Vasoactive intestinal peptide as a bronchodilator in asthmatic subjects

Vasoactive intestinal peptide (V.I.P.) caused bronchodilatation in 7 asthmatic volunteers when given intravenously at the rate of 6 pmol kg-1 min-1 for 15 minutes during a double blind study. Mean baseline FEV1 was 2.8 (+/- 0.3 S.E.) which was 81% of predicted and increased by 0.21 (range 0.1-0.45) l after 15 minutes infusion (p greater than 0.02). Tachycardia and cutaneous flushing were also observed during the infusion. Subsequent induced bronchoconstriction with a predetermined dose of histamine was ameliorated at 180 seconds following challenge when compared with placebo. Mean fall in FEV1 0.26 compared with 0.741 when pre-infusion FEV1 was taken on baseline. Mean fall in FEV1 0.49 l compared with 0.75 l when the FEV1 immediately preceding challenge was used on baseline (p greater than 0.02). The demonstration that V.I.P. is a bronchodilator in asthmatics and ameliorates histamine induced bronchoconstriction has important implications for the pharmacology of asthma.     

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögren’s syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.     

Vasoactive intestinal peptide receptor activity in human fetal enterocytes

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind ¹²⁵I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of ¹²⁵I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 × 10⁻¹⁰ M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10⁻⁷M and above) in the following order of potency: VIP> PHI> GRF> secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9–12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.     

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

Vasoactive intestinal peptide stimulation of pineal serotonin-N-acetyltransferase activity: general characteristics

Vasoactive intestinal polypeptide (VIP) stimulates basal serotonin-N-acetyltransferase (NAT) activity in pineals in organ culture and enhances the effects of catecholamines in inducing the enzyme. VIP appears to act postsynaptically; its action is independent of the beta receptor and is dependent upon protein synthesis. Its effects may be mediated by a receptor. The magnitude of the pineal response to VIP varies with age, is greater in pineals maintained in 48-h organ culture than in those in acute culture, and can be detected in pineals from newborns after 48-h organ culture. Intravenous administration of VIP can increase pineal NAT activity in vivo.     

Ultrastructure of cerebrospinal fluid-contacting neurons immunoreactive to vasoactive intestinal peptide and properties of the blood-brain barrier in the lateral septal organ of the duck

Immuno-electron-microscopic investigations of cerebrospinal fluid (CSF)-contacting neurons immunoreactive to vasoactive intestinal peptide in the duck lateral septum have revealed that this cell type gives rise to an adventricular dendrite terminating with a bulbous swelling in the lateral ventricle. The swelling bears a cilium and contains mitochondria and immunolabeled dense-core vesicles. Two types of processes emerge from the basal part of the perikaryon. The first has a large diameter, contains diffusely distributed immunoreaction, and receives synaptic input, indicating that this process is a basal dendrite. The other type is of a beaded appearance, displays immunolabeled dense-core vesicles, and represents the axon of the CSF-contacting neuron. VIP-immunoreactive terminal formations are located within the neuropil of the lateral septum and the nucleus accumbens. Some of them form synaptic contacts with immunonegative profiles. No VIP-immunoreactive terminal formations are seen in the perivascular spaces of the lateral septum. Tracer experiments with horseradish peroxidase have revealed that the blood-brain barrier is lacking in the lateral septal organ and nucleus accumbens of the duck. Capillaries, arterioles, and venoles of this region are coated by nonfenestrated endothelial cells connected by leaky junctions, allowing the tracer to penetrate from the lumen into the perivascular space and further into the intercellular clefts of the neuropil. Our immuno-electron-microscopic investigations show that VIP-immunoreactive CSF-contacting neurons of the lateral septum closely resemble CSF-contacting neurons occurring in other brain regions, e.g., the hypothalamus. The arrangement of VIP-immunoreactive terminal formations suggests that, in the lateral septum, the VIP-like neuropeptide serves as a neurotransmitter (-modulator). The lack of a blood-brain barrier in the lateral septal organ and the nucleus accumbens raises the possibility that this region is a window in the avian brain allowing exchange of information between the central nervous system and the bloodstream; it thus resembles a circumventricular organ.     

Correlation-associated peptide networks of human cerebrospinal fluid

Profiling of peptides and small proteins from either human body fluids or tissues by chromatography and subsequent mass spectrometry reveals several thousand individual peptide signals per sample. Any peptide is an intermediate in the course of biosynthesis, post-translational modification (PTM), proteolytic processing and degradation. Changes in the concentration of one peptide often affects the concentration of the other, hence a challenge consists in the development of suitable tools to turn this large amount of data into biologically relevant information. Comprehensive statistical analysis of the peptide profiling data allows associating peptides, which are closely related in terms of peptide biochemistry. Here, the bioinformatic concept of peptide networks, correlation-associated peptide networks (CANs), is introduced. Peptides with statistical similarity of their concentrations are grouped in form of networks, and these networks are interpreted in terms of peptide biochemistry. The spectrum of functional relationships found in cerebrospinal fluid CAN covers PTM and proteolytic degradation of peptides, clearance processing in the complement cascade, common secretion of peptides by neuroendocrine cells as well as ubiquitin-mediated degradation. Our results indicate that CAN is a powerful bioinformatic tool for the systematic analysis and interpretation of large peptidomics and proteomics data and helps to discover novel bioactive and diagnostic peptides.     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

Cholinergic Regulation of Vasoactive Intestinal Peptide Content and Release in Rat Frontal Cortex and Hippocampus

