Chemical Modifications to Enhance the Drug Properties of a VIP Receptor Antagonist (ANT) Peptide.

Antagonist peptides (ANTs) of vasoactive intestinal polypeptide receptors (VIP-Rs) are shown to enhance T cell activation and proliferation in vitro, as well as improving T cell-dependent anti-tumor response in acute myeloid leukemia (AML) murine models. However, peptide therapeutics often suffer from poor metabolic stability and exhibit a short half-life/fast elimination in vivo. In this study, we describe efforts to enhance the drug properties of ANTs via chemical modifications. The lead antagonist (ANT308) is derivatized with the following modifications: N-terminus acetylation, peptide stapling, and PEGylation. Acetylated ANT308 exhibits diminished T cell activation in vitro, indicating that N-terminus conservation is critical for antagonist activity. The replacement of residues 13 and 17 with cysteine to accommodate a chemical staple results in diminished survival using the modified peptide to treat mice with AML. However, the incorporation of the constraint increases survival and reduces tumor burden relative to its unstapled counterpart. Notably, PEGylation has a significant positive effect, with fewer doses of PEGylated ANT308 needed to achieve comparable overall survival and tumor burden in leukemic mice dosed with the parenteral ANT308 peptide, suggesting that polyethylene glycol (PEG) incorporation enhances longevity, and thus the antagonist activity of ANT308.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Administration of a VIP-antagonist in vivo modifies ovarian hormone secretion in a rat model with polycystic ovary syndrome.

AIMS: In the cyclic rat in estrus, the vasoactive intestinal peptide (VIP) has an impact on ovarian function, which depends on the endocrine status of the animal. In this work, we aimed to clarify the participation of VIP in the pathophysiological condition of polycystic ovary syndrome (PCOS) using a model of PCOS induced by estradiol valerate (EV-PCOS) in rats. MAIN METHODS: In the cyclic rat in estrus and in the EV-PCOS model, we analyzed the acute effects of blocking VIP receptors with the use of an antagonist (Ant-VIP) injected into the left or right ovarian bursa on the steroidogenic response and ovarian catecholamine levels. KEY FINDINGS: In the cyclic animal in estrus, the treatment with Ant-VIP in the left ovarian bursa resulted in a reduction in testosterone serum levels and in ovarian levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), without changes in 4-hydroxy-3-methoxyphenyl (MHPG) and norepinephrine (NE). When the treatment was applied on the right side, only MHPG levels increased. In the EV-PCOS model, the treatment with Ant-VIP in the left ovarian bursa increased testosterone, estradiol, MHPG, and NE levels. When the treatment was performed on the right side, progesterone levels decreased and estradiol increased, without changes in ovarian catecholamines. SIGNIFICANCE: The binding of VIP to its receptors differentially regulates steroidogenesis in the cyclic animal in estrus and in the EV-PCOS model. The blocking of VIP signaling produces changes in ovarian catecholamines.

Vasoactive intestinal peptide regulates ileal goblet cell production in mice.

Innervation of the intestinal mucosa has gained more attention with demonstrations of tuft and enteroendocrine cell innervation. However, the role(s) these fibers play in maintaining the epithelial and mucus barriers are still poorly understood. This study therefore examines the proximity of mouse ileal goblet cells to neuronal fibers, and the regulation of goblet cell production by vasoactive intestinal peptide (VIP). An organotypic intestinal slice model that maintains the cellular diversity of the intestinal wall ex vivo was used. An ex vivo copper-free click-reaction to label glycosaminoglycans was used to identify goblet cells. Pharmacological treatment of slices was used to assess the influence of VIP receptor antagonism on goblet cell production and neuronal fiber proximity. Goblet cells were counted and shown to have at least one peripherin immunoreactive fiber within 3 µm of the cell, 51% of the time. Treatment with a VIP receptor type I and II antagonist (VPACa) resulted in an increase in the percentage of goblet cells with peripherin fibers. Pharmacological treatments altered goblet cell counts in intestinal crypts and villi, with tetrodotoxin and VPACa substantially decreasing goblet cell counts. When cultured with 5-Ethynyl-2'-deoxyuridine (EdU) as an indicator of cell proliferation, colocalization of labeled goblet cells and EdU in ileal crypts was decreased by 77% when treated with VPACa. This study demonstrates a close relationship of intestinal goblet cells to neuronal fibers. By using organotypic slices from mouse ileum, vasoactive intestinal peptide receptor regulation of gut wall goblet cell production was revealed.

Vasoactive intestinal peptide receptor-based imaging and treatment of tumors (Review).

Vasoactive intestinal peptide receptors (VIPRs) are members of the G-protein-coupled receptor superfamily. These receptors are overexpressed in many common malignant tumors and play a major role in the progression and angiogenesis of a number of malignancies. Therefore, VIPRs may be a valuable target for the molecular imaging of tumors and therapeutic interventions. The specific natural ligand or its analogs can be labeled with a radionuclide and used for tumor receptor imaging, which could be used to visualize VIPR-related surface protein expression in vivo and to monitor the in vivo effects of molecular drugs on tumors. Moreover, the involvement of VIPRs in malignant transformation and angiogenesis renders them potential therapeutic targets for cancer treatment. A variety of VIP antagonists and cytotoxic VIP conjugates have been synthesized and evaluated for VIPR-targeted molecular therapy. The importance of VIPRs in tumor biology and the ability to predict responses to targeted therapy and monitor drug interventions suggest that VIP receptor-based imaging and treatment will be critical for the early diagnosis and management of cancer. Here, we review the current literature regarding VIPRs and their natural ligands and the involvement of VIPRs in tumor growth and angiogenesis, with an emphasis on the present use of VIPRs for the molecular imaging of tumors and therapies targeting VIPRs.

VPAC receptor signaling modulates grouping behavior and social responses to contextual novelty in a gregarious finch: a role for a putative prefrontal cortex homologue.

In both mammals and birds, vasoactive intestinal polypeptide (VIP) neurons and fibers are present in virtually every brain area that is important for social behavior. VIP influences aggression in birds, social recognition in rodents, and prolactin secretion in both taxa, but other possible functions in social modulation remain little explored. VIP effects are mediated by VPAC receptors, which bind both VIP and pituitary adenylate cyclase activating peptide. Within the lateral septum and medial bed nucleus of the stria terminalis, VPAC receptors are found at higher densities in gregarious finch species relative to territorial species, suggesting that VPAC receptor activation promotes social contact and/or preference for larger groups. Here we here test this hypothesis in zebra finches (Taeniopygia guttata), and also examine the relevance of VPAC receptors to anxiety-like processes. Intraventricular infusions of the VPAC receptor antagonist, neurotensin6-11 mouseVIP7-28, strongly reduce social contact when animals are tested in a novel environment, and exert sex-specific effects on grouping behavior. Specifically, VPAC receptor antagonism reduces gregariousness in females but increases gregariousness in males. Interestingly, VPAC antagonism in the medial pallium (putative prefrontal cortex homologue) significantly reduces gregariousness in both sexes, suggesting site-specific effects of VIP signaling. However, VPAC antagonism does not modulate novel-familiar social preferences in a familiar environment or general anxiety-like behaviors. The current results suggest that endogenous activation of VPAC receptors promotes social contact under novel environmental conditions, a function that may be accentuated in gregarious species. Moreover, endogenous VIP modulates gregariousness in both males and females.

VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice.

Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-γ- and TNF-α-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.

Vasoactive intestinal polypeptide regulates barrier function via mast cells in human intestinal follicle-associated epithelium and during stress in rats.

