Vasoactive Intestinal Peptide mRNA Research Articles

The role of vasoactive intestinal peptide (VIP) in atropine-related inhibition of the progression of myopia

ObjectiveThis study aimed to investigate the potential involvement of vasoactive intestinal polypeptide (VIP) in myopia development and its contribution to the mechanism of action of the anti-myopia drug, atropine.MethodsThirty-three-week-old guinea pigs were randomly divided into normal control (NC, n = 10), monocularly form-deprived (FDM, n = 10), and FDM treated with 1% atropine (FDM + AT, n = 10) groups. The diopter and axial length were measured at 0, 2, and 4 weeks. Guinea pig eyeballs were removed at week four, fixed, and stained for morphological changes. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to evaluate VIP protein and mRNA levels.ResultsThe FDM group showed an apparent myopic shift compared to the control group. The results of the H&E staining were as follows: the cells of the inner/outer nuclear layers and retinal ganglion cells were disorganized; the choroidal thickness (ChT), blood vessel lumen, and area were decreased; the sclera was thinner, with disordered fibers and increased interfibrillar space. IHC and ISH revealed that VIP's mRNA and protein expressions were significantly up-regulated in the retina of the FDM group. Atropine treatment attenuated FDM-induced myopic shift and fundus changes, considerably reducing VIP's mRNA and protein expressions.ConclusionsThe findings of elevated VIP mRNA and protein levels observed in the FDM group indicate the potential involvement of VIP in the pathogenesis and progression of myopia. The ability of atropine to reduce this phenomenon suggests that this may be one of the molecular mechanisms for atropine to control myopia.

Immunomodulatory role of vasoactive intestinal peptide and ghrelin in Oncorhynchus mykiss

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1β) and anti-inflammatory (IL-10 and TGF-β) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

Biosynthesis and function of VIP and oxytocin: mechanisms of C-terminal amidation, oxytocin secretion and transport.

In this review, we provide the current status of research on vasoactive intestinal peptide (VIP) and oxytocin, typical C-terminal α-amidated peptide hormones, including their precursor protein structures, processing and C-terminal α-amidation, and the recently identified mechanisms of regulation of oxytocin secretion and its transportation through the blood brain barrier. More than half of neural and endocrine peptides, such as vasoactive intestinal peptide (VIP) and oxytocin, have the α-amide structure at their C-terminus, which is generally essential for biological activities. We have studied the synthesis and function of C-terminal α-amidated peptides, including VIP and oxytocin, since the 1980s. Human VIP mRNA encoded not only VIP but also another related C-terminal α-amidated peptide, PHM-27 (peptide having amino-terminal histidine, carboxy-terminal methionine amide and 27 amino acid residues). The human VIP/PHM-27 gene was composed of seven exons and regulated synergistically by cyclic AMP and protein kinase C pathways. VIP has an essential role in glycemic control using transgenic mouse technology. The peptide C-terminal α-amidation proceeded through a two-step mechanism catalyzed by two different enzymes encoded in a single mRNA. In the oxytocin secretion from the hypothalamus/the posterior pituitary, the CD38-cyclic ADP-ribose signal system, that was first established in the insulin secretion from pancreatic β cells of the islets of Langerhans, was found to be essential. A possible mechanism involving receptor for advanced glycation end-products (RAGE) of the oxytocin transportation from the blood stream into the brain through the blood-brain barrier has also been suggested.

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.

