Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).     

Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands

We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.     

Surface-active properties of vasoactive intestinal peptide*

The purpose of this study was to determine whether human vasoactive intestinal peptide (VIP) aggregates in aqueous solution and, if so, whether the peptide interacts with a biomimetic phospholipid monolayer and increases surface pressure. Using a custom-made Teflon trough containing HEPES buffer (pH 7.4) at room temperature and a surface tensiometer, we found that the critical micellar concentration (CMC) of VIP is 0.4 microM. Surface pressure of a dipalmitoylphosphatidylcholine (DPPC) monolayer spread over the HEPES buffer declined significantly over 120 min because of phospholipid decomposition. However, injection of VIP at concentrations above CMC into the subphase of the monolayer elicited a significant concentration-dependent increase in surface pressure that persisted for 120 min (P < 0.05). Unlike VIP, injection of [(8)Arg]-vasopressin at an equimolar concentration only prevented the time-dependent decline in DPPC monolayer surface pressure. Taken together, these data indicate that human VIP aggregates in aqueous solution and expresses surface-active properties at physiological concentrations in vitro. We suggest that these attributes could have a role in modulating the bioactive effects of the peptide in vivo.     

Decreased binding of vasoactive intestinal peptide to intestinal epithelial cells from hypothyroid rats

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase by VIP were studied in intestinal epithelial cells during hypothyroidism. Experimental hypothyroidism was induced in rats by the administration of KC10(4). The binding capacity, but not the affinity, of VIP receptors decreased in the hypothyroid rats. Besides, the stimulation of cyclic AMP production by VIP was also diminished in cells from hypothyroid rats. These observations indicate a decrease of the responsiveness of intestinal epithelial cells to VIP in the hypothyroid status, suggesting a role of the peptide in the pathophysiologic mechanism of intestinal manifestations during hypothyroidism.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.     

Characterization of vasoactive intestinal peptide receptors by a photoaffinity label. Site-specific modification of vasoactive intestinal peptide by derivatization of the receptor-bound peptide

The biological effects of vasoactive intestinal peptide (VIP) are mediated by binding to a membrane-bound receptor. Probes designed to trap this receptor by binding to it in a covalent way may suffer from a greatly reduced affinity. We report here, for the VIP receptor, the use of a photoaffinity probe obtained by derivatization of receptor-bound VIP with para-azidophenylglyoxal. This method protected the parts of the molecule essential for receptor binding. The VIP derivative thus obtained became covalently linked when irradiated. In the dark, however, it exhibited normal VIP-like behavior and retained its biological activity. This derivatization method might be generally applicable when hormone analogues have to be prepared without loss of receptor affinity. Receptor characterization studies on liver plasma membranes showed the presence of high- and low-affinity binding sites with KD = 0.1 and 5 nM, respectively. Treatment of membranes with dithiothreitol causes loss of high-affinity binding. The high-affinity site, trapped by the photoaffinity probe, resolved into two molecular mass forms, 50 and 200-250 kDa. Reduction of the receptor-probe complex left the 50-kDa form intact, whereas the amount of the 200-250-kDa form greatly diminished. We demonstrate the importance of the presence of disulfide bonds in one of the molecular forms involved in high-affinity binding.     

Immunoeffector and immunoregulatory activities of vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1R and VPAC2R, are prominent in the immune system and potently affect T cells and macrophages. VPAC1Rs are expressed constitutively by blood and tissue T cells, with an order of prevalence of Th2>Th1>Ts, and transmit signals suppressive for migration, proliferation and cytokine production. Immune activation of T cells downregulates VPAC1Rs and upregulates VPAC2Rs. VPAC2Rs mediate T cell chemotaxis, stimulation of some Th2-type cytokines, and inhibition of some Th1-type cytokines. A tentative hypothesis that the VIP-VPAC2R axis is the major neuroregulator of Th2/Th1 balance has been confirmed by finding an increased ratio in CD4 T cells of transgenic (TG) mice, expressing high levels of VPAC2Rs, and a decreased ratio in CD4 T cells of VPAC2R-null (K/O) mice. VPAC2R TG mice exhibit an allergic phenotype, whereas the K/O mice are hypoallergic and have heightened delayed-type hypersensitivity. The mechanisms of VIP-VPAC2R effects include decreased Th2 apoptosis, increased Th2-type cytokine production, and greater generation of Th2 memory cells. VPAC2R antagonists are being developed to alleviate allergic diseases and strengthen effector Th1 cell-mediated immunoprotection.     

