High levels of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor mRNA expression in primary and tumor lymphoid cells

Neuropeptides exert a variety of putative immunomodulatory actions. Despite the molecular cloning of multiple forms of receptors for several neuropeptides with putative immunomodulatory effects, including vasoactive intestinal peptide (VIP), the related peptide pituitary adenylate cyclase-activating peptide (PACAP), the opiate peptides, tachykinins, somatostatin and corticotropin-releasing factor, it has not been reported that any of the receptor genes are expressed at significant levels in cells of the immune system. The low level of expression of these receptors and lack of knowledge concerning receptor subtype has impeded progress in understanding how neuropeptides regulate immune function. For example, it is not understood why VIP produces immunomodulatory effects at concentrations far below its receptor-binding affinity. Receptors for VIP and PACAP have recently been cloned. We show here by Northern blot analysis that the VIP/PACAP₁ receptor mRNA is present in total RNA prepared from mouse spleen B- and T-lymphocytes. The VIP/PACAP₁ receptor mRNA was also present in human peripheral blood lymphocytes, and in a B-lymphocyte and a myelocytic cell line. The mRNA for a second form of the receptor, the VIP/PACAP₂ receptor, was not expressed at detectable levels in normal cells, but was detected in several human T-cell lines and a murine mast cell line. The results indicate that VIP/PACAP₁ and perhaps VIP/PACAP₂ receptors mediate the diverse effects of VIP and PACAP on immune cells.     

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.     

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.     

Detection of vasoactive intestinal peptide and localization of its mRNA within granulomas of murine schistosomiasis

Schistosomiasis mansoni is a parasitic disease resulting in the deposition of ova predominantly in the liver and intestines. These ova secrete antigens which induce host sensitization and evoke focal granulomas. The granulomas are intricate delayed-hypersensitivity reactions governed by numerous cellular and humoral interactions. They displace or destroy normal tissue. Vasoactive intestinal peptide (VIP) is one of several neuropeptides which exert a broad range of biologic actions that may include modulation of immune responses. In this study, VIP was sought within liver granulomas isolated from Schistosoma mansoni-infected, CBA/J mice. Granuloma extracts contained appreciable amounts of immunoreactive VIP as detected by radioimmunoassay. Immunoreactive VIP was shown, by each of two chromatographic methods, to elute as a single peak coinciding with that of synthetic VIP. In situ hybridization was performed with an oligonucleotide probe complementary to a portion of the nucleotide sequence encoding VIP on preproVIP mRNA (antisense probe). Radiolabeled VIP probe adhered exclusively to granuloma eosinophils and to eosinophils within a peritonitis induced in normal mice by proteose peptone. Hybridization of radiolabeled VIP probe in the presence of unlabeled probe substantially attenuated binding. A sense probe failed to bind. These data suggest that the granulomas contain authentic VIP and that eosinophils express the gene for this molecule.     

Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat

The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited a specific pattern of accumulation, suggesting proper regulation steps for the expression of the corresponding digestive enzymes.     

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.     

猕猴发育过程中肠肝组织血管活性肠肽及其受体的变化

本文旨在探讨猕猴发育过程中血管活性肠肽(vasoactive intestinal polypeptide,VIP)及其受体在肠肝组织的变化。通过手术途径获得胚胎6月、新生2 d、新生45 d和成年猕猴的回肠、肝脏、门静脉和外周血等标本,应用放射免疫分析法测定各标本中的VIP含量;通过免疫组化方法观察VIP在肠、肝组织内的分布;利用原位杂交法检测VIP受体1(VIP receptor 1,VIPR1)的表达。结果显示:(1)胚胎6月的猕猴小肠VIP含量为(20．7±14．3)ng／mg蛋白;小肠绒毛根部及黏膜下层可见少量的VIP阳性染色颗粒;在发育过程中,小肠VIP含量逐渐增加,成年期时达(514．8±49．2)ng／mg蛋白,较胚胎6月显著增加(P<0．01)。(2)成年猕猴小肠VIP主要分布于绒毛隐窝部、黏膜下层神经及环、纵行肌间神经丛及环行肌,在发育过程中相应部位的VIPR1表达逐渐上调。(3)肝脏在发育过程中VIP及VIPR1含量逐渐降低。(4)发育的各个时期,小肠组织的VIP含量均明显高于肝脏组织,门静脉VIP水平也始终高于外周血。结果提示,小肠绒毛隐窝部、黏膜下层神经及环、纵行肌间神经内VIP及VIPR1含量足在出生以后才迅速增加的;不论是在胚胎还是成年期,VIP均不在肝中代谢和分解,VIPR1仅见于胚胎肝脏血管。     

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.     