Abstract 5571: Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling

Abstract Pancreatic ductal adenocarcinoma (PDAC) constitutes more than 90% of all pancreatic cancers and has a 5-year survival rate of only 5-7%. A dense stroma, an immunosuppressive tumor microenvironment (TME) and scarce tumor infiltrating T cells, contribute toward the poor responsiveness of PDAC to conventional and FDA-approved checkpoint therapies. In this study, we show that vasoactive intestinal peptide (VIP) signaling is a novel immune checkpoint pathway in PDAC, which when inhibited in combination with anti-PD1 checkpoint inhibitor, significantly decreases tumor burden and enhances T cell infiltration in mouse models of PDAC. VIP is a 28-amino acid neuropeptide secreted at the nerve terminals, pancreas and the GI tract, and is commonly associated with regulating gut motility and blood pressure. Interestingly, VIP is also secreted by immune cells and is known to have immunosuppressive properties such as decreasing T cell proliferation and polarizing T cells towards Th2 phenotype. Intriguingly, according to the cancer genome atlas (TCGA), human exocrine pancreatic cancers expresses the highest level of VIP mRNA expression when compared to other solid malignancies. Similarly, we have generated preliminary data that shows enhanced VIP levels both systemically and locally in mouse models of PDAC and in human PDAC patients. Thus, taking all this into consideration, we hypothesized that enhanced VIP signaling is a potential mechanism of immune escape in PDAC and that inhibiting VIP signaling either alone or in combination with checkpoint inhibitors would significantly enhance treatment response in mouse models of PDAC. We tested the effect of inhibiting VIP signaling by using a VIP receptor antagonist (VIPR antagonist) in orthotopic mouse models of PDAC, where luciferase transfected KPC cells (mouse PDAC cell line), were implanted in the tail of the pancreas. Mice were then treated with VIPR antagonist and/or anti-PD1 seven days after tumor implantation. Our results showed that the combination treatment significantly reduced tumor burden and growth rate, as well as, enhanced the infiltration of both CD4+ and CD8+ T cells into the tumor microenvironment (as per immunohistochemistry of tumor tissues). Furthermore, analysis of tumor infiltrating T cells via Nanostring analysis showed that the combination treatment also significantly enhanced levels of IFN gamma and TNF alpha secreting T cells and costimulatory and effector molecules, including CD27 and FasL. In summary, we have identified that VIP signaling is a novel and targetable immune checkpoint pathway in PDAC, which when inhibited in combination with checkpoint inhibitors, significantly improves treatment response in mouse PDAC models. As the VIP sequence is conserved across different species including mouse and human, and since we have observed VIPR antagonists stimulate proliferation of human T cells, there is an increased potential for clinical translation of VIPR antagonists in the treatment of human PDAC. Citation Format: Sruthi Ravindranathan, Brandon Ware, Mohammad Zaidi, Jingru Zhu, Rohan K. Dhamsania, Shuhua Wang, Yiwen Li, Ishani H. Rao, Susan N. Thomas, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5571.

Function study of vasoactive intestinal peptide on chick embryonic bone development

Embryonic bone development is a complicated procedure and modulated by neuro-osteogenic interaction. Vasoactive intestinal peptide (VIP) was first identified as a neural vasodilator and further proved to possess multiple biological functions such as neurotransmitter and immune regulator. However, as a key peptide regulator presented in skeletal nerve fibers, the function of VIP on innervation and early bone development regulation has not fully been uncovered yet. In this study, the chick embryo has been used as an experimental model to address the effect of VIP on embryonic bone development. Our study results confirmed the innervation of peripheral nerve fibers into limb bone tissue, which was revealed by the detection of neurofilament (NF) and class III β-tubulin (TUJ-1) in bone tissue at various developing stages. The VIP mRNA and peptide expression level in bone tissue were also increased upon innervation progress. A chick embryonic chemical sympathectomy model was constructed by exposing chick embryos with neurotoxin 6-OHDA. The 6-OHDA exposure of the early chick embryo caused the reduction of neural crest formation and NF expression. 6-OHDA treatment also inhibited distal limb bone development as well as VIP expression. Furthermore, co-application of VIP with 6-OHDA exposure could rescue the inhibited osteogenesis activity and delayed bone development during embryogenesis. Taken together, these results reveal that VIP played an important role during innervation at early stage of bone development. VIP could restore chemical sympathectomy induced osteogenesis inhibition and bone development impair in chick embryos.

Vasoactive intestinal peptide as a mediator of the effects of a supergene on social behaviour.

Supergenes, or linked groups of alleles that are inherited together, present excellent opportunities to understand gene-behaviour relationships. In white-throated sparrows (Zonotrichia albicollis), a supergene on the second chromosome associates with a more aggressive and less parental phenotype. This supergene includes the gene for vasoactive intestinal peptide (VIP), a neuropeptide known to play a causal role in both aggression and parental behaviour. Here, using a free-living population, we compared the levels of VIP mRNA between birds with and without the supergene. We focused on the anterior hypothalamus and infundibular region, two brain regions containing VIP neurons known to play a causal role in aggression and parental behaviour, respectively. First, we show that the supergene enhances VIP expression in the anterior hypothalamus and that expression positively predicts vocal aggression independently of genotype in both sexes. Next, we show that the supergene reduces VIP expression in the infundibular region, which suggests reduced secretion of prolactin, a pro-parental hormone. Thus, the patterns of VIP expression in these two regions are consistent with the enhanced aggression and reduced parental behaviour of birds with the supergene allele. Our results illustrate mechanisms by which elements of genomic architecture, such as supergenes, can contribute to the evolution of alternative behavioural phenotypes.

Pituitary adenylate cyclase activating polypeptide concentrations in the sheep mammary gland, milk, and in the lamb blood plasma after suckling.

Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in development and reproduction. We previously described elevated PACAP levels in the milk compared to the plasma, and the presence of its specific PAC1 receptor in the mammary gland. This study aimed to determine PACAP and vasoactive intestinal peptide (VIP) levels in female suckling lambs compared to ewe plasma and mammary gland, as well as their age-dependent alterations. mRNA expressions of PACAP, VIP, PAC1 receptor and brain-derived neurotrophic factor (BDNF) were quantified in the milk whey and mammary gland. PACAP38-like immunoreactivity (PACAP38-LI) was measured in plasma, milk whey and mammary gland by radioimmunoassay, VIP-LI by enzyme-linked immunoassay. PACAP38-LI was 5, 6 times higher in the milk compared to the plasma of lactating sheep. It significantly increased in the lamb plasma 1 h, but returned to basal level 2 h after suckling. However, VIP mRNA was not present in the mammary gland, we detected the VIP protein in the milk whey. BDNF mRNA significantly decreased with age to approximately 60% and 25% in the 3- and 10-year-old sheep respectively, compared to the 3-month-old lambs. No differences were found between mammary and jugular vein plasma PACAP and VIP concentrations, or during the daily cycle. We propose a rapid absorption of PACAP38 from the milk and/or its release in suckling lambs. PACAP accumulated in the milk might be synthesized in the mammary gland or secreted from the plasma of the mothers. PACAP is suggested to have differentiation/proliferation promoting and immunomodulatory effects in the newborns and/or a local function in the mammary gland.

Therapeutic effect of vasoactive intestinal peptide on form-deprived amblyopic kittens

BackgroundExploring the role of vasoactive intestinal peptide (VIP) in the lateral geniculate body (LGBd) in visual development and studying the therapeutic effect of VIP on amblyopic kittens.MethodsThree-week-old domestic cats were divided into a control group (n = 10) and a monocular deprivation group (n = 20), with an eye mask covering the right eye of those in the deprived group. After pattern visual evoked potential (PVEP) recording confirmed the formation of monocular amblyopia, the left LGBd was isolated from 5 kittens in each group. The remaining control kittens continued to be raised, and the remaining deprivation group was divided into a VIP intervention group (n = 5), Sefsol (caprylic acid monoglyceride, VIP solution) intervention group (n = 5) and amblyopia non-intervention group (n = 5) after removal of the eye mask. Three weeks later, PVEPs, VIP immunohistochemistry and VIP mRNA expression in the left LGBd were compared across groups.ResultsAt 6 weeks of age, there were significant differences in P100 wave latency and amplitude and VIP immunohistochemistry and in situ hybridization between the control group and the deprivation group (P < 0.05). After 3 weeks of the corresponding interventions, the latency and amplitude in the VIP intervention group were better than that in the Sefsol intervention group and amblyopia non-intervention group (P < 0.05). Furthermore, VIP treatment increased the number of immunohistochemical VIP-positive cells (P < 0.05) and the average optical density of positive cells (P > 0.05), as well as the number (P < 0.05) and average optical density of VIP mRNA-positive cells (P < 0.05).ConclusionsVIP plays an important role in visual development. Nasal administration of VIP can improve the function of neurons in the LGBd of kittens and has a certain therapeutic effect on amblyopia.

