Mismatch novelty exploration training shifts VPAC1 receptor-mediated modulation of hippocampal synaptic plasticity by endogenous VIP in male rats.

Novelty influences hippocampal-dependent memory through metaplasticity. Mismatch novelty detection activates the human hippocampal CA1 area and enhances rat hippocampal-dependent learning and exploration. Remarkably, mismatch novelty training (NT) also enhances rodent hippocampal synaptic plasticity while inhibition of VIP interneurons promotes rodent exploration. Since VIP, acting on VPAC1 receptors (Rs), restrains hippocampal LTP and depotentiation by modulating disinhibition, we now investigated the impact of NT on VPAC1 modulation of hippocampal synaptic plasticity in male Wistar rats. NT enhanced both CA1 hippocampal LTP and depotentiation unlike exploring an empty holeboard (HT) or a fixed configuration of objects (FT). Blocking VIP VPAC1Rs with PG 97269 (100 nM) enhanced both LTP and depotentiation in naïve animals, but this effect was less effective in NT rats. Altered endogenous VIP modulation of LTP was absent in animals exposed to the empty environment (HT). HT and FT animals showed mildly enhanced synaptic VPAC1R levels, but neither VIP nor VPAC1R levels were altered in NT animals. Conversely, NT enhanced the GluA1/GluA2 AMPAR ratio and gephyrin synaptic content but not PSD-95 excitatory synaptic marker. In conclusion, NT influences hippocampal synaptic plasticity by reshaping brain circuits modulating disinhibition and its control by VIP-expressing hippocampal interneurons while upregulation of VIP VPAC1Rs is associated with the maintenance of VIP control of LTP in FT and HT animals. This suggests VIP receptor ligands may be relevant to co-adjuvate cognitive recovery therapies in aging or epilepsy, where LTP/LTD imbalance occurs.

Impact Assessment of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Hemostatic Sponge on Vascular Anastomosis Regeneration in Rats.

The proper regeneration of vessel anastomoses in microvascular surgery is crucial for surgical safety. Pituitary adenylate cyclase-activating polypeptide (PACAP) can aid healing by decreasing inflammation, apoptosis and oxidative stress. In addition to hematological and hemorheological tests, we examined the biomechanical and histological features of vascular anastomoses with or without PACAP addition and/or using a hemostatic sponge (HS). End-to-end anastomoses were established on the right femoral arteries of rats. On the 21st postoperative day, femoral arteries were surgically removed for evaluation of tensile strength and for histological and molecular biological examination. Effects of PACAP were also investigated in tissue culture in vitro to avoid the effects of PACAP degrading enzymes. Surgical trauma and PACAP absorption altered laboratory parameters; most notably, the erythrocyte deformability decreased. Arterial wall thickness showed a reduction in the presence of HS, which was compensated by PACAP in both the tunica media and adventitia in vivo. The administration of PACAP elevated these parameters in vitro. In conclusion, the application of the neuropeptide augmented elastin expression while HS reduced it, but no significant alterations were detected in collagen type I expression. Elasticity and tensile strength increased in the PACAP group, while it decreased in the HS decreased. Their combined use was beneficial for vascular regeneration.

lncRNA-AC079061.1/VIPR1 axis may suppress the development of hepatocellular carcinoma: a bioinformatics analysis and experimental validation.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant tumors to threaten human life, and the survival rate remains low due to delayed diagnosis. Meanwhile, lncRNAs have great potential for application in tumor prognosis, therefore relevant research in hepatocellular carcinoma is indispensable. METHODS: Based on the EZH2 expression, the differentially expressed lncRNAs DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) were identified in hepatocellular carcinoma by using the TCGA database. Bioinformatics technology was utilized to determine the effect of key genes in HCC progression. The methylation and immune infiltration analyses were performed to explore the underlying function of hub genes. Finally, cellular function experiments were performed to investigate the association between identified genes and biological phenotypes in HCC. RESULTS: lncRNA-AC079061.1, hsa-miR-765, and VIPR1 were identified as independent factors that affect the prognosis of hepatocellular carcinoma. The immune infiltration analyses revealed that lncRNA-AC079061.1 can alter the immune microenvironment and thus inhibit the development of HCC by regulating the expression of an immune-related gene (VIPR1). Methylation analyses demonstrated that VIPR1 expression is negatively related to the methylation level in HCC. Experimental results suggested that lncRNA-AC079061.1 and VIPR1 were frequently downregulated in HCC cells, while hsa-miR-765 was significantly upregulated. Moreover, the lncRNA-AC079061.1/VIPR1 axis suppressed the proliferation and invasion of HCC cells. CONCLUSION: The present study identified the lncRNA-AC079061.1/VIPR1 axis as a novel biomarker that inhibited the proliferation and invasion of hepatocellular carcinoma, affecting the ultimate disease outcome.