BACKGROUND: Vasoactive intestinal polypeptide (VIP) has been implicated as a regulator of intestinal barrier function and inflammation. Our aim was to elucidate the role of VIP in follicle-associated epithelium (FAE) and villus epithelium (VE) permeability following stress in rats and on human intestinal barrier function. METHODS: Rats were injected intraperitoneally (i.p.) with VIP receptor-antagonists (anti-VPACs), a mast cell stabilizer, doxantrazole (DOX), or NaCl, and submitted to acute water avoidance stress. Ileal segments were mounted in Ussing chambers to assess (51) chromium-edta ((51) Cr-edta) and Escherichia (E.) coli (strain K-12) permeability. Rat ileal and human ileal and colonic segments were exposed to VIP ± anti-VPACs or DOX. An in vitro co-culture model of human FAE was used to study epithelial-VIP effects. VIP/VPACs distribution was assessed by microscopy. KEY RESULTS: Stress increased (51) Cr-edta and E. coli permeability in VE and FAE. The increases were abolished by i.p. injection of DOX or anti-VPACs. Ileal VIP-exposure ex vivo increased bacterial passage and this was reduced by DOX. In human FAE ex vivo, VIP treatment doubled bacterial uptake, which was normalized by DOX or anti-VPACs. No barrier effects were observed in human colonic tissue. VPACs were found in rat and human ileal follicles, with partial mast cell co-localization. The co-culture model confirmed VIP-mast cell-epithelial interactions in the regulation of barrier function. CONCLUSIONS & INFERENCES: Stress affects the FAE barrier by mechanisms involving VIP and VPACs on mucosal mast cells. We suggest a regulatory role for VIP in the control of ileal permeability that may be relevant to bacterial-epithelial interactions in stress-related intestinal disorders.

Pharmacological characterization and expression of VIP and PACAP receptors in isolated cranial arteries of the rat.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) are potent vasodilators in animals and humans. PACAP infusion but not VIP infusion precipitates migraine attacks in migraine patients. The vascular effects of VIP and the two varieties of PACAP (PACAP-27 and PACAP-38) were investigated versus selective antagonists in segments of rat middle cerebral arteries (MCA), basilar arteries (BA) and middle meningeal arteries (MMA) using myographs. The luminal and abluminal effects of VIP were studied using perfusion myograph. mRNA expression of the relevant receptors (VPAC(1), VPAC(2) and PAC(1)) was examined by in situ hybridization. There was no significant difference in relaxant potency of the peptides in the MCA. In BA the relaxant potency was VIP>PACAP-27=PACAP-38. Relaxant responses were either absent or very weak in MMA. VIP was found to be somewhat more potent in BA than in the MCA. Maxadilan, a selective PAC(1)-receptor agonist, showed no relaxant effect in either vessel. The VPAC(2)-antagonist PG 99-465 alone proved ineffective in the MCA, while it had a weak effect on BA. The VPAC(1)-antagonist PG 97-269 inhibited relaxation induced by both VIP and the PACAPs in cerebral vessels. In combination, the two antagonists demonstrated better effect than either alone. VIP applied luminally via perfusion myograph caused no dilatation, indicating lack of endothelial involvement. In situ hybridization demonstrated the presence of mRNA for all three receptors in the smooth muscle cells of the vessels. In conclusion, migraine-like headache induced by PACAP-38 infusion is unlikely to be caused by direct vasodilator action on intracranial vessels.

Umami receptor activation increases duodenal bicarbonate secretion via glucagon-like peptide-2 release in rats.

