frequently cross-reacted with αCGRP and are therefore not suitable for use in tissues that highly express CGRP. Earlier work using these antibodies should be revisited in light of our findings.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcitonina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Masculino , Páncreas/metabolismo , Hormonas Peptídicas/metabolismoRESUMEN
Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Donantes de Óxido Nítrico/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Antineoplásicos/química , Bencilaminas/química , Bencilaminas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/química , Femenino , Células HCT116 , Células HEK293 , Humanos , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/química , Compuestos Nitrosos/química , Compuestos Nitrosos/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Marine bromopyrrole alkaloids are a diverse family of natural products with a large array of biological applications. The mukanadin family is a group of molecules consisting of seven members (mukanadin A-G) that possess a range of biological activities. Inhibition of serotonergic signaling has been demonstrated by mukanadin B derivatives, presenting this chemical scaffold as a candidate for further SAR exploration. A library of thirteen novel mukanadin B and D derivatives with structural variation targeted at the pyrrole ring, central linker and hydantoin ring, were synthesized. These analogues were subsequently assessed for serotonergic antagonism, in addition to natural products, mukanadin B, D, F and 9-hydroxy mukanadin B. A collection of compounds exhibited significant 5-HT1A signaling, including five of the novel derivatives and two of the naturally occurring bromopyrroles, mukanadin B and F. Particular SAR information could be determined from these results, such as modification of the pyrrole ring being a well-tolerated strategy for improving serotonergic inhibition. Other changes to the pharmacophore led to significant reduction in activity such as saturation of the linker region, or no conclusive improvement in inhibitory activity such as a 9-OH group or replacement of the hydantoin ring with a triazole moiety.
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Pirroles , Receptor de Serotonina 5-HT1A , Transducción de Señal , Relación Estructura-Actividad , Pirroles/farmacología , Pirroles/química , Pirroles/síntesis química , Humanos , Transducción de Señal/efectos de los fármacos , Receptor de Serotonina 5-HT1A/metabolismo , Estructura Molecular , Animales , Antagonistas del Receptor de Serotonina 5-HT1/farmacología , Antagonistas del Receptor de Serotonina 5-HT1/química , Antagonistas del Receptor de Serotonina 5-HT1/síntesis química , Relación Dosis-Respuesta a DrogaRESUMEN
BACKGROUND AND PURPOSE: The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. EXPERIMENTAL APPROACH: Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. KEY RESULTS: PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.
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Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Médula Espinal , Péptido Intestinal Vasoactivo , Animales , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/agonistas , Humanos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Ratones , Ratas , Transducción de Señal/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Células Cultivadas , Ratas Sprague-Dawley , Masculino , Ratones Endogámicos C57BL , AMP Cíclico/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/agonistasRESUMEN
The neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) and their receptors are linked to migraine neurobiology. Recent antimigraine therapeutics targeting the signaling of these neuropeptides are effective; however, some patients respond suboptimally, indicating an incomplete understanding of migraine pathophysiology. The CGRP- and PACAP-responsive receptors can be differentially spliced. It is known that receptor splice variants can have different pathophysiological effects in other receptor-mediated pain pathways. Despite considerable knowledge on the structural and pharmacological differences of the CGRP- and PACAP-responsive receptor splice variants and their expression in migraine-relevant tissues, their role in migraine is rarely considered. Here we shine a spotlight on the calcitonin and PACAP (PAC1) receptor splice variants and examine what implications they may have for drug activity and design.
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Péptido Relacionado con Gen de Calcitonina , Trastornos Migrañosos , Humanos , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcitonina , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido alfa Relacionado con Calcitonina , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/genéticaRESUMEN
The related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) have diverse biological functions in peripheral tissues and the central nervous system. Therefore, these peptides and their three receptors represent potential drug targets for several conditions, including neurological and pain-related disorders. However, very little is known about how these peptides regulate their receptors through processes such as internalization. Therefore, we developed tools to study receptor regulation through the synthesis of fluorescently labeled analogues of PACAP-38, PACAP-27, and VIP using copper-mediated 1,3-dipolar cycloaddition of the Cy5 fluorophore. The functionality of Cy5-labeled peptides at their receptors was confirmed in cAMP accumulation assays. Internalization of the Cy5-labeled peptides was then examined and quantified at two distinct PAC1 receptor splice variants, VPAC1 and VPAC2 receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manner. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC1n, PAC1s, VPAC1, and VPAC2 receptors. Interestingly, the pattern of Cy5-labeled peptide activity differed for cAMP accumulation and internalization, indicating that these peptides differentially stimulate cAMP accumulation and internalization and therefore display biased agonism. This novel insight into PACAP-responsive receptor signaling and internalization may provide a unique avenue for future therapeutic development. The fluorescently labeled PACAP and VIP peptides described herein, which we validated as tools to study receptor internalization, will have utility across a broad range of applications and provide greater insight into this receptor family.
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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.
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The CGRP system has emerged as a key pharmacological target for the treatment of migraine. However, some individuals who suffer from migraine have low or no response to anti-CGRP or other treatments, suggesting the need for additional clinical targets. CGRP belongs to the calcitonin family of peptides, which includes calcitonin, amylin, adrenomedullin and adrenomedullin 2. These peptides display a range of pro-nociceptive and anti-nociceptive actions, in primary headache conditions such as migraine. Calcitonin family peptides also show expression at sites relevant to migraine and pain. This suggests that calcitonin family peptides and their receptors, beyond CGRP, may be therapeutically useful in the treatment of migraine and other pain disorders. This review considers the localisation of the calcitonin family in peripheral pain pathways and discusses how they may contribute to migraine and pain. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.
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Trastornos Migrañosos , Hormonas Peptídicas , Adrenomedulina , Calcitonina , Péptido Relacionado con Gen de Calcitonina/metabolismo , Cefalea , Humanos , Trastornos Migrañosos/tratamiento farmacológico , Dolor/tratamiento farmacológico , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismoRESUMEN
Calcitonin gene-related peptide (CGRP) is a neuropeptide that is involved in the transmission of pain. Drugs targeting CGRP or a CGRP receptor are efficacious in the treatment of migraine. The canonical CGRP receptor is a complex of a G protein-coupled receptor, the calcitonin-like receptor (CLR), with an accessory protein, receptor activity-modifying protein 1 (RAMP1). A second receptor, the AMY1 receptor, a complex of the calcitonin receptor with RAMP1, is a dual high-affinity receptor for CGRP and amylin. Receptor regulatory processes, such as internalization, are crucial for controlling peptide and drug responsiveness. Given the importance of CGRP receptor activity in migraine we compared the internalization profiles of both receptors for CGRP using novel fluorescent probes and a combination of live cell imaging, fixed cell imaging, and ELISA. This revealed stark differences in the regulation of each receptor with the AMY1 receptor unexpectedly showing little internalization.
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The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C-terminus and receptor "capture" and "activation" regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide-receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity.