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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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Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Medula Espinal , Peptídeo Intestinal Vasoativo , Animais , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Humanos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Camundongos , Ratos , Transdução de Sinais/efeitos dos fármacos , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Células Cultivadas , Ratos Sprague-Dawley , Masculino , Camundongos Endogâmicos C57BL , AMP Cíclico/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/agonistasRESUMO
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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Peptídeo Relacionado com Gene de Calcitonina , Receptores da Calcitonina , Ratos , Feminino , Masculino , Humanos , Camundongos , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Gânglios Espinais , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Hibridização in Situ Fluorescente , Dor , RNA MensageiroRESUMO
Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.
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Amiloide , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Humanos , Ratos , Animais , Receptores de Polipeptídeo Amiloide de Ilhotas PancreáticasRESUMO
We read with great interest the recent article by Yoo and colleagues [...].
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Neurologia , Hormônios Peptídicos , Animais , Ratos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , EncéfaloRESUMO
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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Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse physiological functions. Its two isoforms (α and ß) are widely expressed throughout the body in sensory neurons as well as in other cell types, such as motor neurons and neuroendocrine cells. CGRP acts via at least two G protein-coupled receptors that form unusual complexes with receptor activity-modifying proteins. These are the CGRP receptor and the AMY1 receptor; in rodents, additional receptors come into play. Although CGRP is known to produce many effects, the precise molecular identity of the receptor(s) that mediates CGRP effects is seldom clear. Despite the many enigmas still in CGRP biology, therapeutics that target the CGRP axis to treat or prevent migraine are a bench-to-bedside success story. This review provides a contextual background on the regulation and sites of CGRP expression and CGRP receptor pharmacology. The physiological actions of CGRP in the nervous system are discussed, along with updates on CGRP actions in the cardiovascular, pulmonary, gastrointestinal, immune, hematopoietic, and reproductive systems and metabolic effects of CGRP in muscle and adipose tissues. We cover how CGRP in these systems is associated with disease states, most notably migraine. In this context, we discuss how CGRP actions in both the peripheral and central nervous systems provide a basis for therapeutic targeting of CGRP in migraine. Finally, we highlight potentially fertile ground for the development of additional therapeutics and combinatorial strategies that could be designed to modulate CGRP signaling for migraine and other diseases.
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Peptídeo Relacionado com Gene de Calcitonina , Transtornos de Enxaqueca , Humanos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/uso terapêutico , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Sistema Nervoso Central/metabolismo , Neurônios MotoresRESUMO
The prevalence of obesity is rising, creating an urgent need for efficacious therapies. Recent clinical trials show that tirzepatide, a dual agonist of receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), yields more weight loss than selective GLP-1 receptor (GLP-1R) agonists. Incretin receptors in the central nervous system (CNS) may contribute to these effects. Yet exactly how each receptor regulates body weight from within the CNS is not clearly understood. It remains especially unclear how GIP receptor (GIPR) signalling contributes to the effects of tirzepatide because both stimulation and inhibition of CNS GIPRs yield weight loss in preclinical models. We summarise current knowledge on CNS incretin receptor pharmacology to provide insight into the potential mechanisms of action of dual GIPR/GLP-1R agonists, with tirzepatide as the exemplar. In addition, we discuss recent developments in incretin-based dual- and tri-agonism for inducing weight loss in obese individuals.