The purpose of this study was to determine whether the cholinergic system might have a regulatory role on vasoactive intestinal peptide (VIP) synthesis and release in the rat hippocampus and frontal cortex. Incubation of hippocampal or frontal cortical slices with the muscarinic agonist oxotremorine or antagonist atropine did not significantly alter VIP release. The nicotinic agonist methylcarbamylcholine (MCC) and the nicotinic antagonist dihydro-beta-erythroidine were also ineffective in altering VIP release. Chronic atropine (20 mg/kg, s.c., b.i.d., 10 days) and nicotine (0.59 mg/kg, s.c., b.i.d., 10 days) treatment significantly decreased the VIP content of the frontal cortex, by 42% and 26%, respectively. In contrast, neither treatment significantly altered the VIP content of the hippocampus. Both drug treatments decreased the amount of VIP released from tissue slices depolarized with veratridine in both cerebral cortex and hippocampus. Therefore, long-term treatment with atropine and nicotine results in changes in the synthesis and release of VIP in the cerebral cortex, whereas in the hippocampus the effect is limited to an alteration of VIP release. These results suggest that the acetylcholine regulates VIP neurotransmission in the rat frontal cortex and hippocampus by an action on muscarinic and nicotinic receptors.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

Vasoactive intestinal polypeptide (VIP) immunoreactivity of endocrine-like cells in the feline pyloric mucosa

Summary Immunoreactivity to VIP by endocrine-like cells in the feline pyloric mucosa was examined by using three kinds of region-specific anti-porcine VIP sera. VIP-immunoreactive endocrine-like cells were detected clearly with all of the VIP antisera used. They were located mainly around the neck of the pyloric glands. Some of these endocrine-like cells showed dilution-dependent immunoreactivity against VIP antisera. The immunostaining intensity of VIP-immunoreactive endocrine-like cells showing dilution-independence could not be distinguished from those of nerve elements. The present results suggest that the immunoreactivity with properties very similar to those of authentic VIP may be present in the endocrine-like cells of the feline pyloric glands.     

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.     

Vasoactive intestinal peptide stimulates luteinizing hormone-releasing hormone release from median eminence synaptosomes

Third ventricular injections of vasoactive intestinal polypeptide (VIP) result in increased circulating levels of luteinizing hormone (LH) in conscious, freely moving, ovariectomized (OVX) rats. This effect of VIP has been hypothesized to be mediated via stimulation of luteinizing hormone-releasing hormone (LH-RH) secretion from hypothalamic neurons since VIP is incapable of stimulating LH release from rat pituitaries in vitro. To test this hypothesis, crude synaptosomes were prepared from OVX rat median eminence (ME) tissue. Release of LH-RH from these preparations displayed time and temperature dependencies. Additionally, depolarization-induced (elevated K⁺) LH-RH release was demonstrated to be Ca²⁺-dependent. VIP, in doses ranging from 1.5 · 10^?9 M, was capable of stimulating significantly greater LH-RH release from ME synaptosomes than that from control preparations. VIP's close structural homolog, glucagon, was incapable at the same doses of stimulating increased LH-RH release. These findings offer an explanation for the effect of third ventricularly injected VIP on LH release and suggest a modulatory role for VIP in the hypothalamic control of LH secretion.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

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 Functional Receptors for Vasoactive Intestinal Peptide in Bovine Cerebral Arteries

This study reports the characterization of receptors for vasoactive intestinal peptide (VIP) on membranes prepared from bovine cerebral arteries. By use of HPLC we prepared two purified monoiodinated VIP radioligands with nearly equivalent cerebral vasorelaxant potency as native VIP, [Tyr(125I)10 )VIP and [Tyr(125I)22]VIP. The former resulted in a higher proportion of specific binding to arterial membranes than the latter and was therefore thought to be the superior radioligand for receptor characterization. The binding of [Tyr(125I)10]VIP to cerebral arterial membranes was saturable, specific, reversible, and dependent on time and temperature. Scatchard analysis suggested the presence of a high- and a low-affinity binding site with KD values of 0.2 and 11 nM and receptor concentrations of 79 and 737 fmol/mg of protein, respectively. The dose-response curves for binding to the VIP receptor by the VIP-homologous peptides PHI, PHM, and rat growth hormone-releasing factor (GRF) were very similar to their dose-response curves for relaxation of cerebral arteries. The order of potency was VIP greater than PHM greater than PHI greater than rat GRF. It is suggested that the characteristics of the vascular VIP binding sites and the close correlation between the binding and vasorelaxant properties of VIP and its related peptides argue for the vascular binding sites being functional receptors for VIP.     

Vasoactive intestinal polypeptide stimulates nonovarian progesterone secretion in rabbits

Recent experiments conducted in this laboratory have shown that intravenous infusions of vasoactive intestinal polypeptide (VIP) induced significant increases in plasma progesterone (P) in female rabbits. The purpose of this study was to determine the organ source of this P and to clarify the mechanisms by which it is induced. Intravenous infusions of VIP (37.5, 75, and 150 pmol/kg per min for 60 min) produced acute dose-dependent increases in plasma P in intact estrous rabbits. In ovariectomized (OVX) animals, VIP infusion (75 pmol/kg per min) produced a P increase of the same magnitude. In animals both OVX and adrenalectomized (ADX), this VIP effect was eliminated. The only significant change noted in luteotropic hormone (LH) or follicle stimulating hormone (FSH) was a decrease in FSH immediately following VIP infusion (150 pmol/kg). VIP infusion significantly increased plasma cortisol in intact and OVX animals, but not in OVX/ADX animals. It is concluded that VIP primarily stimulates the adrenal component of P secretion in the rabbit, via mechanisms independent of LH or FSH.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone

We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea-pigs, cattle, and pigs all responded potently to bovine (b) PTH-(1-34), whereas considerable between-species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken-cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH-(1-34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI greater than bPTH-(1-34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP-(10-28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, inhibited bPTH-(1-34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH-(1-34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH-(1-34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.