Cholecystokinin, vasoactive intestinal peptide and peptide histidine methionine responses to feeding in anorexia nervosa.

Anorexia nervosa (AN) is a syndrome of unknown cause characterized by voluntary starvation. Cholecystokinin has been implicated as a neuroendocrine regulatory factor in control of satiety. Relatively little information is known about gastrointestinal hormone responses to feeding in subjects with anorexia nervosa. In the present studies, we examine fasting and postprandial levels of cholecystokinin (CCK), vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in anorexia nervosa subjects and in control individuals. Results of these studies indicate that plasma CCK response to a liquid meal (Ensure Plus) in untreated AN subjects was distinctly different from that observed in healthy controls, both in terms of temporal pattern of peptide released and the amount of CCK secreted into the circulation. Peak levels of CCK release occurred at 30 min following meal ingestion in AN patients and at 60 min in control subjects. Integrated CCK release in untreated AN patients was approximately twice that measured in control individuals. Renutrition therapy was associated with reversion of the pattern of CCK release to that observed in control subjects. Plasma VIP levels were unchanged following meal ingestion in both control and anorexic subjects. In contrast, PHM levels in AN subjects were significantly greater than that observed in control individuals. The pattern of PHM release following liquid meal ingestion was similar to that observed with plasma CCK; namely, peak release of peptide was observed at 30 min which was significantly greater than corresponding control values (P less than 0.05). In conclusion, these results demonstrate distinctive differences in plasma CCK and PHM levels in response to feeding in AN subjects when compared to control individuals. These findings suggest that the earlier and greater rise in plasma CCK levels in AN subjects following meal ingestion may contribute to the abnormal sensation of satiety in this condition.     

Stimulation of prolactin release in turkeys by vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a potent releasor of prolactin in birds. The main purpose of this study was to identify its site of action. Synthetic porcine VIP administered intraatrially to freely moving ovariectomized (OVX) turkeys induced an elevation of circulating PRL within 15 min in a dose-related manner. Removal of hypothalamic control of PRL release by surgical disconnection of the neurohemal regions of the median eminence did not significantly diminish the PRL response to VIP. Intraatrial injection of eledoisin or bradykinin into OVX hens did not influence PRL secretion, indicating that the PRL releasing activity of VIP is probably not attributable to its vasodilatory action. These results support the possibility that VIP is an authentic prolactin releasing factor (PRF) in birds.     

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5-,-imidotriphosphate (Gpp(NH)p) and guanosine 5-O-(3-thiotriphosphate) (GTP--S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP--S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.     

IL-4 inhibits vasoactive intestinal peptide production by macrophages

In schistosomiasis, eggs induce granulomas that have a vasoactive intestinal peptide (VIP) immunoregulatory circuit. This study explored the regulation of VIP production at sites of inflammation. Splenocytes from uninfected C57BL/6 mice expressed VIP mRNA and protein, which stopped following egg deposition. Eggs induce a Th2 response, suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP. To address this issue, splenocytes from uninfected mice were incubated for 4 h with or without recombinant IL-4. IL-4 inhibited VIP mRNA expression. F4/80+ macrophages were the source of constitutively expressed VIP, subject to IL-4 regulation. In IL-4 knockout mice, splenic VIP production did not downmodulate during schistosome infection, suggesting that IL-4 is a critical cytokine regulating VIP production in wild-type mouse spleen. IL-4-producing granulomas in schistosomiasis made VIP. Experiments showed that granuloma VIP derived from F4/80- (nonmacrophage) cell populations, explaining this paradox. Granuloma F4/80+ cells from IL-4 knockout mice expressed VIP. Thus macrophages can make VIP, which is subject to IL-4 regulation. However, in the Th2 granulomas, other cell types produce VIP, which compensates for loss of macrophages as a source of this molecule.     