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Diabetes Mellitus Tipo 2 , Incretinas , Humanos , Incretinas/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Obesidade/tratamento farmacológico , Redução de Peso , Diabetes Mellitus Tipo 2/tratamento farmacológicoRESUMO
Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the trigeminal ganglia (TG). The TG conducts nociceptive signals in the head and may play roles in migraine. PACAP infusion provokes headaches in healthy individuals and migraine-like attacks in patients; however, it is not clear whether targeting this system could be therapeutically efficacious. To effectively target the PACAP system, an understanding of PACAP receptor distribution is required. Therefore, this study aimed to characterize commercially available antibodies and use these to detect PACAP-responsive receptors in the TG. Antibodies were initially validated in receptor transfected cell models and then used to explore receptor expression in rat and human TG. Antibodies were identified that could detect PACAP-responsive receptors, including the first antibody to differentiate between the PAC1n and PAC1s receptor splice variants. PAC1, VPAC1, and VPAC2 receptor-like immunoreactivity were observed in subpopulations of both neuronal and glial-like cells in the TG. In this study, PAC1, VPAC1, and VPAC2 receptors were detected in the TG, suggesting they are all potential targets to treat migraine. These antibodies may be useful tools to help elucidate PACAP-responsive receptor expression in tissues. However, most antibodies exhibited limitations, requiring the use of multiple methodologies and the careful inclusion of controls.
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Transtornos de Enxaqueca , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Humanos , Ratos , Animais , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Gânglio Trigeminal/metabolismo , Expressão Gênica , Anticorpos , Transtornos de Enxaqueca/genéticaRESUMO
OBJECTIVE: To summarize the pharmacology of the calcitonin peptide family of receptors and explore their relationship to migraine and current migraine therapies. BACKGROUND: Therapeutics that dampen calcitonin gene-related peptide (CGRP) signaling are now in clinical use to prevent or treat migraine. However, CGRP belongs to a broader peptide family, including the peptides amylin and adrenomedullin. Receptors for this family are complex, displaying overlapping pharmacologic profiles. Despite the focus on CGRP and the CGRP receptor in migraine research, recent evidence implicates related peptides and receptors in migraine. METHODS: This narrative review summarizes literature encompassing the current pharmacologic understanding of the calcitonin peptide family, and the evidence that links specific members of this family to migraine and migraine-like behaviors. RESULTS: Recent work links amylin and adrenomedullin to migraine-like behavior in rodent models and migraine-like attacks in individuals with migraine. We collate novel information that suggests females may be more sensitive to amylin and CGRP in the context of migraine-like behaviors. We report that drugs designed to antagonize the canonical CGRP receptor also antagonize a second CGRP-responsive receptor and speculate as to whether this influences therapeutic efficacy. We also discuss the specificity of current drugs with regards to CGRP isoforms and how this may influence therapeutic profiles. Lastly, we emphasize that receptors related to, but distinct from, the canonical CGRP receptor may represent underappreciated and novel drug targets. CONCLUSION: Multiple peptides within the calcitonin family have been linked to migraine. The current focus on CGRP and its canonical receptor may be obscuring pathways to further therapeutics. Drug discovery schemes that take a wider view of the receptor family may lead to the development of new anti-migraine drugs with favorable clinical profiles. We also propose that understanding these related peptides and receptors may improve our interpretation regarding the mechanism of action of current drugs.
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Peptídeo Relacionado com Gene de Calcitonina , Transtornos de Enxaqueca , Feminino , Humanos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Adrenomedulina/uso terapêutico , Calcitonina/uso terapêutico , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismoRESUMO
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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Peptídeo Relacionado com Gene de Calcitonina , Transtornos de Enxaqueca , Animais , Encéfalo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Camundongos , Ratos , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas PancreáticasRESUMO
Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.