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.

VIP/VPAC Axis Expression in Immune-Mediated Inflammatory Disorders: Associated miRNA Signatures.

Few studies have considered immune-mediated inflammatory disorders (IMID) together, which is necessary to adequately understand them given they share common mechanisms. Our goal was to investigate the expression of vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 in selected IMID, analyze the effect of biological therapies on them, and identify miRNA signatures associated with their expression. Serum VIP levels and mRNA of VPAC and miRNA expression in peripheral blood mononuclear cells were analyzed from 52 patients with psoriasis, rheumatoid arthritis, Graves' disease, or spondyloarthritis and from 38 healthy subjects. IMID patients showed higher levels of VIP and increased expression of VPAC2 compared to controls (p < 0.0001 and p < 0.0192, respectively). Receiver operating characteristic curve analysis showed that the levels of VIP or VPAC2 expression were adequate discriminators capable of identifying IMID. Treatment of IMID patients with anti-TNFα and anti-IL12/23 significantly affected serum VIP levels. We identified miRNA signatures associated with levels of serum VIP and VPAC2 expression, which correlated with IMID diagnosis of the patients. The results indicate that the expression of VIP/VPAC2 is able of identify IMIDs and open up a line of research based on the association between the VIP/VPAC axis and miRNA signatures in immune-mediated diseases.

Inhibition of PACAP/PAC1/VPAC2 signaling impairs the consolidation of social recognition memory and nitric oxide prevents this deficit.

Social recognition memory (SRM) forms the basis of social relationships of animals. It is essential for social interaction and adaptive behavior, reproduction and species survival. Evidence demonstrates that social deficits of psychiatric disorders such as autism and schizophrenia are caused by alterations in SRM processing by the hippocampus and amygdala. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptors PAC1, VPAC1 and VPAC2 are highly expressed in these regions. PACAP is a pleiotropic neuropeptide that modulates synaptic function and plasticity and is thought to be involved in social behavior. PACAP signaling also stimulates the nitric oxide (NO) production and targets outcomes to synapses. In the present work, we investigate the effect of the infusion of PACAP-38 (endogenous neuropeptide and potent stimulator of adenylyl cyclase), PACAP 6-38 (PAC1/VPAC2 receptors antagonist) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) in the CA1 region of the hippocampus and in the basolateral amygdala (BLA) on the consolidation of SRM. For this, male Wistar rats with cannulae implanted in CA1 or in BLA were subjected to a social discrimination paradigm, which is based on the natural ability of rodents to investigate unfamiliar conspecifics more than familiar one. In the sample phase (acquisition), animals were exposed to a juvenile conspecific for 1 h. Immediately, 60 or 150 min after, animals received one of different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. Animals that received infusions of PACAP 6-38 (40 pg/side) into CA1 immediately after the sample phase or into BLA immediately or 60 min after the sample phase were unable to recognize the familiar juvenile during the retention test. This impairment was abolished by the coinfusion of PACAP 6-38 plus SNAP (5 µg/side). These results show that the blockade of PACAP/PAC1/VPAC2 signaling in the CA1 and BLA during a restricted post-acquisition time window impairs the consolidation of SRM and that the SNAP is able to abolish this deficit. Findings like this could potentially be used in the future to influence studies of psychiatric disorders involving social behavior.