Luminal nutrient chemosensing during meal ingestion is mediated by intestinal endocrine cells, which regulate secretion and motility via the release of gut hormones. We have reported that luminal coperfusion of L-Glu and IMP, common condiments providing the umami or proteinaceous taste, synergistically increases duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers, taste receptor type 1, member 1 (T1R1)/R3. We hypothesized that glucose-dependent insulinotropic peptide (GIP) or glucagon-like peptide (GLP) is released by duodenal perfusion with L-Glu/IMP. We measured DBS with pH and CO(2) electrodes through a perfused rat duodenal loop in vivo. GIP, exendin (Ex)-4 (GLP-1 receptor agonist), or GLP-2 was intravenously infused (0.01-1 nmol/kg/h). l-Glu (10 mM) and IMP (0.1 mM) were luminally perfused with or without bolus intravenous injection (3 or 30 nmol/kg) of the receptor antagonists Pro(3)GIP, Ex-3(9-39), or GLP-2(3-33). GIP or GLP-2 infusion dose-dependently increased DBS, whereas Ex-4 infusion gradually decreased DBS. Luminal perfusion of l-Glu/IMP increased DBS, with no effect of Pro(3)GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited L-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6-28) intravenously or N(G)-nitro-L-arginine methyl ester coperfusion inhibited the effect of L-Glu/IMP. Perfusion of L-Glu/IMP increased portal venous concentrations of GLP-2, followed by a delayed increase of GLP-1, with no effect on GIP release. GLP-1/2 and T1R1/R3 were expressed in duodenal endocrine-like cells. These results suggest that luminal L-Glu/IMP-induced DBS is mediated via GLP-2 release and receptor activation followed by VIP and nitric oxide release. Because GLP-1 is insulinotropic and GLP-2 is intestinotrophic, umami receptor activation may have additional benefits in glucose metabolism and duodenal mucosal protection and regeneration.

Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter.

BACKGROUND & AIMS: To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS: We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS: The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS: Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.

Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery.

OBJECTIVE: We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries. METHODS: Concentration response curves to PACAP38, PACAP27, VIP and the VPAC(1) receptor agonist ([Lys15,Arg16,Leu27]-VIP[1-7]-GRF[8-27]) were constructed in the absence or presence of the PAC(1) receptor antagonist PACAP6-38 or the VPAC(1) receptor antagonist, PG97269. mRNA expression was measured using qPCR. RESULTS: PACAP38 was less potent than VIP in both arteries. Both peptides had lower potency and efficacy in meningeal than in coronary arteries, while mRNA expression of VPAC(1) receptor was more pronounced in meningeal arteries. PACAP6-38 reduced the E(max) of PACAP27, while PG97269 right-shifted the VIP-induced relaxation curve only in the coronary arteries. CONCLUSION: The direct vasodilatory effect of VIP and PACAP might be less relevant than the central effect of this compound in migraine pathogenesis.

The vasoactive intestinal peptide-receptor system is involved in human glioblastoma cell migration.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor in adults. This cancer has an infiltrative nature and the median survival of patients is about one year. Vasoactive intestinal peptide (VIP) belongs to a structurally related family of polypeptides and is a major regulatory factor in the central and peripheral nervous systems. VIP regulates proliferation of astrocytes and of numerous cancer cell lines and modulates migration in prostatic and colonic cancer cell lines. Little is known about the involvement of VIP and its receptors (VIP-receptor system) in proliferation or migration of GBM cells. The effects of VIP, PACAP and of synthetic VIP antagonists were tested in two human GBM cell lines, M059K and M059J, established from two different parts of a single tumor. In these cells, the data revealed that the VIP-receptor system did not affect proliferation but controlled cell migration. Indeed, in M059K cells which express components of the VIP receptor system, the VIP receptor antagonists and a PACAP antibody enhanced migration. The VIP receptor antagonists increased generation of typical migration-associated processes: filopodia and lamellipodia, and activation of Rac1 and Cdc42 GTPases. Reciprocally, in M059J cells which poorly express the VIP-receptor system, treatments with the agonists VIP and PACAP resulted in decreased cell migration. Furthermore, the peptides appeared to act through a subclass of binding sites displaying an uncommon very high affinity for these ligands. Taken together, these observations suggest that components of the VIP-receptor system negatively regulate cell migration, thus showing potential anti-oncogenic properties.

Involvement of vasoactive intestinal polypeptide in the parasympathetic vasodilatation of the rat masseter muscle.