Expression of Transcripts Selective for GABA Neuron Subpopulations across the Cortical Visuospatial Working Memory Network in the Healthy State and Schizophrenia.

Visuospatial working memory (WM), which is impaired in schizophrenia, depends on a distributed network including visual, posterior parietal, and dorsolateral prefrontal cortical regions. Within each region, information processing is differentially regulated by subsets of γ-aminobutyric acid (GABA) neurons that express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP). In schizophrenia, WM impairments have been associated with alterations of PV and SST neurons in the dorsolateral prefrontal cortex. Here, we quantified transcripts selectively expressed in GABA neuron subsets across four cortical regions in the WM network from comparison and schizophrenia subjects. In comparison subjects, PV mRNA levels declined and SST mRNA levels increased from posterior to anterior regions, whereas VIP mRNA levels were comparable across regions except for the primary visual cortex (V1). In schizophrenia subjects, each transcript in PV and SST neurons exhibited similar alterations across all regions, whereas transcripts in VIP neurons were unaltered in any region except for V1. These findings suggest that the contribution of each GABA neuron subset to inhibitory regulation of local circuitry normally differs across cortical regions of the visuospatial WM network and that in schizophrenia alterations of PV and SST neurons are a shared feature across these regions, whereas VIP neurons are affected only in V1.

VIP is involved in peripheral CRF-induced stimulation of propulsive colonic motor function and diarrhea in male rats.

We investigated whether vasoactive intestinal peptide (VIP) and/or prostaglandins contribute to peripheral corticotropin-releasing factor (CRF)-induced CRF1 receptor-mediated stimulation of colonic motor function and diarrhea in rats. The VIP antagonist, [4Cl-D-Phe6, Leu17]VIP injected intraperitoneally completely prevented CRF (10 µg/kg ip)-induced fecal output and diarrhea occurring within the first hour after injection, whereas pretreatment with the prostaglandins synthesis inhibitor, indomethacin, had no effect. In submucosal plexus neurons, CRF induced significant c-Fos expression most prominently in the terminal ileum compared with duodenum and jejunum, whereas no c-Fos was observed in the proximal colon. c-Fos expression in ileal submucosa was colocalized in 93.4% of VIP-positive neurons and 31.1% of non-VIP-labeled neurons. CRF1 receptor immunoreactivity was found on the VIP neurons. In myenteric neurons, CRF induced only a few c-Fos-positive neurons in the ileum and a robust expression in the proximal colon (17.5 ± 2.4 vs. 0.4 ± 0.3 cells/ganglion in vehicle). The VIP antagonist prevented intraperitoneal CRF-induced c-Fos induction in the ileal submucosal plexus and proximal colon myenteric plexus. At 60 min after injection, CRF decreased VIP levels in the terminal ileum compared with saline (0.8 ± 0.3 vs. 2.5 ± 0.7 ng/g), whereas VIP mRNA level detected by qPCR was not changed. These data indicate that intraperitoneal CRF activates intestinal submucosal VIP neurons most prominently in the ileum and myenteric neurons in the colon. It also implicates VIP signaling as part of underlying mechanisms driving the acute colonic secretomotor response to a peripheral injection of CRF, whereas prostaglandins do not play a role. NEW & NOTEWORTHY Corticotropin-releasing factor (CRF) in the gut plays a physiological role in the stimulation of lower gut secretomotor function induced by stress. We showed that vasoactive intestinal peptide (VIP)-immunoreactive neurons in the ileal submucosal plexus expressed CRF1 receptor and were prominently activated by CRF, unlike colonic submucosal neurons. VIP antagonist abrogated CRF-induced ileal submucosal and colonic myenteric activation along with functional responses (defecation and diarrhea). These data point to VIP signaling in ileum and colon as downstream effectors of CRF.