Chemical modification of guanidinium groups of vasoactive intestinal peptide

Molecular characterization of receptors depends on the availability of ligand derivatives carrying a reactive group to covalently link the active sites. Two vasoactive intestinal peptide (VIP) derivatives, each labeled either at the two arginine residues 12 and 14 or singly in position 14, were prepared. In the first case, this was achieved by a selective chemical modification using azidophenylglyoxal. In the second, the amino acids of VIP, buried in the active site of the receptor, were protected and one arginine residue of bound VIP was successfully modified using azidophenylglyoxal. The two molecules were resolved by radioimmunocompetition and reversed phase high performance liquid chromatography. Identification of sites of labeling was achieved by tryptic peptide mapping and amino acid analysis. One derivative (Az-Bz-Arg14-VIP) retains a high binding affinity for the receptor and was found to be biologically active. The present method yields a derivative which is useful in structural analysis of the receptor.     

Passage of vasoactive intestinal peptide across the blood-brain barrier

We investigated the ability of vasoactive intestinal peptide (VIP) to cross the blood-brain barrier (BBB), the interface between the peripheral circulation and central nervous system (CNS). VIP labeled with 131I (I-VIP) and injected intravenously into mice was taken up by brain as determined by multiple-time regression analysis. Excess unlabeled VIP was unable to impede the entry of I-VIP, indicating that passage is by nonsaturable transmembrane diffusion. High pressure liquid chromatography (HPLC) showed the radioactivity entering the brain to be intact I-VIP. After intracerebroventricular (i.c.v.) injection, I-VIP was sequestered by brain, slowing its efflux from the CNS. In summary, VIP crosses the BBB unidirectionally from blood to brain by transmembrane diffusion.     

Intracerebroventricular administration of vasoactive intestinal peptide inhibits food intake

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide expressed throughout the peripheral and central nervous systems. VIP and the VIP receptor VPAC(2)R are expressed in hypothalamic nuclei involved in the regulation of energy homeostasis. VIP has been shown to be involved in the regulation of energy balance in a number of non-mammalian vertebrates. We therefore examined the effects of intracerebroventricular (ICV) administration of VIP on food intake, energy expenditure and activity in adult male Wistar rats. VIP administration caused a potent short lived decrease in food intake and an increase in activity and energy expenditure. The pathways potentially involved in the anorexigenic effects of VIP were investigated by measuring the release of neuropeptides involved in the regulation of food intake from hypothalamic explants treated with VIP. VIP significantly stimulated the release of the anorexigenic peptide alpha-melanocyte stimulating hormone (αMSH). These studies suggest that VIP may have an endogenous role in the hypothalamic control of energy homeostasis.     

A recombinant adenoviral vector encoding functional vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a small neuropeptide, which exerts pleiotropic functions. Based on its immunomodulatory, secretory, and possibly trophic effects, VIP is a valuable candidate molecule for the management of autoimmune disease. The purpose of this study was to develop a recombinant viral vector capable of directing the expression of functional VIP. The vector rAd5CMVhVIP was constructed and used to infect 293 cells. VIP expression was measured by an ELISA and function was evaluated by measurement of intracellular cAMP formation. rAd5CMVhVIP directed VIP expression and the transgenic VIP elicited a dose-dependent increase of intracellular cAMP, mediated through the VIP receptor VPAC(1). This is the first report showing the construction of a recombinant viral vector encoding biologically active VIP.     

Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.     

The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs in vitro and in vivo. The VIP/PACAP-generated DCs are CD11c(low)CD45RB(high), do not up-regulate CD80, CD86, and CD40 following LPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptor and protein kinase A, and correlates with the inhibition of IkappaB phosphorylation and of NF-kappaBp65 nuclear translocation. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previously described Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensitivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege.     

Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection

Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated c y c l i c AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine . The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation.