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.

PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide's stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide's stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

Bordetella spp. utilize the type 3 secretion system to manipulate the VIP/VPAC2 signaling and promote colonization and persistence of the three classical Bordetella in the lower respiratory tract.

Introduction: Bordetella are respiratory pathogens comprised of three classical Bordetella species: B. pertussis, B. parapertussis, and B. bronchiseptica. With recent surges in Bordetella spp. cases and antibiotics becoming less effective to combat infectious diseases, there is an imperative need for novel antimicrobial therapies. Our goal is to investigate the possible targets of host immunomodulatory mechanisms that can be exploited to promote clearance of Bordetella spp. infections. Vasoactive intestinal peptide (VIP) is a neuropeptide that promotes Th2 anti-inflammatory responses through VPAC1 and VPAC2 receptor binding and activation of downstream signaling cascades. Methods: We used classical growth in vitro assays to evaluate the effects of VIP on Bordetella spp. growth and survival. Using the three classical Bordetella spp. in combination with different mouse strains we were able to evaluate the role of VIP/VPAC2 signaling in the infectious dose 50 and infection dynamics. Finally using the B. bronchiseptica murine model we determine the suitability of VPAC2 antagonists as possible therapy for Bordetella spp. infections. Results: Under the hypothesis that inhibition of VIP/VPAC2 signaling would promote clearance, we found that VPAC2-/- mice, lacking a functional VIP/VPAC2 axis, hinder the ability of the bacteria to colonize the lungs, resulting in decreased bacterial burden by all three classical Bordetella species. Moreover, treatment with VPAC2 antagonists decrease lung pathology, suggesting its potential use to prevent lung damage and dysfunction caused by infection. Our results indicate that the ability of Bordetella spp. to manipulate VIP/VPAC signaling pathway appears to be mediated by the type 3 secretion system (T3SS), suggesting that this might serve as a therapeutical target for other gram-negative bacteria. Conclusion: Taken together, our findings uncover a novel mechanism of bacteria-host crosstalk that could provide a target for the future treatment for whooping cough as well as other infectious diseases caused primarily by persistent mucosal infections.

Overexpression of VIPR2 in mice results in microencephaly with paradoxical increased white matter volume.

Large scale studies in populations of European and Han Chinese ancestry found a series of rare gain-of-function microduplications in VIPR2, encoding VPAC2, a receptor that binds vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide with high affinity, that were associated with an up to 13-fold increased risk for schizophrenia. To address how VPAC2 receptor overactivity might affect brain development, we used a well-characterized Nestin-Cre mouse strain and a knock-in approach to overexpress human VPAC2 in the central nervous system. Mice that overexpressed VPAC2 were found to exhibit a significant reduction in brain weight. Magnetic resonance imaging analysis confirmed a decrease in brain size, a specific reduction in the hippocampus grey matter volume and a paradoxical increase in whole-brain white matter volume. Sex-specific changes in behavior such as impaired prepulse inhibition and contextual fear memory were observed in VPAC2 overexpressing mice. The data indicate that the VPAC2 receptor may play a critical role in brain morphogenesis and suggest that overactive VPAC2 signaling during development plays a mechanistic role in some forms of schizophrenia.

VPAC2 receptor mediates VIP-potentiated insulin secretion via ion channels in rat pancreatic ß cells.

Vasoactive intestinal peptide (VIP), a small neuropeptide composing of 28 amino acids, functions as a neuromodulator with insulinotropic effect on pancreatic ß cells, in which it is of vital importance in regulating the levels of blood glucose. VIP potently agonizes VPAC2 receptor (VPAC2-R). Agonists of VPAC2-R stimulate glucose-dependent insulin secretion. The purpose of this study was to further investigate the possible ion channel mechanisms in VPAC2-R-mediated VIP-potentiated insulin secretion. The results of insulin secretion experiments showed that VIP augmented insulin secretion in a glucose-dependent manner. The insulinotropic effect was mediated by VPAC2-R rather than VPAC1 receptor (VPAC1-R), through the adenylyl cyclase (AC)/protein kinase A (PKA) signalling pathway. The calcium imaging analysis demonstrated that VIP increased intracellular Ca2+ concentration ([Ca2+]i). In addition, in the whole-cell voltage-clamp mode, we found that VIP blocked the voltage-dependent potassium (Kv) channel currents, while this effect was reversed by inhibiting the VPAC2-R, AC or PKA respectively. Taken together, these findings suggest that VIP stimulates insulin secretion by inhibiting the Kv channels, activating the Ca2+ channels, and increasing [Ca2+]i through the VPAC2-R and AC/PKA signalling pathway. These findings provide theoretical basis for the research of VPAC2-R as a novel therapeutic target.