Reproductive axis gene regulation during photostimulation and photorefractoriness in Yangzhou goose ganders

BackgroundThe Yangzhou goose is a long-day breeding bird that has been increasingly produced in China. Artificial lighting programs are used for controlling its reproductive activities. This study investigated the regulations of photostimulation and photorefractoriness that govern the onset and cessation of the breeding period.ResultsIncreasing the daily photoperiod from 8 to 12 h rapidly stimulated testis development and increased plasma testosterone concentrations, with peak levels being reached 2 months after the photoperiod increase. Subsequently, testicular activities, testicular weight, spermatogenesis, and plasma testosterone concentrations declined steadily and reached to the nadir at 5 months after the 12-hour photoperiod. Throughout the experiment, plasma concentrations of triiodothyronine and thyroxine changed in reciprocal fashions to that of testosterone. The stimulation of reproductive activities caused by the increasing photoperiod was associated with increases in gonadotropin-releasing hormone (GnRH), but decreases in gonadotropin-inhibitory hormone (GnIH) and vasoactive intestinal peptide (VIP) gene messenger RNA (mRNA) levels in the hypothalamus. In the pituitary gland, the levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mRNA abruptly increased during the longer 12-hour photoperiod. The occurrence of photorefractoriness was associated with increased GnIH gene transcription by over 250-fold, together with increased VIP mRNA levels in the hypothalamus, and then prolactin and thyroid-stimulating hormone in the pituitary gland. FSH receptor, LH receptor, and StAR mRNA levels in the testis changed in ways paralleling those of testicular weight and testosterone concentrations.ConclusionsThe seasonal reproductive activities in Yangzhou geese were directly stimulated by a long photoperiod via upregulation of GnRH gene transcription, downregulation of GnIH, VIP gene transcription, and stimulation of gonadotrophin. Development of photorefractoriness was characterized by hyper-regulation of GnIH gene transcription in the hypothalamus, in addition of upregulation of VIP and TRH gene transcription, and that of their receptors, in the pituitary gland.

LRRK2: An Emerging New Molecule in the Enteric Neuronal System That Quantitatively Regulates Neuronal Peptides and IgA in the Gut

Leucine-rich repeat kinase 2 (LRRK2) is a recently discovered molecule associated with familial and sporadic Parkinson's disease. It regulates many central neuronal functions such as cell proliferation, apoptosis, autophagy, and axonal extension. However, in contrast to the well-documented function of LRRK2 in central neurons, it is unclear whether LRRK2 is expressed in enteric neurons and affects the physiology of the gut. By examining LRRK2-KO mice, this study investigated whether enteric neurons express LRRK2 and whether intestinal neuronal peptides and IgA are quantitatively changed. Intestinal protein lysates and sections prepared from male C57BL/6J mice were analyzed by Western blotting and immunostaining using anti-LRRK2 antibody, respectively. Intestinal neuronal peptide-mRNAs were quantified by real-time PCR in wild-type mice and LRRK2-KO mice. Intestinal IgA was quantified by ELISA. Lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry to evaluate the ratio of B1 to B2 B cells. Western analysis and immunostaining revealed that LRRK2 is expressed in enteric neurons. The amounts of mRNA for vasoactive intestinal peptide, neuropeptide Y, and substance P were increased in LRRK2-KO mice accompanied by an increment of IgA. However, the intestinal B cell subpopulations were not altered in LRRK2-KO mice. For the first time, we have revealed that LRRK2 is expressed in enteric neurons and related to quantitative alterations of neuronal peptide and IgA. Our study highlights the importance of LRRK2 in enteric neurons as well as central neurons.

NFATc3 and VIP in Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease.