The parasympathetic vasodilatory fibres are known to innervate vessels in a rat masseter muscle via both cholinergic and non-cholinergic mechanisms. However, the non-cholinergic mechanisms are still unclear. Recently, vasoactive intestinal polypeptide (VIP) was convincingly shown to be involved in the parasympathetic vasodilatation in orofacial areas, such as submandibular glands and lower lip. However, very little is known about the rat masseter muscle. The present study was designed in the rat masseter muscle to assess (1) whether the parasympathetic nerve innervating vessels have VIP immunoreactivities, (2) whether intravenous administration of VIP induces the vasodilatation, and (3) effects of selective VIP receptor antagonist ([4Cl-d-Phe(6), Leu(17)] VIP) in the presence or absence of atropine on the parasympathetic vasodilatation. The VIP immunoreactivities were found at two sites of the parasympathetic otic ganglion and nerve fibres located around vessels. The intravenous administration of VIP induced the vasodilatation, and [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the vasodilatation evoked by VIP administration. The parasympathetic vasodilatation was not inhibited by [4Cl-d-Phe(6), Leu(17)] VIP. However, treatment with [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the parasympathetic vasodilatation when [4Cl-d-Phe(6), Leu(17)] VIP was administered together with atropine. These results suggest that (1) VIP exists in the postganglionic parasympathetic nerve innervating the vessels in the masseter muscle, (2) the intravenous administration of VIP induces the vasodilatation in the masseter muscle, and (3) VIP may be involved in the parasympathetic vasodilatation in the masseter muscle when muscarinic cholinergic receptors are deactivated by either atropine or the suppression of the ACh release.

Role of CREB in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells.

Vasoactive intestinal peptide (VIP) is one of the most important sensory neuropeptides in respiratory system. We previously reported that VIP enhances wound healing and proliferation of human bronchial epithelial cells (HBECs), and these effects are mediated by intracellular signaling molecules such as protein kinase A (PKA), protein kinase C (PKC), Calmodulin (CaM), and extracellular signal-regulated kinase (ERK). In the present study, we further investigated the role of cAMP Response Element Binding Protein (CREB) in VIP-promoted protective effects in mechanical-damaged HBECs. VIP-mediated wound healing and cell proliferation in HBECs was inhibited by CREB antisense oligonucleotides (ASO) in a time-dependent manner. VIP increased the CREB DNA binding activity and expression of the p-CREB that were inhibited by VIP receptor antagonist. Increased CREB DNA binding activity and expression of the p-CREB were also partially inhibited by PKA and ERK inhibitors. These results suggest that the VIP-mediated wound repair of HBECs is associated with activation of CREB via PKA and ERK dependent pathway.

Vasoactive intestinal peptide acts via multiple signal pathways to regulate hippocampal NMDA receptors and synaptic transmission.

Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide, which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase-activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1-R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1-R only if it is used at relatively high concentrations (e.g., 100 nM); however, at lower concentrations (e.g., 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1-R activation signals through PKC/CAKbeta/Src pathway to regulate NMDA receptors; however, there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1 nM VIP enhanced NMDA currents by stimulating the VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac-Tyr(1), D-Phe(2)]GRF (1-29). This enhancement of NMDA currents was blocked by both Rp-cAMPS and PKI(14-22) (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP-induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g., 100 nM) was mediated by PAC1-R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways.

[Effect of vasoactive intestinal peptide receptor antagonist VIPhybrid on the development of form deprivation myopia in chicks].

