The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

Microglia cells treated with synthetic vasoactive intestinal peptide or transduced with LentiVIP protect neuronal cells against degeneration.

A common pathological hallmark of neurodegenerative disorders is neuronal cell death, accompanied by neuroinflammation and oxidative stress. The vasoactive intestinal peptide (VIP) is a pleiotropic peptide that combines neuroprotective and immunomodulatory actions. The gene therapy field shows long-term promise for treating a wide range of neurodegenerative diseases (ND). In this study, we aimed to investigate the in vitro efficacy of transduction of microglia using lentiviral gene therapy vectors encoding VIP (LentiVIP). Additionally, we tested the protective effects of the secretome derived from LentiVIP-infected "immortalized human" microglia HMC3 cells, and cells treated with Synthetic VIP (SynVIP), against toxin-induced neurodegeneration. First, LentiVIP, which stably expresses VIP, was generated and purified. VIP secretion in microglial conditioned media (MG CM) for LentiVIP-infected HMC3 microglia cells was confirmed. Microglia cells were activated with lipopolysaccharide, and groups were formed as follows: 1) Control, 2) SynVIP-treated, or 3) LentiVIP-transduced. These MG CM were applied on an in vitro neurodegenerative model formed by differentiated (d)-SH-SY5Y cells. Then, cell survival analysis and apoptotic nuclear staining, besides measurement of oxidative/inflammatory parameters in CM of cells were performed. Activated MG CM reduced survival rates of both control and toxin-applied (d)-SH-SY5Y cells, whereas LentiVIP-infected MG CM and SynVIP-treated ones exhibited better survival rates. These findings were supported by apoptotic nuclear evaluations of (d)-SH-SY5Y cells, alongside oxidative/inflammatory parameters in their CM. LentiVIP seems worthy of further studies for the treatment of ND because of the potential of gene therapy to treat diseases effectively with a single injection.

Estrogen-mediated coupling via gap junctions in the suprachiasmatic nucleus.

The circadian clock orchestrates many physiological and behavioural rhythms in mammals with 24-h periodicity, through a hierarchical organisation, with the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus. The circuits of the SCN generate circadian rhythms with precision, relying on intrinsic coupling mechanisms, for example, neurotransmitters like arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), neuronal gamma-aminobutyric acid (GABA) signalling and astrocytes connected by gap junctions composed of connexins (Cx). In female rodents, the presence of estrogen receptors (ERs) in the dorsal SCN suggests an influence of estrogen (E2) on the circuit timekeeping that could regulate circadian rhythm and coupling. To investigate this, we used SCN explants together with hypothalamic neurons and astrocytes. First, we showed that E2 stabilised the circadian amplitude in the SCN when rAVPs (receptor-associated vasopressin peptides) were inhibited. However, the phase delay induced by VIPAC2 (VIP receptors) inhibition remained unaffected by E2. We then showed that E2 exerted its effects in the SCN via ERß (estrogen receptor beta), resulting in increased expression of Cx36 and Cx43. Notably, specific inhibition of both connexins resulted in a significant reduction in circadian amplitude within the SCN. Remarkably, E2 restored the period with inhibited Cx36 but not with Cx43 inhibition. This implies that the network between astrocytes and neurons, responsible for coupling in the SCN, can be reinforced through E2. In conclusion, these findings provide new insights into how E2 regulates circadian rhythms ex vivo in an ERß-dependent manner, underscoring its crucial role in fortifying the SCN's rhythm.

The vasoactive effects of bradykinin, vasoactive intestinal peptide, calcitonin gene-related peptide and neuropeptide Y depend on the perivascular tissue in porcine retinal arterioles in vitro.

PURPOSE: The retina contains a number of vasoactive neuropeptides and corresponding receptors, but the role of these neuropeptides for tone regulation of retinal arterioles has not been studied in detail. METHODS: Porcine arterioles with preserved perivascular retinal tissue were mounted in a wire myograph, and the tone was measured after the addition of increasing concentrations of bradykinin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and brain natriuretic peptide (BNP). The experiments were performed during inhibition of the synthesis of nitric oxide (NO), prostaglandins and dopamine and were repeated after removal of the perivascular retinal tissue. RESULTS: Bradykinin, VIP and CGRP induced significant concentration-dependent dilatation and NPY significant concentration-dependent contraction of the arterioles in the presence of perivascular retinal tissue (p < 0.03 for all comparisons) but not on isolated arterioles. BNP and SP had no effect on vascular tone. The NOS inhibitor L-NAME reduced bradykinin- and VIP-induced relaxation (p < 0.001 for both comparisons), whereas none of the other inhibitors influenced the vasoactive effects of the studied neuropeptides. CONCLUSION: The effects of neuropeptides on the tone of retinal arterioles depend on the perivascular retinal tissue and may involve effects other than those mediated by nitric oxide, prostaglandins and adrenergic compounds. Investigation of the mechanisms underlying the vasoactive effect of neuropeptides may be important for understanding and treating retinal diseases where disturbances in retinal flow regulation are involved in the disease pathogenesis.

