Early Alterations of PACAP and VIP Expression in the Female Rat Brain Following Spinal Cord Injury.

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.

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.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice.

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.

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.

Vasoactive intestinal peptide receptor 2 signaling promotes breast cancer cell proliferation by enhancing the ERK pathway.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a class B G protein-coupled receptor with the neuropeptide VIP as a ligand. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been documented in several cancers including breast carcinoma. However, the pathophysiological role of increased VIPR2 in the proliferation of breast cancer cells remains largely unknown. In this study, we found that VIPR2 overexpression in MCF-7 and MDA-MB-231 cells, human breast cancer cell lines, promoted cell proliferation. Increased VIPR2 also exacerbated intraperitoneal proliferation of breast cancer MDA-MB-231 cells in a tumor nude mouse model in vivo. Treatment with KS-133, a VIPR2-selective antagonist peptide, significantly inhibited VIP-induced cell proliferation in VIPR2-overexpressing MCF-7 and MDA-MB-231 cells. Overexpressed VIPR2 caused increases in the levels of cAMP and phosphorylated extracellular signal-regulated kinase (ERK), which involves a VIPR2 signaling pathway through Gs protein. Additionally, phosphorylation of vasodilator-stimulated phosphoprotein (Ser157) and cAMP response element binding protein (Ser133) in VIPR2-overexpressing MCF-7 cells was greater than that in control cells, suggesting the increased PKA activity. Moreover, an inhibitor of mitogen-activated protein kinase kinase, U0126, attenuated tumor proliferation in exogenous VIPR2-expressing MCF-7 and MDA-MB-231 cells at the same level as observed in EGFP-expressing cells treated with U0126. Together, these findings suggest that VIPR2 controls breast tumor growth by regulating the cAMP/PKA/ERK signaling pathway, and the excessive expression of VIPR2 may lead to an exacerbation of breast carcinoma.

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.

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.

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.

Genetic labeling of axo-axonic cells in the basolateral amygdala.

GABAergic neurons are classified into multiple subtypes based on morphology, physiological properties, and gene expression profiles. Although traditionally defined axo-axonic cells (AACs) are a unique type of interneuron that expresses parvalbumin and innervates the axon initial segment (AIS) of pyramidal neurons, a genetic marker for AACs in the basolateral amygdala (BLA) has not been identified. Here, we show that vasoactive intestinal peptide receptor 2 (Vipr2)-expressing interneurons exhibit anatomical and electrophysiological properties of AACs in the BLA. Using a reporter mouse expressing fluorescent proteins specifically in Vipr2+ cells, we analyzed the distribution, postsynaptic targeting and electrophysical properties of Vipr2+ cells in the BLA. More than half of the Vipr2+ cells showed parvalbumin immunoreactivity and innervated the AIS of pyramidal neurons in the BLA of Vipr2-tdTomato mice. Notably, most of the Vipr2+ cells showed fast-spiking properties. Furthermore, the use of a Cre-dependent adeno-associated virus led to more selective labeling of AACs in the BLA. These results suggest that AACs are genetically identifiable in the BLA without anatomical or physiological analysis.

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide.

Naїve CD4+ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4+CD45RA+ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFß mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC2 receptor increased significantly with respect to the VPAC1 receptor from day 4 of CD4+CD45RA+ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4+CD45RA+ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.

Dysfunction of VIPR2 leads to myopia in humans and mice.

BACKGROUND: Myopia is the leading cause of refractive errors. As its pathogenesis is poorly understood, we determined if the retinal VIP-VIPR2 signalling pathway axis has a role in controlling signalling output that affects myopia development in mice. METHODS: Association analysis meta-study, single-cell transcriptome, bulk RNA sequencing, pharmacological manipulation and VIPR2 gene knockout studies were used to clarify if changes in the VIP-VIPR2 signalling pathway affect refractive development in mice. RESULTS: The SNP rs6979985 of the VIPR2 gene was associated with high myopia in a Chinese Han cohort (randomceffect model: p=0.013). After either 1 or 2 days' form deprivation (FD) retinal VIP mRNA expression was downregulated. Retinal single-cell transcriptome sequencing showed that VIPR2 was expressed mainly by bipolar cells. Furthermore, the cAMP signalling pathway axis was inhibited in some VIPR2+ clusters after 2 days of FD. The selective VIPR2 antagonist PG99-465 induced relative myopia, whereas the selective VIPR2 agonist Ro25-1553 inhibited this response. In Vipr2 knockout (Vipr2-KO) mice, refraction was significantly shifted towards myopia (p<0.05). The amplitudes of the bipolar cell derived b-waves in 7-week-old Vipr2-KO mice were significantly larger than those in their WT littermates (p<0.05). CONCLUSIONS: Loss of VIPR2 function likely compromises bipolar cell function based on presumed changes in signal transduction due to altered signature electrical wave activity output in these mice. As these effects correspond with increases in form deprivation myopia (FDM), the VIP-VIPR2 signalling pathway axis is a viable novel target to control the development of this condition.

Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of 'tyrosine toggle switch' required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist - cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

The Circadian Clock Is Sustained in the Thyroid Gland of VIP Receptor 2 Deficient Mice.

VIP/VPAC2-receptor signaling is crucial for functioning of the circadian clock in the suprachiasmatic nucleus (SCN) since the lack results in disrupted synchrony between SCN cells and altered locomotor activity, body temperature, hormone secretion and heart rhythm. Endocrine glands, including the thyroid, show daily oscillations in clock gene expression and hormone secretion, and SCN projections target neurosecretory hypothalamic thyroid-stimulating hormone (TSH)-releasing hormone cells. The aim of the study was to gain knowledge of mechanisms important for regulation of the thyroid clock by evaluating the impact of VIP/VPAC2-receptor signaling. Quantifications of mRNAs of three clock genes (Per1, Per2 and Bmal1) in thyroids of wild type (WT) and VPAC2-receptor deficient mice were done by qPCR. Tissues were taken every 4th h during 24-h 12:12 light-dark (LD) and constant darkness (DD) periods, both genders were used. PER1 immunoreactivity was visualized on sections of both WT and VPAC2 lacking mice during a LD cycle. Finally, TSH and the thyroid hormone T4 levels were measured in the sera by commercial ELISAs. During LD, rhythmic expression of all three mRNA was found in both the WT and knockout animals. In VPAC2-receptor knockout animals, the amplitudes were approximately halved compared to the ones in the WT mice. In the WT, Per1 mRNA peaked around "sunset", Per2 mRNA followed with approximately 2 h, while Bmal1 mRNA was in antiphase with Per1. In the VPAC2 knockout mice, the phases of the mRNAs were advanced approximately 5 h compared to the WT. During DD, the phases of all the mRNAs were identical to the ones found during LD in both groups of mice. PER1 immunoreactivity was delayed compared to its mRNA and peaked during the night in follicular cells of both the thyroid and parathyroid glands in the WT animals. In WT animals, TSH was high around the transition to darkness compared to light-on, while T4 did not change during the 24 h cycle. In conclusion, sustained and identical rhythms (phases and amplitudes) of three clock genes were found in VPAC2 deficient mice during LD and DD suggesting high degree of independence of the thyroid clock from the master SCN clock.

Modified BuShenYiQi formula alleviates experimental allergic asthma in mice by negative regulation of type 2 innate lymphoid cells and CD4+ type 9 helper T cells and the VIP-VPAC2 signalling pathway.

CONTEXT: Modified BuShenYiQi formula (M-BYF) is derived from BuShenYiQi formula, used for the treatment of allergic asthma. The exact effect and mechanism of M-BYF on the improvement of asthma remain unclear. OBJECTIVE: We investigated the mechanism underlying the therapeutic effect of M-BYF on allergic asthma. MATERIALS AND METHODS: The asthma model was established in female BALB/c mice that were sensitized and challenged with ovalbumin (OVA). Mice in the treated groups were orally treated once a day with M-BYF (7, 14 and 28 g/kg/d) or dexamethasone before OVA challenge. Control and Model group received saline. Pathophysiological abnormalities and percentages of lung type 2 innate lymphoid cells (ILC2s) and Th9 cells were measured. Expression levels of type 2 cytokines and transcription factors required for these cells function and differentiation were analysed. Expression of vasoactive intestinal polypeptide (VIP)-VPAC2 signalling pathway-related proteins, and percentages of VIP expressing (VIP+) cells and VPAC2, CD90 co-expressing (VPAC2+CD90+) cells were detected. RESULTS: M-BYF alleviated airway hyperresponsiveness, inflammation, mucus hypersecretion and collagen deposition in asthmatic mice. M-BYF down-regulated percentages of ILC2s and Th9 cells with lower expression of GATA3, PU.1 and IRF4, reduced IL-5, IL-13, IL-9 and VIP production. The decrease in the expression of VIP-VPAC2 signalling pathway and percentages of VIP+ cells, VPAC2+CD90+ cells were observed after M-BYF treatment. The LD50 value of M-BYF was higher than 90 g/kg. DISCUSSION AND CONCLUSIONS: M-BYF alleviated experimental asthma by negatively regulating ILC2s and Th9 cells and the VIP-VPAC2 signalling pathway. These findings provide the theoretical basis for future research of M-BYF in asthma patient population.

Altered interaction between enteric glial cells and mast cells in the colon of women with irritable bowel syndrome.