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Agonistas dos Receptores da Amilina/química , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/química , Animais , Microscopia Crioeletrônica , Humanos , Fenótipo , Conformação Proteica , Multimerização Proteica , SalmãoRESUMO
BACKGROUND AND PURPOSE: The pituitary adenylate cyclase-activating peptide (PACAP) family is of clinical interest for the treatment of migraine. These peptides activate three different PACAP-responsive class B G protein-coupled receptors: the PAC1 , VPAC1 and VPAC2 receptors. The PAC1 receptor may be alternatively spliced, generating variants that can differ in their pharmacological or signalling profiles. To inform drug discovery efforts targeting migraine, we need to better understand how the different PACAP-responsive receptors signal and how effectively these responses can be blocked by antagonists. EXPERIMENTAL APPROACH: The signalling profiles of the human PAC1n , PAC1s , VPAC1 and VPAC2 receptors were examined in transfected Cos7 cells for cAMP, IP1 , pAkt, pERK and pCREB. Biased signalling was then quantified. The ability of antagonists to block PACAP-38, PACAP-27 or VIP stimulated cAMP accumulation at PACAP-responsive receptors was also determined. KEY RESULTS: PACAP-responsive receptors exhibited varied pharmacological profiles but activated signalling in a similar manner. The PAC1n and PAC1s receptors displayed distinct pharmacology. At the PAC1s receptor, VIP and PHM were more potent than at the PAC1n receptor. PACAP-responsive receptors displayed agonist-dependent antagonism where PACAP-38 was less effectively antagonised compared to PACAP-27 and VIP. CONCLUSIONS AND IMPLICATIONS: The distinct pharmacological profile displayed by the PAC1s receptor suggests that it can act as a dual receptor for VIP and PACAP. Furthermore, the effectiveness of blocking a signalling pathway can be influenced by which endogenous PACAP family agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PACAP system. 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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Transtornos de Enxaqueca , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Descoberta de Drogas , Humanos , Transtornos de Enxaqueca/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Peptídeo Intestinal VasoativoRESUMO
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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Transtornos de Enxaqueca , Hormônios Peptídicos , Adrenomedulina , Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cefaleia , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Dor/tratamento farmacológico , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismoRESUMO
BACKGROUND AND PURPOSE: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice. EXPERIMENTAL APPROACH: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists. KEY RESULTS: We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and ßCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity. CONCLUSION AND IMPLICATIONS: Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes. 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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Transtornos de Enxaqueca , Hormônios Peptídicos , Adrenomedulina/metabolismo , Adrenomedulina/farmacologia , Animais , Calcitonina/metabolismo , Calcitonina/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/química , Humanos , Ligantes , Camundongos , Ratos , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina , Receptores da Calcitonina/químicaRESUMO
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 dos Receptores da Amilina/efeitos adversos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/efeitos adversos , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/efeitos adversos , Estudos Cross-Over , Método Duplo-Cego , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/metabolismoRESUMO
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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Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Calcitonina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Masculino , Pâncreas/metabolismo , Hormônios Peptídicos/metabolismoRESUMO
The corticotropin-releasing factor (CRF) receptors represent potential drug targets for the treatment of anxiety, stress, and other disorders. However, it is not known if endogenous CRF receptor agonists display biased signaling, how effective CRF receptor antagonists are at blocking different agonists and signaling pathways or how receptor activity-modifying proteins (RAMPs) effect these processes. This study aimed to address this by investigating agonist and antagonist action at CRF1 and CRF2 receptors. We used CRF1 and CRF2 receptor transfected Cos7 cells to assess the ability of CRF and urocortin (UCN) peptides to activate cAMP, inositol monophosphate (IP1 ), and extracellular signal-regulated kinase 1/2 signaling and determined the ability of antagonists to block agonist-stimulated cAMP and IP1 accumulation. The ability of RAMPs to interact with CRF receptors was also examined. At the CRF1 receptor, CRF and UCN1 activated signaling in the same manner. However, at the CRF2 receptor, UCN1 and UCN2 displayed similar signaling profiles, whereas CRF and UCN3 displayed bias away from IP1 accumulation over cAMP. The antagonist potency was dependent on the receptor, agonist, and signaling pathway. CRF1 and CRF2 receptors had no effect on RAMP1 or RAMP2 surface expression. The presence of biased agonism and agonist-dependent antagonism at the CRF receptors offers new avenues for developing drugs tailored to activate a specific signaling pathway or block a specific agonist. Our findings suggest that the already complex CRF receptor pharmacology may be underappreciated and requires further investigation.