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both debilitating lung diseases which can lead to hypoxemia and pulmonary hypertension (PH). Nuclear Factor of Activated T-cells (NFAT) is a transcription factor implicated in the etiology of vascular remodeling in hypoxic PH. We have previously shown that mice lacking the ability to generate Vasoactive Intestinal Peptide (VIP) develop spontaneous PH, pulmonary arterial remodeling and lung inflammation. Inhibition of NFAT attenuated PH in these mice suggesting a connection between NFAT and VIP. To test the hypotheses that: 1) VIP inhibits NFAT isoform c3 (NFATc3) activity in pulmonary vascular smooth muscle cells; 2) lung NFATc3 activation is associated with disease severity in IPF and COPD patients, and 3) VIP and NFATc3 expression correlate in lung tissue from IPF and COPD patients. NFAT activity was determined in isolated pulmonary arteries from NFAT-luciferase reporter mice. The % of nuclei with NFAT nuclear accumulation was determined in primary human pulmonary artery smooth muscle cell (PASMC) cultures; in lung airway epithelia and smooth muscle and pulmonary endothelia and smooth muscle from IPF and COPD patients; and in PASMC from mouse lung sections by fluorescence microscopy. Both NFAT and VIP mRNA levels were measured in lungs from IPF and COPD patients. Empirical strategies applied to test hypotheses regarding VIP, NFATc3 expression and activity, and disease type and severity. This study shows a significant negative correlation between NFAT isoform c3 protein expression levels in PASMC, activity of NFATc3 in pulmonary endothelial cells, expression and activity of NFATc3 in bronchial epithelial cells and lung function in IPF patients, supporting the concept that NFATc3 is activated in the early stages of IPF. We further show that there is a significant positive correlation between NFATc3 mRNA expression and VIP RNA expression only in lungs from IPF patients. In addition, we found that VIP inhibits NFAT nuclear translocation in primary human pulmonary artery smooth muscle cells (PASMC). Early activation of NFATc3 in IPF patients may contribute to disease progression and the increase in VIP expression could be a protective compensatory mechanism.

Effects of Vasoactive Intestinal Polypeptide and Forskolin on mRNA Expression of Prolactin and Prolactin Regulatory Element-Binding Protein in the Anterior Pituitary Gland of Chicken Embryo and Laying Hens.

Vasoactive intestinal peptide (VIP) treatment induced mRNA expression of Prolactin (PRL) in the chicken anterior pituitary gland. VIP responsive element (VRE) of the PRL promoter was identified in the various bird species. However, transcription factor, which binds to VRE, has not yet been identified. Prolactin regulatory element-binding protein (PREB) gene cloned as a candidate transcription factor binds to VRE. Increases of mRNA levels of PRL and PREB during embryogenesis were identified. However, whether VIP affects levels of PRL and PREB mRNA during embryogenesis remains unknown. The effects of VIP and forskolin on mRNA expression of PRL and PREB in the embryonic anterior pituitary gland were assessed. Furthermore, administration of VIP to laying hens was conducted to examine the relationship between VIP and PREB mRNA expression. At day 14 of the embryonic growth stage, VIP treatment did not affect mRNA levels of either PRL or PREB, whereas forskolin treatment induced the increase of these mRNA levels. At day 20, both VIP and forskolin induced an increase of PRL and PREB mRNA levels. The administration of VIP significantly increased mRNA levels of PRL and PREB in the anterior pituitary gland of White Leghorn and Nagoya. These results indicate that the effects of VIP on PRL and PREB mRNA expression levels of VIP receptor may in turn affect PRL and PREB mRNA levels in the chicken anterior pituitary gland.

Effect of breeding stage and photoperiod on gonadal and serotonergic axes in domestic ganders