VPAC1 and VPAC2 receptor deficiencies negatively influence pregnancy outcome through distinct and overlapping modulations of immune, trophoblast and vascular functions.

Pregnancy outcome relies on the maintenance of immune and metabolic homeostasis at the maternal fetal interface. Maternal and perinatal morbidity and mortality is associated with impaired placental development. Multiple regulatory effects of the endogenous-produced vasoactive intestinal peptide (VIP) on vascular, metabolic and immune functions at the maternal-fetal interface have been reported. Here we studied the involvement of the two primary high affinity receptors for VIP (VPAC1 and VPAC2) on maternal immune response, placental homeostasis and pregnancy outcome. Targeted disruption of each receptor gene led to altered placental structure, vascular and trophoblast functional markers and shaped the functional profiles of macrophages and neutrophils towards a proinflammatory state. Several changes in pregnant mice were receptor specific: ROS production elicited by VIP on neutrophils was selectively dependent on the presence of VPAC1 whereas apoptosis rate was associated with the VPAC2 deletion. In peritoneal macrophages from pregnant mice, levels of MHC-II, TLR2, and IL-10 were selectively altered in VPAC2 receptor-deficient mice, whereas IL-6 gene expression was reduced only in mice lacking VPAC1 receptors. Additionally, MMP9 mRNA in isolated TGCs was reduced in VPAC2 receptor deleted mice, while the percentage of IL-12 cells in post-phagocytosis macrophage cultures was selectively reduced in VPAC2 receptor deficient mice. The results indicate that manipulation of VPAC1 and VPAC2 receptor affects immune, vascular and metabolic environment at the maternal fetal interface. These mouse models offer new approaches to study pregnancy complications adding new perspectives to the development of VPAC receptor-selective drugs.

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.

Localization of the neuropeptides pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and their receptors in the basal brain blood vessels and trigeminal ganglion of the mouse CNS; an immunohistochemical study.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally related neuropeptides that are widely expressed in vertebrate tissues. The two neuropeptides are pleiotropic and have been associated with migraine pathology. Three PACAP and VIP receptors have been described: PAC1, VPAC1, and VPAC2. The localization of these receptors in relation to VIP and PACAP in migraine-relevant structures has not previously been shown in mice. In the present study, we used fluorescence immunohistochemistry, well-characterized antibodies, confocal microscopy, and three-dimensional reconstruction to visualize the distribution of PACAP, VIP, and their receptors in the basal blood vessels (circle of Willis), trigeminal ganglion, and brain stem spinal trigeminal nucleus (SP5) of the mouse CNS. We demonstrated a dense network of circularly oriented VIP fibers on the basal blood vessels. PACAP nerve fibers were fewer in numbers compared to VIP fibers and ran along the long axis of the blood vessels, colocalized with calcitonin gene-related peptide (CGRP). The nerve fibers expressing CGRP are believed to be sensorial, with neuronal somas localized in the trigeminal ganglion and PACAP was found in a subpopulation of these CGRP-neurons. Immunostaining of the receptors revealed that only the VPAC1 receptor was present in the basal blood vessels, localized on the surface cell membrane of vascular smooth muscle cells and innervated by VIP fibers. No staining was seen for the PAC1, VPAC1, or VPAC2 receptor in the trigeminal ganglion. However, distinct PAC1 immunoreactivity was found in neurons innervated by PACAP nerve terminals located in the spinal trigeminal nucleus. These findings indicate that the effect of VIP is mediated via the VPAC1 receptor in the basal arteries. The role of PACAP in cerebral arteries is less clear. The localization of PACAP in a subpopulation of CGRP-expressing neurons in the trigeminal ganglion points toward a primary sensory function although a dendritic release cannot be excluded which could stimulate the VPAC1 receptor or the PAC1 and VPAC2 receptors on immune cells in the meninges, initiating neurogenic inflammation relevant for migraine pathology.

Therapeutic potential of vasoactive intestinal peptide and its receptor VPAC2 in type 2 diabetes.