Sympathetic Neurotransmitter, VIP, Delays Intervertebral Disc Degeneration via FGF18/FGFR2-Mediated Activation of Akt Signaling Pathway.

Neuromodulation-related intervertebral disc degeneration (IVDD) is a novel IVDD pattern and are proposed recently. However, the mechanistic basis of neuromodulation and intervertebral disc (IVD) homeostasis remains unclear. Here, this study aimed to investigate the expression of postganglionic sympathetic nerve fiber-derived vasoactive intestinal peptide (VIP) system in human IVD tissue, and to assess the role of VIP-related neuromodulation in IVDD. Patient samples and in vitro cell experiments showed that the expression of receptors for VIP is negatively correlated with the severity of IVDD, and the administration of exogenous VIP can ameliorate interleukin 1ß-induced nucleus pulposus (NP) cell apoptosis and inflammation. Further mRNA-seq analysis revealed that fibroblast growth factor 18- (FGF18)-mediated activation of V-akt murine thymoma viral oncogene homolog signaling pathway is involved in the protective effects of VIP on inflammation-induced NP cell degeneration. Further analysis identified VIP via its receptor vasoactive intestinal peptide receptor 2 can directly result in decreased expression of miR-15a-5p, which targeted FGF18. Finally, in vivo mice lumbar IVDD model confirmed that focally exogenous administration of VIP can effectively ameliorated the progression of IVDD, as shown by the radiological and histological analysis. In conclusion, these results indicated that sympathetic neurotransmitter, VIP, delayed IVDD via FGF18/FGFR2-mediated activation of V-akt murine thymoma viral oncogene homolog signaling pathway, which will broaden the horizon concerning how the neuromodulation correlates with IVDD and shed new light on novel therapeutical alternatives to IVDD.

Changes in the Phenotype of Intramural Inhibitory Neurons of the Porcine Descending Colon Resulting from Glyphosate Administration.

Environmental contamination and the resulting food contamination represent a serious problem and pose a major threat to animal and human health. The gastrointestinal tract is directly exposed to a variety of substances. One is glyphosate, whose presence in the soil is commonly observed. This study demonstrates the effects of low and high glyphosate doses on the populations of intramural neurons of the porcine descending colon. An analysis was performed on neurons ex-pressing the vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, a neuronal isoform of nitrogen oxide synthase, and galanin. Even a low dose of glyphosate increased the number of neurons immunoreactive against the studied substances. However, the changes depended on both the plexus analysed and the substance tested. Meanwhile, a high glyphosate dose resulted in quantitative changes (an increase in the number) within neurons immunoreactive against all the studied neuropeptides/enzymes in the myenteric plexus and both submucosal plexuses. The response of the enteric nervous system in the form of an increase in the number of neurons immunoreactive against neuroprotective substances may suggest that glyphosate has a toxic effect on enteric neurons which attempt to increase their survivability through the released neuroprotective substances.

Baizhu-Baishao herb pair ameliorates functional constipation and intestinal microflora disorder in rats.

BACKGROUND: In China, Rhizoma atractylodis macrocephalae-Paeonia lactiflora Pall (Biazhu-Baishao, BZBS) is a classic herb pair used to treat intestinal stress syndrome, ulcerative colitis and other diseases. However, the mechanism of BZBS in the treatment of functional constipation (FC) has been little studied and remains unclear. In this study, a behavioral investigation, colon tissue morphology, enzyme-linked immunosorbent assay (Elisa) and intestinal microflora analysis have been used to illuminate the potential mechanism of the effects of BZBS on FC in a rat model. METHODS: A FC rat model was constructed and BZBS was given as treatment. Observations and recordings were made of the fecal moisture content, the defecation time of the first black stool, and the rate of intestinal propulsion. Elisa was used to detect the expression levels of substance P (SP), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine (5-HT) in the colon. To ascertain the composition of the microbial community, a high throughput 16S ribosomal RNA (16S rRNA) gene sequencing technique was employed. RESULTS: Oral administration of BZBS significantly ameliorated several key excretion parameters, including the time to first black stool defecation, stool water content, and the propulsion rate in the small intestine in FC rats. It increased the expression of SP, VIP and 5-HT in the colon. 16S rRNA gene sequencing results showed that BZBS changed the microbial community structure, decreased the Bacteroidetes/Firmicutes ratio, increased the relative abundance of Blautia and Fusicatenibacter, and decreased the relative abundance of Ruminococcus and Roseburia. CONCLUSIONS: BZBS effectively alleviates FC and improves dysbacteriosis.