The regulatory role of vasoactive intestinal peptide in lacrimal gland ductal fluid secretion: A new piece of the puzzle in tear production.

Purpose: Vasoactive intestinal peptide (VIP) is an important regulator of lacrimal gland (LG) function although the effect of VIP on ductal fluid secretion is unknown. Therefore, the aim of the present study was to investigate the role of VIP in the regulation of fluid secretion of isolated LG ducts and to analyze the underlying intracellular mechanisms. Methods: LGs from wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) knockout (KO) mice were used. Immunofluorescence was applied to confirm the presence of VIP receptors termed VPAC1 and VPAC2 in LG duct cells. Ductal fluid secretion evoked by VIP (100 nM) was measured in isolated ducts using videomicroscopy. Intracellular Ca2+ signaling underlying VIP stimulation was investigated with microfluorometry. Results: VIP stimulation resulted in a robust and continuous fluid secretory response in isolated duct segments originated from WT mice. In contrast, CFTR KO ducts exhibited only a weak pulse-like secretion. A small but statistically significant increase was detected in the intracellular Ca2+ level [Ca2+]i during VIP stimulation in the WT and in CFTR KO ducts. VIP-evoked changes in [Ca2+]i did not differ considerably between the WT and CFTR KO ducts. Conclusions: These results suggest the importance of VIP in the regulation of ductal fluid secretion and the determining role of the adenylyl cyclase-cAMP-CFTR route in this process.

Immunomodulation by vasoactive intestinal peptide is associated with increased survival and growth of Salmonella Typhimurium in mice.

Studies have shown that administration of vasoactive intestinal peptide (VIP) in mice rescues them from lethal endotoxaemia and that this is correlated with decreased concentration of inflammatory cytokines. VIP has, therefore, been proposed as a novel anti-inflammatory which could be used in the treatment of Gram negative sepsis. However, the effect of VIP has not been reported in mice infected with viable Gram negative bacteria. Here, we show that Salmonella enterica serovar Typhimurium 4/74 significantly increased expression of mRNA of a type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in murine ileum and mesenteric lymph nodes at day 6 post-infection (d6 pi) and in the spleen at d3 pi. When VIP (5 nmol/ml) was administered to S. Typhimurium-infected mice, there was a significant increase in the number of S. Typhimurium cultured from murine faeces and ileum at d3 and 6 pi and in MLN and spleen at d3 dpi, compared to faeces and tissues examined from mice infected with S. Typhimurium (without VIP administration). Administration of VIP to S. Typhimurium-infected mice also altered the splenic architecture, resulting in a lack of discernable periarterial lymphoid sheaths or marginal zones at d6 pi but liver histology appeared similar on both d3 and d6 pi. The effects of VIP administration were correlated with a significant decrease in expression of inflammatory cytokine mRNA, associated with systemic inflammatory response syndrome (SIRS) of bacteraemia and acute sepsis. We conclude that VIP inhibits expression of diagnostic/prognostic cytokine biomarkers of sepsis in S. Typhimurium-infected mice. However, this occurred with a concomitant increase in Salmonella growth in tissues and increased bacterial shedding in faeces. Thus, VIP may have potential as an adjunctive therapy to antibiotics in sepsis.

Vasoactive intestinal peptide increases apoptosis of hepatocellular carcinoma by inhibiting the cAMP/Bcl-xL pathway.

Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10  M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.

Xenin-25 induces anion secretion by activating noncholinergic secretomotor neurons in the rat ileum.

Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl--free and HCO3- -free solutions. The responses were almost completely blocked by TTX (10-6 M) but not by atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT3 and 5-HT4), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors (Vipr) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl-/ HCO3- secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl-/ HCO3- secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25. NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl-/ HCO3- secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.

3D structure prediction of VAPC1 and identification of dual natural inhibitors for VPAC1 and EGFR.