Owing to the increasing prevalence of type 2 diabetes, the development of novel hypoglycemic drugs has become a research hotspot, with the ultimate goal of developing therapeutic drugs that stimulate glucose-induced insulin secretion without inducing hypoglycemia. Vasoactive intestinal peptide (VIP), a 28-amino-acid peptide, can stimulate glucose-dependent insulin secretion, particularly by binding to VPAC2 receptors. VIP also promotes islet ß-cell proliferation through the forkhead box M1 pathway, but the specific molecular mechanism remains to be studied. The clinical application of VIP is limited because of its short half-life and wide distribution in the human body. Based on the binding properties of VIP and VPAC2 receptors, VPAC2-selective agonists have been developed to serve as novel hypoglycemic drugs. This review summarizes the physiological significance of VIP in glucose homeostasis and the potential therapeutic value of VPAC2-selective agonists in type 2 diabetes.

Messenger RNA Gene Expression Screening of VIP and PACAP Neuropeptides and Their Endogenous Receptors in Ruminants.

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate-Cyclase-Activating Peptide (PACAP) are anti-inflammatory neuropeptides that play important roles in human and rodent gut microbiota homeostasis and host immunity. Pharmacologically regulating these neuropeptides is expected to have significant health and feed efficiency benefits for agriculturally relevant animals. However, their expression profile in ruminant tissues is not well characterized. To this end, we screened for VIP and PACAP neuropeptides and their endogenous GPCRs using 15 different tissues from wethers and steers by RT-qPCR. Our results revealed relatively similar expression profiles for both VIP and PACAP neuropeptide ligands in the brain and intestinal tissue of both species. In contrast, the tissue expression profiles for VPAC1, VPAC2, and PAC1 were more widespread and disparate, with VPAC1 being the most diversely expressed receptor with mRNA detection in the brain and throughout the gastrointestinal tract. These data are an important first step to allow for future investigations regarding the VIP and PACAP signaling pathways in livestock ruminant species.

Alterations in Gut Microbiota and Upregulations of VPAC2 and Intestinal Tight Junctions Correlate with Anti-Inflammatory Effects of Electroacupuncture in Colitis Mice with Sleep Fragmentation.

The relationship between inflammatory bowel disease and sleep disturbances is complicated and of increasing interest. We investigated the inflammatory and immunological consequences of EA in sleep-deprived colitis and found that dextran sulfate sodium (DSS)-induced colitis in sleep-fragmented (SF) mice was more severe than that in mice with normal sleep. This increase in the severity of colitis was accompanied by reduced body weight, shortened colon length, and deteriorated disease activity index. DSS with SF mice presented obvious diminished intestinal tight junction proteins (claudin-1 and occludin), elevated proinflammatory cytokines (CRP, IFN-γ, IL-6), lowered melatonin and adiponectin levels, downregulated vasoactive intestinal peptide (VIP) type 1 and 2 receptor (VPAC1, VPAC2) expression, and decreased diversity of gut bacteria. EA ameliorated colitis severity and preserved the performance of the epithelial tight junction proteins and VIP receptors, especially VPAC2. Meanwhile, the innate lymphoid cells-derived cytokines in both group 2 (IL-4, IL5, IL-9, IL-13) and group 3 (IL-22, GM-CSF) were elevated in mice colon tissue. Furthermore, dysbiosis was confirmed in the DSS group with and without SF, and EA could maintain the species diversity. Firmicutes could be restored, such as Lachnospiraceae, and Proteobacteria become rebalanced, mainly Enterobacteriaceae, after EA intervention. On the other hand, SF plays different roles in physiological and pathological conditions. In normal mice, interrupted sleep did not affect the expression of claudin-1 and occludin. But VPAC1, VPAC2, and gut microbiota diversity, including Burkholderiaceae and Rhodococcus, were opposite to mice in an inflamed state.

Role of VPAC1 and VPAC2 receptors in the etiology of pregnancy rhinitis: an experimental study in rats / O papel dos receptores VPAC1 e VPAC2 na etiologia da rinite gestacional: um estudo experimental em ratos

Abstract Introduction: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. Objectives: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. Methods: Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2°C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400mg/kg Na-pentobarbitone. Then, 10 - 15 mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. Results: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p = 0.023) and VPAC2 (p = 0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. Conclusions: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.

Resumo Introdução: A rinite gestacional é um distúrbio comum da otorrinolaringologia relacionado a hormônios sexuais. Existem alguns estudos epidemiológicos e fisiológicos sobre rinite gestacional, mas as alterações histopatológicas e biomoleculares ainda não foram estudadas completamente. Objetivo: Os receptores VPAC1 e VPAC2 são conhecidos por seu papel na rinite alérgica. Por outro lado, a ativação da alergia subclínica tem sido sugerida na fisiopatologia da rinite gestacional. Portanto, objetivamos comparar o padrão fisiológico e gestacional da expressão de VPAC1 e VPAC2 na mucosa nasal de ratos. Método: Vinte ratas fêmeas Wistar albinas adultas foram incluídas no estudo. Os dois grupos foram divididos em 10 ratas; controle (grupo A) e 10 ratas prenhes (grupo B). Elas foram alimentadas ad libitum e abrigadas em temperatura ambiente (22° ±2° C). Sacrificamos as ratas no 20° dia de gestação por injeção intraperitoneal de 400 mg/kg de sódio-pentobarbital. Em seguida, foram coletados 10 a 15 mL de sangue e as amostras foram reservadas para a detecção dos níveis séricos de estradiol e progesterona pelo método Elisa. O septo nasal foi ressecado e dividido em 2 para análises imuno-histoquímicas e testes de reação em cadeia da polimerase em tempo real, RT-PCR, de VPAC1 e VPAC2. Resultados: VPAC1 e VPAC2 foram encontrados em todas as camadas da amostra septal, mas a imunocoloração do epitélio de superfície foi mais distinta nas amostras de ambos os grupos. Demonstramos maior intensidade geral de coloração no grupo gestante. A reação de polimerase em cadeia revelou aumento significante na expressão de VPAC1 (p = 0,023) e VPAC2 (p = 0,021) no grupo gestante quando comparado ao grupo controle. Além disso, demonstramos um efeito up-regulador do estradiol e progesterona na expressão do receptor peptídeo intestinal vasoativo. Conclusão: A up-regulação gestacional dos receptores VPAC1 e VPAC2 nasais foi demonstrada tanto por reação de polimerase em cadeia quanto por análise imuno-histoquímica. Esses achados corroboram a hipótese de que a rinite gestacional é causada pela ativação de alergia subclínica presente antes da gestação.

Targeting VIP and PACAP Receptor Signaling: New Insights into Designing Drugs for the PACAP Subfamily of Receptors.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) are neuropeptides involved in a diverse array of physiological and pathological processes through activating the PACAP subfamily of class B1 G protein-coupled receptors (GPCRs): VIP receptor 1 (VPAC1R), VIP receptor 2 (VPAC2R), and PACAP type I receptor (PAC1R). VIP and PACAP share nearly 70% amino acid sequence identity, while their receptors PAC1R, VPAC1R, and VPAC2R share 60% homology in the transmembrane regions of the receptor. PACAP binds with high affinity to all three receptors, while VIP binds with high affinity to VPAC1R and VPAC2R, and has a thousand-fold lower affinity for PAC1R compared to PACAP. Due to the wide distribution of VIP and PACAP receptors in the body, potential therapeutic applications of drugs targeting these receptors, as well as expected undesired side effects, are numerous. Designing selective therapeutics targeting these receptors remains challenging due to their structural similarities. This review discusses recent discoveries on the molecular mechanisms involved in the selectivity and signaling of the PACAP subfamily of receptors, and future considerations for therapeutic targeting.

Opposing reduced VPAC1 and enhanced VPAC2 VIP receptors in the hippocampus of the Li2+-pilocarpine rat model of temporal lobe epilepsy.

VIP binding sites are upregulated in mesial temporal lobe epilepsy (MTLE) patients, also suffering from severe cognitive deficits. Although altered VIP and VIP receptor levels were described in rodent models of epilepsy, the VIP receptor subtype(s) were never identified. We now investigated how VPAC1 and VPAC2 receptor levels change in the Li2+-pilocarpine rat model of MTLE. Cognitive decline and altered synaptic plasticity as estimated from phosphorylation of AMPA GluA1 subunit on Ser831 and Ser845 and AMPA GluA1/GluA2 ratio was also probed. Animals showing spontaneous recurrent seizures (SRSs) for at least 4 weeks showed impaired learning in the radial arm maze (RAM) and presented decreased VPAC1 and increased VPAC2 receptor levels. In addition, SRSs rats showed increased AMPA GluA1 phosphorylation in Ser831 and Ser845, marked decrease in GluA1 levels and a milder decrease in GluA2 levels. Consequently, the GluA1/GluA2 ratio was also decreased in SRSs rats. Altered VIP receptor levels may differentially prevent or contribute to MTLE pathology, since VPAC1 receptors promote the endogenous control of LTP, mediate endogenous VIP neuroprotection against altered synaptic plasticity following epileptiform activity, and mediate anti-inflammatory actions in microglia, while VPAC2 receptors mediate VIP endogenous neuroprotection against neonatal excitotoxicity and prevent reactive astrogliosis. This discovery imposes a different mindset for considering VIP receptors as therapeutic targets in MTLE, allowing a differential targeting of the cellular events contributing to epileptogenesis.