Pineal vasoactive intestinal peptide is reduced during the proestrous stage of the rat estrous cycle

Immunoreactive levels of vasoactive intestinal peptide (IR-VIP) in the rat pineal gland were examined during the estrous cycle. IR-VIP was shown to be identical to the synthetic porcine material by its similarity in competitive binding studies, and by both gel filtration and high pressure liquid chromatography. Pineal IR-VIP decreased at early proestrus (0300 h, dark), partially as a function of the rise in serum estradiol levels. Although the functional role of VIP in the pineal remains to be clearly elucidated, these results suggest that it might be involved in the reproductive function of the female rat, and that estrogens could partially modulate its pineal concentration.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.     

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.     

Release of vasoactive intestinal peptide in the dumping syndrome.

To determine the effect of gastric surgery on the plasma vasoactive intestinal peptide (VIP) concentration, 13 patients with gastrectomy and seven controls were given an oral hypertonic load (200 ml 50% glucose solution). Blood was taken at intervals during the test for measurement of VIP and blood glucose concentrations and packed cell volume. At the same time observations were made on the occurrence of dumping symptoms and a record kept of the pulse rate. VIP values in the patients with gastrectomy were significantly increased by glucose ingestion, while these did not alter in controls (p less than 0.001). There was a highly significant correlation between the rate of rise in plasma VIP concentration and the rates of rise in packed cell volume (r = 0.85; p less than 0.001) and blood glucose concentration (r = 0.76; p less than 0.01) in patients with gastrectomy. Changes in packed cell volume and blood glucose values and the occurrence of dumping symptoms during the test were significantly different when postoperative patients were compared with controls (p less than 0.001, p less than 0.005, and p less than 0.001 respectively). Furthermore, when the patients with gastrectomy were divided into those without symptoms and those with dumping after meals the latter group showed a significantly greater rise of VIP (p less than 0.05). Despite the increased plasma VIP concentrations observed during dumping, VIP cannot be taken as the sole factor in the pathogenesis of the dumping syndrome.     

Autoantibody to vasoactive intestinal peptide in human circulation

In a radioassay for Vasoactive Intestinal Peptide (VIP)-binding, eight out of 33 plasma samples from healthy human subjects exhibited specific binding ranging from 2.6% to 46.7% of total [125 I]VIP. This binding was competitively displaced by unlabeled VIP. The structurally homologous peptides, Peptide Histidine Isoleucine (PHI) and secretin, were, respectively, 72-fold and 413-fold less potent than VIP in displacing bound [125 I]VIP, whereas the unrelated peptides, neurotensin, eledoisin, bombesin and metenkephalin, were without effect on the binding. The antibody nature of the VIP-binding factor was suggested by its precipitation with ammonium sulfate, attenuation after absorption with Staphylococcus aureus preparations, precipitation with antisera against human IgG and IgM, and coelution with standard IgG and IgM on anion-exchange and high-performance gel-filtration columns. Pepsin treatment of purified IgG fraction yielded a VIP-binding species with apparent molecular weight of 108 +/- 13 kDa that was precipitated by antiserum against the F(ab)2 fragment of the IgG molecule. These results demonstrate the existence in some human plasmas of an autoantibody that binds VIP.     

Pulmonary vasodilator responses to vasoactive intestinal peptide in the cat

We investigated the effects of vasoactive intestinal peptide (VIP) in the feline pulmonary vascular bed under conditions of controlled pulmonary blood flow when pulmonary vascular tone was at base-line levels and when vascular resistance was elevated. Under base-line conditions, VIP caused small but significant reductions in lobar arterial pressure without affecting left atrial pressure. Decreases in lobar arterial pressure in response to VIP were greater and were dose related when lobar vascular resistance was increased by intralobar infusion of U 46619, a stable prostaglandin endoperoxide analogue. Acetylcholine and isoproterenol also caused significant decreases in lobar arterial pressure under base-line conditions, and responses to these agents were enhanced when lobar vascular tone was elevated. Moreover, when doses of these agents are expressed in nanomoles, acetylcholine and isoproterenol were more potent than VIP in decreasing lobar arterial pressure. Responses to VIP were longer in duration with a slower onset than were responses to acetylcholine or isoproterenol. Pulmonary vasodilator responses to VIP were unchanged by indomethacin, atropine, or propranolol. The present data demonstrate that VIP has vasodilator activity in the pulmonary vascular bed and that responses are dependent on the existing level of vasoconstrictor tone. These studies indicate that this peptide is less potent than acetylcholine or isoproterenol in dilating the feline pulmonary vascular bed and that responses to VIP are not dependent on a muscarinic or beta-adrenergic mechanism or release of a dilator prostaglandin.     

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).     

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.     

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.     

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.