Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor.

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.

Calcitonin receptor, calcitonin gene-related peptide and amylin distribution in C1/2 dorsal root ganglia.

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.

Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology.

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.

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

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.

PAC1, VPAC1, and VPAC2 Receptor Expression in Rat and Human Trigeminal Ganglia: Characterization of PACAP-Responsive Receptor Antibodies.

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.

Production of extracellular vesicles from equine embryo-derived mesenchymal stromal cells.

In brief: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs. Abstract: Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may also provide therapeutic benefits with advantage of being an 'off-the-shelf' solution, provided they can be produced in large enough quantities, without contamination from bovine EVs contained in fetal bovine serum that is a common component of cell culture media. Toward this aim, we demonstrated the successful isolation and characterization of equine MSCs from preimplantation embryos. We also demonstrate that many of these lines can be propagated long-term in culture while retaining their differentiation potential and conducted a head-to-head comparison of two bioreactor systems for scalable EV production including in serum-free conditions. Based on our findings, the CELLine AD 1000 flasks enabled higher cell density cultures and significantly more EV production than the FiberCell system or conventional culture flasks. These findings will enable future isolation of equine MSCs and the scalable culture of their EVs for a wide range of applications in this rapidly growing field.

Calcitonin receptor antibody validation and expression in the rodent brain.

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.

Characterisation of agonist signalling profiles and agonist-dependent antagonism at PACAP-responsive receptors: Implications for drug discovery.

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.

Agonist bias and agonist-dependent antagonism at corticotrophin releasing factor receptors.

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.