Vasoactive intestinal peptide exerts therapeutic action by regulating PTEN in a model of Sjögren's disease.

INTRODUCTION: Sjögren's disease (SjD) is a chronic autoimmune disease characterized by the loss of the secretory function of the exocrine glands. At present, drugs that can both correct the immune imbalance and improve exocrine gland function are needed. Meanwhile, vasoactive intestinal peptide (VIP) has been reported as a candidate with anti-inflammatory and immunoregulatory properties for treating autoimmune diseases. METHODS: Nonobese diabetic (NOD) mice and the primary splenic lymphocyte cells (SPLCs) were used to construct the SS model. The therapeutic effects of VIP for SjD by evaluating water consumption, histopathology, T cell subsets, and related cytokines. RT-qPCR and Western blot analysis were used to identify the expression of the PTEN/PI3K/AKT pathway. RESULTS: We found that VIP therapy in NOD mice could increase the expression of PTEN and VIP/VPAC1 receptor, as well as decrease the PI3K/AKT pathway. In vitro, the results showed that the PTEN knockdown decreased the Treg/Th17 ratio and enhanced the phosphorylated PI3K/AKT pathway, which were reversed with VIP treatment. CONCLUSIONS: VIP exerts potential therapeutic action in SjD by upregulating PTEN through the PI3K/AKT pathway and Treg/Th17 cell balance.

Acute alcohol and chronic drinking bidirectionally regulate the excitability of prefrontal cortex vasoactive intestinal peptide interneurons.

The prefrontal cortex (PFC) regulates drinking behaviors and affective changes following chronic alcohol use. PFC activity is dynamically modulated by local inhibitory interneurons (INs), which can be divided into non-overlapping groups with distinct functional roles. Within deeper layers of neocortex, INs that express either parvalbumin or somatostatin directly inhibit pyramidal cells. By contrast, the plurality of all remaining INs express vasoactive intestinal peptide (VIP), reside within superficial layers, and preferentially target other types of INs. While recent studies have described adaptations to PFC parvalbumin-INs and somatostatin-INs in alcohol use models, whether ethanol or drinking affect the physiology of PFC VIP-INs has not been reported. To address this gap, we used genetically engineered female and male mice to target VIP-INs in layers 1-3 of prelimbic PFC for whole-cell patch-clamp electrophysiology. We found that ethanol (20 mM, ∼0.09 BEC/90 mg/dL) application to PFC brain slices enhances VIP-IN excitability. We next examined effects following chronic drinking by providing mice with 4 weeks of intermittent access (IA) ethanol two-bottle choice in the home cage. In these studies, VIP-INs from female and male IA ethanol mice displayed reduced excitability relative to cells from water-only controls. Finally, we assessed whether these effects continue into abstinence. After 7-13 days without ethanol, the hypo-excitability of VIP-INs from male IA ethanol mice persisted, whereas cells from female IA ethanol mice were not different from their controls. Together, these findings illustrate that acute ethanol enhances VIP-IN excitability and suggest these cells undergo pronounced homeostatic changes following long-term drinking.

[Development of the vasoactive intestinal peptide receptor 2 (VIPR2) antagonist peptide for the treatment of schizophrenia].