Reduction in reproductive potential of ganders with progress in seasonal breeding is a known problem in commercial geese production. The role of changes in hypothalamic–pituitary–gondal axis and testis functions in this process is not clear. This article presents studies on the hypothalamic (GnRH-I, vasoactive intestinal peptide), pituitary (LHβ, prolactin [PRL], PRL receptor [PRLR]), testis (PRLR) axis messenger RNA (mRNA) expression during different stages of the breeding period and photoperiodic conditions. Testis mass; histologic and functional (testosterone [T]) parameters; and plasma concentrations of T, LH, and PRL were evaluated. We collected (six times) samples from 2-year-old ganders (n = 48) maintained in short day (10L:14D) during the period from November to July. Moreover, in the peak of sexual activity (March), an additional group was on exposure (6 weeks) to long day (LD; 16L:8D). During the first half of reproduction (January, March; photosensitive period), GnRH-I (1.9 vs. 0.3 relative quantity [RQ]) and LHβ (3.0 vs. 0.7 RQ) mRNA transcript expression and concentrations of T (1.9 - 2.9 vs. 0.3 ng/mL), LH (13.6 - 7.4 vs. 0.7 ng/mL) were found to be higher (P < 0.05) than at the end of breeding (July). With progress in breeding, marked elevation (P < 0.05) in PRL (22.0 - 387.1 ng/mL) concentration related to similar changes in vasoactive intestinal peptide (0.9 - 3.0 RQ) and PRL mRNA abundance (1.3 - 11.5 RQ; May, July) was observed. However, testis PRLR mRNA increased (P < 0.05) only at the end of reproduction (1.2 RQ) compared to the peak of sexual activity (0.4 RQ; March). Furthermore, changes in mRNA transcript expression of the lactotrophic axis were accompanied with reduction of testis weight (left: 11.1 - 5.8 g), spermatogenesis (spermatogenic index: 5.4 - 3.0), and steroidogenesis (T: 24.8 - 1.3 ng/g testis), which may suggest their pivotal inhibitory modulation role in the regression of seasonal reproductive activity in ganders. The LD conditions (similar to spring-summer) resulted in earlier peripheral changes in T (0.9 vs. 1.8 ng/mL), LH (1.1 vs. 3.8 ng/mL), and PRL (296.1 vs. 161.2 ng/mL) concentrations than in short day, and this may be related to the advance in the timing of the sexual activity failure observed under natural light regimes. The lack of differences in gonadal and lactotrophic axis mRNA expression after LD treatment suggested a regulation based on the posttranslational mechanisms or modification of transcript or protein.

Protective Effect of Zengye Decoction () on Submandibular Glands in Nonobese Diabetic Mice.

To investigate the protective effect of Zengye Decoction (, ZYD) on the submandibular glands (SMGs) in nonobese diabetic (NOD) mice. Twenty-seven female NOD mice were randomly equally divided into 3 groups: the model group, the hydroxychloroquine (HCQ) group, and the ZYD group. Nine C57/B6 mice served as the normal group. After 1-week acclimation, the HCQ and ZYD groups were intragastrically administered with HCQ and ZYD, respectively, and the normal and model groups were administered with normal saline. Changes in the salivary flow rate were observed. Mice from all 4 groups were sacrificed at the age of 20 weeks. The serum and SMGs were collected. Serum cytokines gamma-interferon (IFN-γ), interleukin-10 (IL-10) were detected by enzyme-linked immunosorbent assay. Histological changes in the submandibular glands were examined by hematoxylin and eosin staining. The mRNA expression of IFN-γ, IL-10 and vasoactive intestinal peptide (VIP) in the submandibular glands were measured by real-time polymerase chain reaction. Compared with the model group, the salivary flow of the ZYD group significantly increased (P<0.05), the extent of the histological changes was ameliorated (P<0.05), and the Th1/Th2 cytokine imbalance was remedied (P<0.05). In the ZYD-treated mice, the VIP mRNA was up-regulated (P<0.05). ZYD is beneficial in protecting structure and function of SMGs in NOD mice. The mechanism may be associated with the correction of the Th1/Th2 cytokine imbalance, and with the prevention of a progressive decline of the VIP level.

Enhancement of catecholamine release from PC12 cells by the traditional Japanese medicine, rikkunshito.

