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.

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.

Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke.

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown. Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model. Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions. Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke. Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Gonadotropin-inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1-7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T-cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c-Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.-Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

Attenuation of stress-induced gastrointestinal motility disorder by gentiopicroside, from Gentiana macrophylla Pall.

AIM: The current study was designed to explore the mechanism of the prokinetic activity of Gentiopicroside (Ge), from Gentiana macrophylla Pall which is widely used to strengthen gastric motility in clinic. METHODS: Gastrointestinal motility disorder rats were induced by stress stimulation and the rats were treated with Ge. The functions of gastric emptying and intestinal propelling were measured after blood was obtained to assay the levels of plasmatic motilin (MTL), vasoactive intestinal peptide (VIP), somatostatin (SST), gastrin (GAS), neurotensin (NT) and substance of P (SP). The expressions of MTL receptor (MTLR), VIP receptor 2 (VIPR2) and SST receptor 2 (SSTR2) were measured also. In addition, an isolated guinea pig ileum was applied to evaluate the influences of Ge on M-R, H1-R, 5-HT4-R and D-R in vitro. RESULTS: Ge increased gastric emptying and intestinal propelling obviously. It also decreased the level of SST and increased GAS in plasma significantly. Moreover, it promoted the expressions of MTLR in gastric antrum, duodenum, jejunum and ileum, and restrained the expression of VIPR2 in duodenum. Piboserod and loratadine had no obvious restrain to Ge' exciting ileum effect and Ge also didn't affect dopamine paralyzing ileum. However, Ge failed to improve the hypofunction of guinea pigs ileums pre-treated with atropine sulfate. CONCLUSION: The mechanisms of Ge' prokinetic effect were associated with modulating the levels of SST and GAS in plasma, raising the expressions of MTLR in gastric antrum, duodenum, ileum and jejunum, reducing the expression of VIPR2 in duodenum and activating M-R.

The selective PAC1 receptor agonist maxadilan inhibits neurogenic vasodilation and edema formation in the mouse skin.

We have earlier shown that PACAP-38 decreases neurogenic inflammation. However, there were no data on its receptorial mechanism and the involvement of its PAC1 and VPAC1/2 receptors (PAC1R, VPAC1/2R) in this inhibitory effect. Neurogenic inflammation in the mouse ear was induced by topical application of the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor activator mustard oil (MO). Consequent neurogenic edema, vasodilation and plasma leakage were assessed by measuring ear thickness with engineer's micrometer, detecting tissue perfusion by laser Doppler scanning and Evans blue or indocyanine green extravasation by intravital videomicroscopy or fluorescence imaging, respectively. Myeloperoxidase activity, an indicator of neutrophil infiltration, was measured from the ear homogenates with spectrophotometry. The selective PAC1R agonist maxadilan, the VPAC1/2R agonist vasoactive intestinal polypeptide (VIP) or the vehicle were administered i.p. 15 min before MO. Substance P (SP) concentration of the ear was assessed by radioimmunoassay. Maxadilan significantly diminished MO-induced neurogenic edema, increase of vascular permeability and vasodilation. These inhibitory effects of maxadilan may be partially due to the decreased substance P (SP) levels. In contrast, inhibitory effect of VIP on ear swelling was moderate, without any effect on MO-induced plasma leakage or SP release, however, activation of VPAC1/2R inhibited the increased microcirculation caused by the early arteriolar vasodilation. Neither the PAC1R, nor the VPAC1/2R agonist influenced the MO-evoked increase in tissue myeloperoxidase activity. These results clearly show that PAC1R activation inhibits acute neurogenic arterial vasodilation and plasma protein leakage from the venules, while VPAC1/2R stimulation is only involved in the attenuation of vasodilation.

Hypothalamic expression of anorexigenic and orexigenic hormone receptors in obese females Neotomodon alstoni: effect of fasting.

Obesity is a world problem that requires a better understanding of its physiological and genetic basis, as well as the mechanisms by which the hypothalamus controls feeding behavior. The volcano mouse Neotomodon alstoni develops obesity in captivity when fed with regular chow diet, providing a novel model for the study of obesity. Females develop obesity more often than males; therefore, in this study, we analysed in females, in proestrous lean and obese, the differences in hypothalamus expression of receptors for leptin, ghrelin (growth hormone secretagogue receptor GHS-R), and VPAC, and correlates for plasma levels of total ghrelin. The main comparisons are between mice fed ad libitum and mice after 24 hours of fasting. Mice above 65 g body weight were considered obese, based on behavioral and physiological parameters such as food intake, plasma free fatty acids, and glucose tolerance. Hypothalamic tissue from obese and lean mice was analysed by western blot. Our results indicate that after ad libitum food access, obese mice show no significant differences in hypothalamic leptin receptors, but a significant increase of 60% in the GHS-R, and a nearly 62% decrease in VPAC2 was noted. After a 24-hour fast, plasma ghrelin increased nearly two fold in both lean and obese mice; increases of hypothalamic leptin receptors and GHS-R were also noted, while VPAC2 did not change significantly; levels of plasma free fatty acids were 50% less after fasting in obese than in lean animals. Our results indicate that in obese N. alstoni mice, the levels of orexigenic receptors in the hypothalamus correlate with overfeeding, and the fact that lean and obese females respond in different ways to a metabolic demand such as a 24-hour fast.