Vasoactive intestinal polypeptide receptor 1 (VPAC1) and epidermal growth factor receptor (EGFR) are associated with signal transduction pathways relevant to neuroblastoma, cancer of breast, prostate and lungs. In order to identify appropriate ligand analogues for simultaneous inhibition of EGFR and VPAC1, in-silico homology modelling of VPAC1 and its characterization by molecular interaction studies have been undertaken. Homology modelling was performed with the Swiss Model and validation of the predicted 3D structure was carried out using PROCHECK and RAMPAGE. Ramachandran's plot of the predicted structure from this two software revealed that 92% and 94% of the residues were in the most favoured region, respectively. Compounds screened from Naturally Occurring Plant-based Anti-Cancerous Compound-Activity-Target (NPACT) database having strong interactions with EGFR were further checked for ADMET properties. Molecular interaction studies revealed four compounds namely Fisetin, Genistein, Tectorigenin, and Tephrosin docked with VPAC1 having respective binding energies of -7.1, -6.98, -6.9 and - 6.61 kcal/mol. Fisetin and Genistein with a rotatable bond and lower molecular weight increased their drug-likeness than the others. Therefore, simultaneous inhibition of VPAC1 and EGFR, in turn, might inhibit the progression of breast carcinoma. The results obtained were further substantiated by comparing them with positive and negative controls. Quercetin was used as positive control, and strong binding energy of -7.54 kcal/mol with EGFR is in accordance with experimental evidence. 3-O-cis-p coumaroyl alphitolic acid was used as negative control, where docking was not possible in absence of binding with either EGFR or VIPR1.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Vasoactive Intestinal Polypeptide and Mast Cells Regulate Increased Passage of Colonic Bacteria in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis and symptoms of IBS develop following gastroenteritis. We aimed to study the passage of live bacteria through the colonic epithelium, and determine the role of mast cells (MCs) and vasoactive intestinal polypeptide (VIP) in barrier regulation in IBS and healthy individuals. METHODS: Colon biopsies from 32 women with IBS and 15 age-matched healthy women (controls) were mounted in Ussing chambers; we measured numbers of fluorescently labeled Escherichia coli HS and Salmonella typhimurium that passed through from the mucosal side to the serosal side of the tissue. Some biopsies were exposed to agents that block the VIP receptors (VPAC1 and VPAC2) or MCs. Levels of VIP and tryptase were measured in plasma and biopsy lysates. Number of MCs and MCs that express VIP or VIP receptors were quantified by immunofluorescence. Biopsies from an additional 5 patients with IBS and 4 controls were mounted in chambers and Salmonella were added; we studied passage routes through the epithelium by transmission electron microscopy and expression of tight junctions by confocal microscopy. RESULTS: In colon biopsies from patients with IBS, larger numbers of E coli HS and S typhimurium passed through the epithelium than in biopsies from controls (P < .0005). In transmission electron microscopy analyses, bacteria were found to cross the epithelium via only the transcellular route. Bacterial passage was reduced in biopsies from patients with IBS and controls after addition of antibodies against VPACs or ketotifen, which inhibits MCs. Plasma samples from patients with IBS had higher levels of VIP than plasma samples from controls. Biopsies from patients with IBS had higher levels of tryptase, larger numbers of MCs, and a higher percentage of MCs that express VPAC1 than biopsies from controls. In biopsies from patients with IBS, addition of Salmonella significantly reduced levels of occludin; subsequent addition of ketotifen significantly reversed this effect. CONCLUSIONS: We found that colonic epithelium tissues from patients with IBS have increased translocation of commensal and pathogenic live bacteria compared with controls. The mechanisms of increased translocation include MCs and VIP.

VPAC1-targeted PET/CT scan: improved molecular imaging for the diagnosis of prostate cancer using a novel cell surface antigen.

PURPOSE: Current approaches to prostate cancer screening and diagnosis are plagued with limitations in diagnostic accuracy. There is a compelling need for biomolecular imaging that will not only detect prostate cancer early but also distinguish prostate cancer from benign lesions accurately. In this topic paper, we review evidence that supports further investigation of VPAC1-targeted PET/CT imaging in the primary diagnosis of prostate cancer. METHODS: A non-systematic review of Medline/PubMed was performed. English language guidelines on prostate cancer diagnosis and management, original articles, and review articles were selected based on their clinical relevance. RESULTS: VPAC1 receptors were overexpressed 1000 times more in prostate cancer than benign prostatic stromal tissue. In vitro and in vivo studies showed that Copper-64 labeled analogs of VPAC1 ligands can be synthesized with high radiochemical efficiency and purity. The radioactive probes had excellent VPAC1 receptor binding specificity and affinity. They had good biochemical stability in vitro and in mouse and human serum. They had minimal urinary excretion, which made them favorable for prostate cancer imaging. Initial feasibility study in men with prostate cancer showed that the probes were safe with no reported adverse reaction. 64Cu-TP3805 PET/CT detected 98% of prostate cancer lesions and nodal metastasis as confirmed with whole mount histopathological evaluation. CONCLUSIONS: VPAC1 receptors are promising targets for biomolecular imaging of primary prostate cancer that can distinguish malignant from benign lesions non-invasively. Further investigations are warranted to validate initial findings and define the clinical utilities of VPAC1-targeted PET imaging for prostate cancer diagnosis and management.

Identification of Dysregulated microRNA Networks in Schwann Cell-Like Cultures Exposed to Immune Challenge: Potential Crosstalk with the Protective VIP/PACAP Neuropeptide System.

Following peripheral nerve injury, dysregulations of certain non-coding microRNAs (miRNAs) occur in Schwann cells. Whether these alterations are the result of local inflammation and/or correlate with perturbations in the expression profile of the protective vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) system is currently unknown. To address these issues, we aimed at profiling the expression of selected miRNAs in the rat RT4 Schwann cell line. Cells exposed to lipopolysaccharide (LPS), to mimic the local inflammatory milieu, were appraised by real-time qPCR, Western blot and ELISAs. We found that upon LPS treatment, levels of pro-inflammatory cytokines (IL-1ß, -6, -18, -17A, MCP-1 and TNFα) increased in a time-dependent manner. Unexpectedly, the expression levels of VIP and PACAP were also increased. Conversely, levels of VPAC1 and VPAC2 receptors were reduced. Downregulated miRNAs included miR-181b, -145, -27a, -340 and -132 whereas upregulated ones were miR-21, -206, -146a, -34a, -155, -204 and -29a, respectively. Regression analyses revealed that a subset of the identified miRNAs inversely correlated with the expression of VPAC1 and VPAC2 receptors. In conclusion, these findings identified a novel subset of miRNAs that are dysregulated by immune challenge whose activities might elicit a regulatory function on the VIP/PACAP system.

PACAP and its role in primary headaches.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.

PACAP and its receptors in cranial arteries and mast cells.

BACKGROUND: In migraineurs pituitary adenylate cyclase activating peptide1-38 (PACAP1-38) is a potent migraine provoking substance and the accompanying long lasting flushing suggests degranulation of mast cells. Infusion of the closely related vasoactive intestinal peptide (VIP) either induces headache or flushing. This implicates the pituitary adenylate cyclase activating peptide type I receptor (PAC1) to be involved in the pathophysiology of PACAP1-38 provoked headaches. Here we review studies characterizing the effects of mainly PACAP but also of VIP on cerebral and meningeal arteries and mast cells. DISCUSSION: PACAP1-38, PACAP1-27 and VIP dilate cerebral and meningeal arteries from several species including man. In rat cerebral and meningeal arteries the dilation seems to be mediated preferably via vasoactive intestinal peptide receptor type 1 (VPAC1) receptors while, in human, middle meningeal artery dilation induced via vasoactive intestinal peptide receptor type 2 (VPAC2) receptors cannot be ruled out. PACAP1-38 is a strong degranulator of peritoneal and dural mast cells while PACAP1-27 and VIP only have weak effects. More detailed characterization studies suggest that mast cell degranulation is not mediated via the known receptors for PACAP1-38 but rather via a still unknown receptor coupled to phospholipase C. CONCLUSION: It is suggested that PACAP1-38 might induce migraine via degranulation of dural mast cells via a yet unknown receptor.