Endogenous VIP VPAC1 Receptor Activation Modulates Hippocampal Theta Burst Induced LTP: Transduction Pathways and GABAergic Mechanisms.

Vasoactive intestinal peptide (VIP), acting on both VPAC1 and VPAC2 receptors, is a key modulator of hippocampal synaptic transmission, pyramidal cell excitability and long-term depression (LTD), exerting its effects partly through modulation GABAergic disinhibitory circuits. Yet, the role of endogenous VIP and its receptors in modulation of hippocampal LTP and the involvement of disinhibition in this modulation have scarcely been investigated. We studied the modulation of CA1 LTP induced by TBS via endogenous VIP release in hippocampal slices from young-adult Wistar rats using selective VPAC1 and VPAC2 receptor antagonists, evaluating its consequence for the phosphorylation of CamKII, GluA1 AMPA receptor subunits and Kv4.2 potassium channels in total hippocampal membranes obtained from TBS stimulated slices. Endogenous VIP, acting on VPAC1 (but not VPAC2) receptors, inhibited CA1 hippocampal LTP induced by TBS in young adult Wistar rats and this effect was dependent on GABAergic transmission and relied on the integrity of NMDA and CaMKII-dependent LTP expression mechanisms but not on PKA and PKC activity. Furthermore, it regulated the autophosphorylation of CaMKII and the expression and Ser438 phosphorylation of Kv4.2 potassium channels responsible for the A-current while inhibiting phosphorylation of Kv4.2 on Thr607. Altogether, this suggests that endogenous VIP controls the expression of hippocampal CA1 LTP by regulating disinhibition through activation of VPAC1 receptors in interneurons. This may impact the autophosphorylation of CaMKII during LTP, as well as the expression and phosphorylation of Kv4.2 K+ channels at hippocampal pyramidal cell dendrites.

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.

Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα q activation and ß-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected ß-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP's poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson's disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.

Signal Transduction by VIP and PACAP Receptors.

Homeostasis of the human immune system is regulated by many cellular components, including two neuropeptides, VIP and PACAP, primary stimuli for three class B G protein-coupled receptors, VPAC1, VPAC2, and PAC1. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) regulate intestinal motility and secretion and influence the functioning of the endocrine and immune systems. Inhibition of VIP and PACAP receptors is an emerging concept for new pharmacotherapies for chronic inflammation and cancer, while activation of their receptors provides neuroprotection. A small number of known active compounds for these receptors still impose limitations on their use in therapeutics. Recent cryo-EM structures of VPAC1 and PAC1 receptors in their agonist-bound active state have provided insights regarding their mechanism of activation. Here, we describe major molecular switches of VPAC1, VPAC2, and PAC1 that may act as triggers for receptor activation and compare them with similar non-covalent interactions changing upon activation that were observed for other GPCRs. Interhelical interactions in VIP and PACAP receptors that are important for agonist binding and/or activation provide a molecular basis for the design of novel selective drugs demonstrating anti-inflammatory, anti-cancer, and neuroprotective effects. The impact of genetic variants of VIP, PACAP, and their receptors on signalling mediated by endogenous agonists is also described. This sequence diversity resulting from gene splicing has a significant impact on agonist selectivity and potency as well as on the signalling properties of VIP and PACAP receptors.

Role of VPAC1 and VPAC2 receptors in the etiology of pregnancy rhinitis: an experimental study in rats.

INTRODUCTION: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. OBJECTIVES: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. METHODS: Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2°C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400mg/kg Na-pentobarbitone. Then, 10-15mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. RESULTS: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p=0.023) and VPAC2 (p=0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. CONCLUSIONS: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.

Generation of KS-133 as a Novel Bicyclic Peptide with a Potent and Selective VIPR2 Antagonist Activity that Counteracts Cognitive Decline in a Mouse Model of Psychiatric Disorders.

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.