OBJECTIVE: To investigate the effect of regulation of VIPhybrid, an unselective antagonist of vasoactive intestinal peptide receptors (VIPR), on the formation and development of form deprivation myopia (FDM) in chick and the expression of protein and mRNA of VIP on the retina and choroids of in chicks. METHODS: Seventy-two 1-day-old yellow healthy leghorn chicks were assigned into 6 groups (12 in each group). Eyes in Group I were covered on the right as a blank control group. Eyes in GroupII were those eyes having been injected with 20 microL saline into vitreous cavity and then covered as a negative control group. Eyes in GroupIII,IV and V were injected with 20 microL VIPhybrid with low (3*10(-12) mol/L), middle (3*10(-10) mol/L) and high (3*10(-8) mol/L) dosage into vitreous cavity and then covered as experimental groups. The above groups had been continuously covered for 1 week. Eyes in Group VI were uncovered and uninjected as a normal control group. Diopter was detected using retinoscopic refraction. The eyeball axis was determined using ophthalmological ultra-A. The expression of protein and mRNA of VIP on retina-choroids-sclera were investigated by SP immunohistochemistry staining and RT-PCR. RESULTS: Form deprivation for 1 week induced high myopia eyes and elongated eyeball axis in GroupI and GroupII, and there was no difference between the 2 groups (P>0.05). The diopter and eyeball axis were significantly reduced in Group III, IV, and V as compared with Group I and II (P<0.01), but the diopter was higher and the eyeball axis was longer than those of Group VI. The diopter and eyeball axis had negative correlation with the concentration gradient of VIPhybrid. The expressions of protein and mRNA of VIP in Group III, IV, and V were down-regulated as compared with those of Group I and I I(P<0.01)and also down-regulated with the increase of concentration of VIPhybrid. CONCLUSION: VIPhybrid can decrease the development of FDM in chicks, which may provide a new pathway for drug therapy of myopia in human beings.

Structure-activity relationship of vasoactive intestinal peptide (VIP): potent agonists and potential clinical applications.

Vasoactive intestinal peptide (VIP) has been identified as one of major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. The general physiologic effects of VIP include vasodilation, anti-inflammatory actions, cell proliferation, hormonal secretion, regulation of gastric motility, and smooth muscle relaxation; therefore, VIP has emerged as a promising drug candidate for the treatment of several diseases. A number of clinical applications of VIP or its derivatives have been developed; however, VIP-based drugs are not yet in clinical use, possibly because of mainly two serious problems: (1) poor metabolic stability and (2) poor penetration to the desired site of action. To overcome these shortcomings, the development of efficacious VIP analogues and several drug delivery systems has been attempted on the basis of numerous structure-activity relationships (SAR) studies and pharmacological experiments. Combination of the use of potent VIP analogues and an appropriate drug delivery system might be advantageous for the VIP-based therapy. We review in this paper SAR studies of VIP for the identification of potent therapeutic agents, describe the development of selective and/or metabolically stable VIP receptor agonists/antagonists, and discuss the potential application for clinical treatment using drug delivery systems.

Pharmacological analysis of components of the change in transmural potential difference evoked by distension of rat proximal small intestine in vivo.

The reflex response to distension of the small intestine in vivo is complex and not well understood. The aim of this study was to characterize the neural mechanisms contributing to the complex time course of the intestinal secretory response to distension. Transmucosal potential difference (PD) was used as a marker for mucosal chloride secretion, which reflects the activity of the secretomotor neurons. Graded distensions (5, 10, and 20 mmHg) of distal rat duodenum with saline for 5 min induced a biphasic PD response with an initial peak (rapid response) followed by a plateau (sustained response). The rapid response was significantly reduced by the neural blockers tetrodotoxin and lidocaine (given serosally) and by intravenous (iv) administration of the ganglionic blocker hexamethonium and the NK(1) receptor antagonist SR-140333. Serosal TTX and iv SR-140333 significantly reduced the sustained response, which was also reduced by the NK(3) receptor antagonist talnetant and by the vasoactive intestinal polypeptide (VPAC) receptor antagonist [4Cl-d-Phe(6), Leu(17)]-VIP. Serosal lidocaine and iv hexamethonium had no significant effect on this component. Inhibition of nitric oxide synthase had no effect on any of the components of the PD response to distension. The PD response to distension thus seems to consist of two components, a rapidly activating and adapting component operating via nicotinic transmission and NK(1) receptors, and a slow component operating via VIP-ergic transmission and involving both NK(1) and NK(3) receptors.

Transactivation of HER2 by vasoactive intestinal peptide in experimental prostate cancer: Antagonistic action of an analog of growth-hormone-releasing hormone.

Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate cancer therapy.