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Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Urocortinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Desenvolvimento de Medicamentos , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptores de Hormônio Liberador da Corticotropina/agonistasRESUMO
The neuropancreatic polypeptide hormone amylin forms pancreatic islet amyloid in type-2 diabetes. Islet amyloid formation contributes to ß-cell death in the disease and to the failure of islet transplants, but the features which influence amylin amyloidogenicity are not understood. We constructed an amino acid sequence alignment of 202 sequences of amylin and used the alignment to design consensus sequences of vertebrate amylins, mammalian amylins, and primate amylins. Amylin is highly conserved, but there are differences between human amylin and each consensus sequence, ranging from one to six substitutions. Biophysical analysis shows that all of the consensus sequences form amyloid but do so more slowly than human amylin in vitro. The rate of amyloid formation by the primate consensus sequence is 3- to 4-fold slower than human amylin; the mammalian consensus sequence is approximately 20- to 25-fold slower, and the vertebrate consensus sequence is approximately 6-fold slower. All of the consensus sequences are moderately less toxic than human amylin toward a cultured ß-cell line, with the vertebrate consensus sequence displaying the largest reduction in toxicity of 3- to 4-fold. All of the consensus sequences activate a human amylin receptor and exhibit only modest reductions in activity, ranging from 3- to 4-fold as judged by a cAMP production assay. The analysis argues that there is no strong selective evolutionary pressure to avoid the formation of islet amyloid and provides information relevant to the design of less amyloidogenic amylin variants.
Assuntos
Amiloide/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Amiloide/química , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Sequência Consenso , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Modelos Moleculares , Ratos , Alinhamento de SequênciaRESUMO
Adrenomedullin 2 (AM2) is a peptide hormone with potent effects in the cardiovascular system. The N-terminal disulfide loop of AM2 is thought to be important for interacting with its receptors to initiate a signaling response. However, the relative contribution of each amino acid within this region is currently unknown. Thus, the region was investigated using an alanine scanning approach. Two AM2 peptides (AM2-47 and AM2-40) were directly compared at the CGRP, AM1, and AM2 receptors in transfected Cos7 cells and found to have equivalent activity. Analogues of AM2-40 were then synthesized, substituting each individual amino acid within the disulfide loop with alanine. The ability of these analogues to stimulate a cAMP response was evaluated at the CGRP, AM1, and AM2 receptors. AM2-40 L12A and T14A were less able to elicit cAMP responses through all tested receptors. In contrast, AM2-40 G13A was slightly more potent than the unmodified peptide at all tested receptors. Thus, it appears that residues within the disulfide loop region play differential roles in the ability of AM2 to stimulate cAMP production. The data provide the first structure-function investigation of AM2 agonism.
Assuntos
Adrenomedulina/química , Adrenomedulina/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/biossíntese , HumanosRESUMO
Islet amyloid formation contributes to ß-cell death and dysfunction in type-2 diabetes and to the failure of islet transplants. Amylin (islet amyloid polypeptide, IAPP), a normally soluble 37 residue polypeptide hormone produced in the pancreatic ß-cells, is responsible for amyloid formation in type-2 diabetes and is deficient in type-1 diabetes. Amylin normally plays an adaptive role in metabolism, and the development of nontoxic, non-amyloidogenic, bioactive variants of human amylin are of interest for use as adjuncts to insulin therapy. Naturally occurring non-amyloidogenic variants are of interest for xenobiotic transplantation and because they can provide clues toward understanding the amyloidogenicity of human amylin. The sequence of amylin is well-conserved among species, but sequence differences strongly correlate with in vitro amyloidogenicity and with islet amyloid formation in vivo. Bovine amylin differs from the human peptide at 10 positions and is one of the most divergent among known amylin sequences. We show that bovine amylin oligomerizes but is not toxic to cultured ß-cells and that it is considerably less amyloidogenic than the human polypeptide and is only a low-potency agonist at human amylin-responsive receptors. The bovine sequence contains several nonconservative substitutions relative to human amylin, including His to Pro, Ser to Pro, and Asn to Lys replacements. The effect of these substitutions is analyzed in the context of wild-type human amylin; the results provide insight into their role in receptor activation, the mode of assembly of human amylin, and the design of soluble amylin analogues.