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not in the trigeminal nucleus caudalis, and no significant differences were found in the expression of the VPAC1 and VPAC2 receptors. Conclusions This study demonstrated the chronic alteration of pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in the rat, which suggests the crucial involvement of pituitary adenylate cyclase-activating polypeptide in the development of migraine. The selective increase in pituitary adenylate cyclase-activating polypeptide-related receptors suggests that the PAC1 receptor pathway is a novel target for the treatment of migraine.

Expression and localization of VPAC1, the major receptor of vasoactive intestinal peptide along the length of the intestine.

Vasoactive intestinal peptide (VIP) is an endogenous neuropeptide with a broad array of physiological functions in many organs including the intestine. Its actions are mediated via G protein-coupled receptors, and vasoactive intestinal peptide receptor 1 (VPAC1) is the key receptor responsible for majority of VIP's biological activity. The distribution of VPAC1 along the length of the gastrointestinal tract and its subcellular localization in intestinal epithelial cells have not been fully characterized. The current studies were undertaken to determine VPAC1 distribution and localization so that VIP-based therapies can be targeted to specific regions of the intestine. The results indicated that the mRNA levels of VPAC1 showed an abundance pattern of colon > ileum > jejunum in the mouse intestine. In parallel, the VPAC1 protein levels were higher in the mouse colon, followed by the ileum and jejunum. Immunofluorescence studies in mouse colon demonstrated that the receptor was specifically localized to the luminal surface, as was evident by colocalization with the apical marker villin but not with the basolateral marker Na+/K+-ATPase. In the human intestine, VPAC1 mRNA expression exhibited a distribution similar to that in mouse intestine and was highest in the sigmoid colon. Furthermore, in the human colon, VPAC1 also showed predominantly apical localization. The physiological relevance of the expression and apical localization of VPAC1 remains elusive. We speculate that apical VPAC1 in intestinal epithelial cells may have relevance in recognizing secreted peptides in the intestinal lumen and therefore supports the feasibility of potential therapeutic and targeting use of VIP formulations via oral route to treat gastrointestinal diseases.NEW & NOTEWORTHY These studies for the first time present comprehensive data on the relative characterization of vasoactive intestinal peptide (VIP) receptors in the intestinal mucosa. Vasoactive intestinal peptide receptor 1 (VPAC1) was identified as the predominant receptor with higher levels in the colon compared with the small intestine and was mainly localized to the apical membrane. In addition, the findings in the human tissues were consistent with VPAC1 expression in the mouse intestine and open possibilities to target colonic tissues with VIP for treating diseases such as inflammatory bowel disease.

In situ proximity of CX3CR1-positive mononuclear phagocytes and VIP-ergic nerve fibers suggests VIP-ergic immunomodulation in the mouse ileum.

Being continuously exposed to a plethora of antigens ranging from food antigens to potential pathogenic organisms, the gastrointestinal (GI) tract harbors the largest collection of immune cells in the mammalian body. This immune system has to maintain a delicate balance between mounting an active immune response and maintaining tolerance. The GI tract is also home to an elaborate intrinsic nervous system, the enteric nervous system (ENS). Various in vitro studies of neuro-immune communication have suggested that vasoactive intestinal peptide (VIP), an important GI neurotransmitter, modulates mononuclear phagocytes (MNPs), i.e., dendritic cells and macrophages. Using a combined approach of reverse transcription plus the polymerase chain reaction, immunofluorescence, three-dimensional maximum intensity projections and immunoelectron microscopy, we investigate the interaction between the enteric innervation and MNPs in the ileal lamina propria (LP). We demonstrate that VIP-ergic fibers of the ENS lie adjacent to CX3CR1+ MNPs and that VPAC1 is constitutively expressed on ileal CX3CR1+ cells in the LP of the mouse. We also identify, for the first time, CX3CR1+ immune cells in the LP at the ultrastructural level. Our data thus reveal the in situ presence of the molecular components that are necessary for a VIP-mediated neuro-immune interaction between the ENS and CX3CR1-expressing immune cells in the LP of the ileum.