Schizophrenia affects approximately 24 million people worldwide. Existing medications for the treatment of schizophrenia work primarily by improving positive symptoms such as agitation, hallucinations, delusions, and aggression. They possess common mechanism of action (MOA), blocking to neurotransmitter receptors such as dopamine, serotonin, and adrenaline receptors. Although multiple agents are available for the treatment of schizophrenia, the majority do not address negative symptoms or cognitive dysfunction. In other cases, patients have drug-related adverse effects. The vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2 receptor) might be an attractive drug target for the treatment of schizophrenia because both clinical and preclinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Despite these backgrounds, the proof-of-concept of VIPR2 inhibitors has not been examined clinically. A reason might be that VIPR2 belongs to class-B GPCRs, and the discovery of small-molecule drugs against class-B GPCRs is generally difficult. We have developed a bicyclic peptide KS-133, which shows VIPR2 antagonist activity and suppresses cognitive decline in a mouse model relevant to schizophrenia. KS-133 has a different MOA from current therapeutic drugs and exhibits high selectivity for VIPR2 and potent inhibitory activity against a single-target molecule. Therefore, it may contribute to both the development of a novel drug candidate for the treatment of psychiatric disorders such as schizophrenia and acceleration of basic studies on VIPR2.

Changes Caused by Bisphenols in the Chemical Coding of Neurons of the Enteric Nervous System of Mouse Stomach.

Bisphenol A (BPA), an organic chemical compound which is widely used in the production of plastics, can severely damage live organisms. Due to these findings, the plastic industry has started to replace it with other substances, most often with bisphenol S (BPS). Therefore, during the present investigation, with the use of double immunofluorescence labeling, we compared the effect of BPA and BPS on the enteric nervous system (ENS) in the mouse corpus of the stomach. The obtained results show that both studied toxins impact the amount of nerve cells immunoreactive to substance P (SP), galanin (GAL), vesicular acetylcholine transporter (VAChT is used here as a marker of cholinergic neurons) and vasoactive intestinal polypeptide (VIP). Changes observed under the impact of both bisphenols depended on the neuronal factor, the type of the enteric ganglion and the doses of bisphenols studied. Generally, the increase in the percentage of neurons immunoreactive to SP, GAL and/or VIP, and the decrease in the percentage of VAChT-positive neurons, was noted. Severity of changes was more visible after BPA administration. However, the study has shown that long time exposure to BPS also significantly affects the ENS.

Research on the mechanism of prednisone in the treatment of ITP via VIP/PACAP-mediated intestinal immune dysfunction.

RATIONALE: Immune thrombocytopenia (ITP) is thought to be a result of immune dysfunction, which is treated by glucocorticoids such as prednisone. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have immunomodulatory properties, but their role in intestinal immune control is unclear. The major goal of this study was to look at the effects of prednisone on platelet, VIP, and PACAP levels in ITP mice, as well as the regulatory system that controls intestinal immunity. METHODS: Eighteen BALB/c mice were randomly divided into three groups: blank control group, model control group, and prednisone group, with six mice in each group. The ITP animal model control group and the prednisone group were injected with anti-platelet serum (APS) to replicate the ITP animal model. The prednisone group began prednisone intervention on the 8th day. Platelet count was dynamically measured before APS injection, on the 4th day of injection, on the 1st day of administration, on the 4th day of administration, and at the end of the experiment. After the experiment, the expression of p53 protein in mouse mesenteric lymph node lymphocytes was detected by immunohistochemistry. The changes in lymphocyte apoptosis rate in mouse mesenteric lymph nodes were detected by in situ terminal transferase labeling (TUNEL). The contents of VIP and PACAP in the mouse brain, colon, and serum were detected by enzyme-linked immunosorbent assay (ELISA). The contents of IFN-γ, IL-4, IL-10, IL-17A in the mouse spleen were detected by ELISA. RESULTS: ①Changes of peripheral platelet count: there was no significant difference in platelet count among the three groups before modeling; on the 4th day, the platelet count decreased in the model control group and prednisone group; on the 8th day, the number of platelets in model control group and prednisone group was at the lowest level; on the 12th day, the platelet count in prednisone group recovered significantly; on the 15th day, the platelet count in prednisone group continued to rise. ②Changes of VIP, PACAP: compared with the blank control group, VIP and PACAP in the model control group decreased significantly in the brain, colon, and serum. Compared with the model control group, the levels of VIP and PACAP in the brain, colon, and serum in the prednisone group were increased except for serum PACAP. ③Changes of mesenteric lymphocytes: the expression of p53 protein in the mesenteric lymph nodes of model control group mice was significantly higher than that of blank control group mice. After prednisone intervention, the expression of p53 protein decreased significantly.④Changes of cytokines in spleen: compared with blank control group, IFN- γ, IL-17A increased and IL-4 and IL-10 decreased in model control group. After prednisone intervention, IFN- γ, IL-17A was down-regulated and IL-4 and IL-10 were upregulated. CONCLUSIONS: Prednisone-upregulated VIP and PACAP levels decreased P53 protein expression and apoptosis rate in mesenteric lymph node lymphocytes and affected cytokine expression in ITP model mice. Therefore, we speculate that the regulation of intestinal immune function may be a potential mechanism of prednisone in treating ITP.