BACKGROUND: Enteric glial cells (EGC) and mast cells (MC) are intimately associated with gastrointestinal physiological functions. We aimed to investigate EGC-MC interaction in irritable bowel syndrome (IBS), a gut-brain disorder linked to increased intestinal permeability, and MC. METHODS: Parallel approaches were used to quantify EGC markers in colonic biopsies from healthy controls (HC) and patients with IBS. Data were correlated with MC, vasoactive intestinal polypeptide (VIP) and VIP receptors (VPAC1/VPAC2) expressions, and bacterial translocation through biopsies mounted in Ussing chambers. In addition, we investigated the effects of EGC mediators on colonic permeability and the pharmacological-induced responses of EGC and MC cell lines. KEY RESULTS: Immunofluorescence of IBS colonic mucosa, as well as Western blotting and ELISA of IBS biopsy lysates, revealed increased glial fibrillary intermediate filament (GFAP) expression, indicating EGC activation. Mucosal GFAP correlated with increased MC and VPAC1+ MC numbers and decreased VIP+ MC, which seemed to control bacterial translocation in HC. In the contrary, EGC activation in IBS correlated with less MC and VPAC1+ MC numbers, and more VIP+ MC. In vitro, MC and EGC cell lines showed intracellular calcium responses to each other's mediators. Furthermore, EGC mediators prevented VIP-induced MC degranulation, while MC mediators induced a reactive EGC phenotype. In Ussing chambers, EGC mediators decreased paracellular passage through healthy colonic biopsies. CONCLUSIONS & INFERENCES: Findings suggest the involvement of EGC and MC in the control of barrier function in the human colon and indicate a potential EGC-MC interaction that seems altered in IBS, with detrimental consequences to colonic permeability. Altogether, results suggest that imbalanced EGC-MC communication contributes to the pathophysiology of IBS.

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.

Alterations in the relative abundance of the vasoactive intestinal peptide receptors (VPAC1 and VPAC2) and functions in uterine contractility during inflammation.

Vasoactive intestinal peptide (VIP) receptor (VPAC1, VPAC2) abundances in the myometrium and functions in the regulation of inflamed uterine contractility in pigs were studied. In the CON group with gilts, only laparotomy was performed. The gilts of SAL- and E. coli-treated groups were administered saline or E. coli into the uterine horns, respectively. The E. coli-induced endometritis resulted in a lesser myometrial relative abundance of VPAC1 and VPAC2 receptor mRNA transcripts and larger abundance of protein for these receptors. In the myometrium, treatment with VIP resulted in a lesser contractility amplitude than in the tissues of the CON- and SAL- and E. coli-treated groups and in frequency in the CON- and E.coli-treated group compared to the period before VIP treatment. Compared to when there was VIP treatment alone, treatment with VPAC1 and VPAC2 receptor antagonists resulted in a lesser inhibitory effect of VIP on contractility amplitude in the myometrium of the CON and SAL-treated groups and there was complete abolishment of the inhibitory VIP effect on frequency of myometrial contractility of the CON group. In the myometrium of E. coli-treated group, treatment with VPAC1 and VPAC2 receptor antagonists resulted in a reversal of the inhibitory effect of VIP on contractility amplitude, while treatment with VPAC2 receptor antagonist resulted in elimination of contractility and a lesser endometrium/myometrium inhibitory effect of VIP on frequency of these contractions. Results indicate VIP functions to decrease myometrial contractility of the inflamed pig uterus by having functions at VPAC1 and VPAC2 receptors.

Impaired cardiorespiratory responses to hypercapnia in neonatal mice lacking PAC1 but not VPAC2 receptors.

The evidence is mounting for a role for abnormal signaling of the stress peptide pituitary adenylate cyclase activating polypeptide (PACAP) and its canonical receptor PAC1 in the pathogenesis of sudden infant death syndrome. In this study, we investigated whether the PACAP receptors PAC1 or VPAC2 are involved in the neonatal cardiorespiratory response to hypercapnic stress. We used head-out plethysmography and surface ECG electrodes to assess cardiorespiratory responses to an 8% hypercapnic challenge in unanesthetized and spontaneously breathing 4-day-old PAC1 or VPAC2 knockout (KO) and wild-type mouse pups. We demonstrate that compared with WTs, breathing frequency (RR) and minute ventilation ([Formula: see text]) in PAC1 KO pups were significantly blunted in response to hypercapnia. Although heart rate was unaltered in PAC1 KO pups during hypercapnia, heart rate recovery posthypercapnia was impaired. In contrast, cardiorespiratory impairments in VPAC2 KO pups were limited to only an overall higher tidal volume (VT), independent of treatment. These findings suggest that PACAP signaling through the PAC1 receptor plays a more important role than signaling through the VPAC2 receptor in neonatal respiratory responses to hypercapnia. Thus deficits in PACAP signaling primarily via PAC1 may contribute to the inability of infants to mount an appropriate protective response to homeostatic stressors in childhood disorders such as SIDS.

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.