BackgroundRikkunshito is a traditional Japanese herbal medicine that is used to treat appetite loss associated with cancer and other disorders. The formulation contains various constituents that influence cell signaling, and rikkunshito may accordingly affect human homeostasis through multiple regulatory pathways, including those governed by the endocrine system. We investigated the actions of rikkunshito on catecholamine release from PC12 cells, an adrenal chromaffin cell line.MethodsThe actions of rikkunshito on PC12 cells were evaluated by measuring intracellular cAMP levels, tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP) mRNA expression levels, and catecholamine levels in the culture medium. The transcriptional activation of VIP gene by rikkunshito was assessed by using a VIP promoter-driven reporter gene assay.ResultsRikkunshito dose-dependently enhanced forskolin-induced elevations in cAMP in PC12 cells, and also increased the gene expression of TH and VIP. The transcriptional activation of VIP gene by rikkunshito was confirmed. Norepinephrine and dopamine secretion into the culture medium of PC12 cells were also dose-dependently augmented by rikkunshito and/or forskolin, but experiments with a protein kinase C (PKC) activator and a phosphodiesterase inhibitor revealed that the effects of rikkunshito were not simply due to the modulation of PKC or phosphodiesterase activity.ConclusionsThese findings suggest that rikkunshito enhances the release of catecholamines by a novel mechanism involving cAMP.

The VIP/VPACR system in the reproductive cycle of male lizard Podarcis sicula

Starting from the knowledge that in the reproductive period the Vasoactive Intestinal Peptide (VIP) is widely distributed in Podarcis sicula testis, we studied VIP expression and the localization of the neuropeptide and its receptors in the testis of the Italian wall lizard P. sicula in the other phases of its reproductive cycle (summer stasis, autumnal resumption, winter stasis, spring resumption). By Real Time-PCR, we demonstrated that testicular VIP mRNA levels change during the reproductive cycle, showing a cyclic trend with two peaks, one in the mid-autumnal resumption and the other in the reproductive period. By in situ hybridization and immunohistochemistry, we demonstrated that both VIP mRNA and protein were widely distributed in the testis in almost all the phases of the cycle, except in the early autumnal resumption. As regards the receptors, the VPAC1R was localized mainly in Leydig cells, while the VPAC2R showed the same distribution of VIP.Our results demonstrate that, differently from mammals, where VIP is present only in nerve fibres innerving the testis, an endotesticular synthesis takes place in the lizard and the VIP synthesis changes throughout the reproductive cycle. Moreover, the VIP/VPAC receptor system distribution observed in germ and somatic cells in various phases of the cycle, and particularly in the autumnal resumption and the reproductive period, strongly suggests its involvement in both spermatogenesis and steroidogenesis. Finally, the wider distribution of VIP in lizards with respect to mammals leads us to hypothesize that during the evolution the synthesis sites have been transferred from the testis to other districts, such as the brain.

Vasoactive Intestinal Peptide Modulation of the Steroid-Induced LH Surge Involves Kisspeptin Signaling in Young but Not in Middle-Aged Female Rats

Age-related LH surge dysfunction in middle-aged rats is characterized, in part, by reduced responsiveness to estradiol (E2)-positive feedback and reduced hypothalamic kisspeptin neurotransmission. Vasoactive intestinal peptide (VIP) neurons in the suprachiasmatic nucleus project to hypothalamic regions that house kisspeptin neurons. Additionally, middle-age females express less VIP mRNA in the suprachiasmatic nucleus on the day of the LH surge and intracerebroventricular (icv) VIP infusion restores LH surges. We tested the hypothesis that icv infusion of VIP modulates the LH surge through effects on the kisspeptin and RFamide-related peptide-3 (RFRP-3; an estradiol-regulated inhibitor of GnRH neurons) neurotransmitter systems. Brains were collected for in situ hybridization analyses from ovariectomized and ovarian hormone-primed young and middle-aged females infused with VIP or saline. The percentage of GnRH and Kiss1 cells coexpressing cfos and total Kiss1 mRNA were reduced in saline-infused middle-aged compared with young females. In young females, VIP reduced the percentage of GnRH and Kiss1 cells coexpressing cfos, suggesting that increased VIP signaling in young females adversely affected the function of Kiss1 and GnRH neurons. In middle-aged females, VIP increased the percentage of GnRH but not Kiss1 neurons coexpressing cfos, suggesting VIP affects LH release in middle-aged females through kisspeptin-independent effects on GnRH neurons. Neither reproductive age nor VIP affected Rfrp cell number, Rfrp mRNA levels per cell, or coexpression of cfos in Rfrp cells. These data suggest that VIP differentially affects activation of GnRH and kisspeptin neurons of female rats in an age-dependent manner.