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.

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.

Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer.

Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 µM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 µM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.

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.

Regulation of CRE-Dependent Transcriptional Activity in a Mouse Suprachiasmatic Nucleus Cell Line.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

Targeting vasoactive intestinal peptide-mediated signaling enhances response to immune checkpoint therapy in pancreatic ductal adenocarcinoma.

A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.

Sympathetic activation leads to Schlemm's canal expansion via increasing vasoactive intestinal polypeptide secretion from trabecular meshwork.

We previously demonstrated vasoactive intestinal polypeptide (VIP) eyedrops reduce intraocular pressure (IOP) and stabilize cytoskeleton of the Schlemm's canal (SC) endothelium in a chronic ocular hypertension rat model. Here we determine if the trabecular meshwork (TM) releases endogenous VIP and affect SC in paracrine manner, and whether this cellular interaction via VIP is strengthened under stimulated sympathetic activity. A rat model of moderate-intensity exercise was established to stimulate sympathetic activation. IOP post exercise was measured by a rebound tonometer. Sympathetic nerve activity at the TM was immunofluorescence-stained with DßH and PGP9.5. Morphological changes of TM and SC were quantitatively measured by hematoxylin-eosin (HE) staining. Further, epinephrine was applied to mimic sympathetic excitation on primary rat TM cells, and ELISA to measure VIP levels in the medium. The cytoskeleton protective effect of VIP in the epinephrine-stimulated conditioned medium (Epi-CM) was evaluated in oxidative stressed human umbilical vein endothelial cells (HUVECs). Elevated sympathetic nerve activity was found at TM post exercise. Changes accompanying the sympathetic excitation included thinned TM, expanded SC and decreased IOP, which were consistent with epinephrine treatment. Epinephrine decreased TM cell size, enhanced VIP expression and release in the medium in vitro. Epi-CM restored linear F-actin and cell junction integrity in H2O2 treated HUVECs. Blockage of VIP receptor by PG99-465 attenuated the protective capability of Epi-CM. VIP expression was upregulated at TM and the inner wall of SC post exercise in vivo. PG99-465 significantly attenuated exercise-induced SC expansion and IOP reduction. Thus, the sympathetic activation promoted VIP release from TM cells and subsequently expanded SC via stabilizing cytoskeleton, which resulted in IOP reduction.

Effects of α2-adrenoceptor stimulation on luminal alkalinisation and net fluid flux in rat duodenum.

The sympathetic nervous system is highly involved in the regulation of gastrointestinal functions such as luminal alkalinisation and fluid absorption. However, the exact mechanisms are not clear. This study aimed to delineate how α2-adrenergic receptor stimulation reduces duodenal luminal alkalinisation and induces net fluid absorption. This was tested by perfusing the duodenum of anesthetized rats with isotonic solutions devoid of Cl- and/or Na+, in the absence and presence of the α2-adrenoceptor agonist clonidine. The clonidine was also studied in rats treated with dimethylamiloride (a Na+/H+ exchange inhibitor), vasoactive intestinal peptide, and the nicotinic receptor antagonist hexamethonium. Clonidine reduced luminal alkalinisation and induced net fluid absorption. The Cl--free solution decreased luminal alkalinisation and abolished net fluid absorption, but did not prevent clonidine from doing so. Both the Na+-free solution and luminal dimethylamiloride increased luminal alkalinisation and abolished net fluid absorption, effects counteracted by clonidine. The NaCl-free solution (D-mannitol) did not affect luminal alkalinisation, but reduced net fluid absorption. Clonidine reduced luminal alkalinisation and induced net fluid absorption in rats perfused luminally with mannitol. However, clonidine did not affect the vasoactive intestinal peptide-induced increase in luminal alkalinisation or fluid secretion. Pre-treatment with hexamethonium abolished the effects of clonidine on luminal alkalinisation and net fluid flux. In summary, our in vivo experiments showed that clonidine-induced reduction in luminal alkalinisation and induction of net fluid absorption was unrelated to luminal Na+ and Cl-, or to apical Na+/H+ or Cl-/HCO3- exchangers. Instead, clonidine seems to exert its effects via suppression of nicotinic receptor-activated acetylcholine secretomotor neurons.