Effect of VIP on intracellular [Ca2+], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.

PURPOSE: To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. RESULTS: Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. CONCLUSIONS: In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

[Effects of Jiaweisinisan on gastric mucosal ultrastructure and brain-gut axis in a rat model of chronic psychological stress].

OBJECTIVE: To study the effect of Jiaweisinisan (JWSNS), a traditional Chinese herbal medicinal recipe, on gastric mucosal ultrastructure and brain-gut axis in rat models of chronic psychological stress and elucidate the mechanism of JWSNS for ameliorating stress-induced gastrointestinal dysfunction. METHODS: Sixty rats were randomly assigned into normal control group, model group, 3 JWSNS groups (high, moderate, and small doses), and omeprazole group (n=10). Rat models of chronic psychological stress were established by random stressful stimulations, and following the corresponding interventions, plasma adrenocorticotropic hormone (ACTH) and cortisol (CORT) levels were detected using radioimmunoassay, and the mRNA expressions of gastrin receptor in the gastric tissue (GASR) and vasoactive intestinal peptide II receptor (VIPR2) in the jejunal tissue were examined using RT-PCR. Transmission electron microscopy was employed to examine the ultrastructural changes in the gastric mucosa tissue cells of the glandular stomach area and alterations in the intercellular junctions. RESULTS: Electron microscopy revealed obvious damages in gastric mucosal epithelial cell organelles and nuclei in the model rats. These damages were ameliorated after treatments with JWSNS and omeprazole. Compared with the model group, the 3 JWSNS groups and omeprazole group all showed significantly lowered plasma ACTH and CORT levels, increased gastrin receptor mRNA expression and decreased jejunal VIPR2 mRNA expression (P<0.05 or 0.01). CONCLUSION: JWSNS can obviously ameliorate the pathologies of the gastric mucosa cells, regulate the state of brain-gut axis, and modulate the gastric gastrin receptor and jejunal VIPR2 mRNA expressions in rats with chronic psychological stress.

[Effects of Jiaweisinisan dispersion on content and gene expression of gastric tissue GASR and jejunal tissue VIPR2 of physical and mental stress model rat].

OBJECTIVE: To observe the effects of Jiaweisini dispersion (JWSNS) on the ultrastructure of gastric mucosa, the content and gene expression of gastric antrum tissue gastrin receptor (GASR) and jejunal tissue vasoactive intestinal peptide receptor 2 (VIPR2) in chronic stress gastric ulcer rats, and to elucidate its mechanism. METHODS: 60 Wistar rats were randomly divided into normal group, model group, JWSNS large, medium, small dose groups, and omeprazole group, 10 rats in each group. Chronic stress method was used to establish the stress ulcer rat model. The every rat in JWSNS small, medium, large dose groups were gavaged with 0.25, 0.5, 1.0 g/ mL Chinese medicine Decoction on 2 mL respectively daily, rats in omeprazole group were gavaged with 0.3 mg/mL omeprazole solution on 2 mL daily, rats in normal group and model group were gavaged 2 mL NS daily. After modeling was end, transmission electron microscopy (TEM) was used to observe gastric mucosa cells and intercellular connections changes of ultrastructure of glandular stomach area and immunohistochemical method and Real time-PCR method were used to detect the protein content and gene expression changes of gastric antrum tissue GASR and jejunal tissue cell VIPR2. RESULTS: TEM observation demonstrated that in the normal group the gastric mucosa epithelial cells connected compact, cell membrane integrity, cell nuclear shape and size was normal; in model group rats the gastric mucosal cells were severely damaged; the rats in the rest treatment groups were better than those in the model group in different degree. After The treatment of JWSNS and omeprazole, the expression of GASR protein and mRNA in gastric antrum tissue were increased when compared with that of model group (P < 0.05), the expression of VIPR2 protein and mRNA in the jejunum tissue were lower than that of the model group (P < 0.05). The expression of GASR, VIPR2 protein and mRNA in the JWSNS large dose group was closed to the normal group with no significant difference (P > 0.05). And compared with omeprazole group and JWSNS small dose group, expression of GASR protein and mRNA in high dose group rats were increased (P < 0.05), and expression of VIPR2 protein and mRNA were decreased (P < 0.05). CONCLUSION: JWSNS can significantly improve microscopic pathologic morphology of the gastric mucosa cell in gastric ulcer of chronic stress rats models, and can through two aspects of inhibiting damage factor and enhancing defense factor to adjust the content and gene expression of gastric tissue GASR and jejunal tissue VIPR2.