Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.     

Identification of noradrenergic nerve terminals immunoreactive for neuropeptide Y and vasoactive intestinal peptide in the rat kidney

Cryostat- and vibratome-cut sections of rat kidneys were singly or doubly labeled to visualize immunoreactive tyrosine hydroxylase (THI), dopamine beta-hydroxylase (DBHI), vasoactive intestinal peptide (VIPI), and neuropeptide Y (NPYI). Rats were perfusion fixed with 2-4% paraformaldehyde with or without 0.15% picric acid and rinsed in buffer for 18-48 hr. Single antigens were labeled with horseradish peroxidase in vibratome sections, whereas cryostat sections were used to label one antigen with peroxidase and another with a fluorophore in the same tissue section. A dense plexus of DBHI noradrenergic nerves innervates the renal arterial tree, and such nerves innervate the interlobar veins and renal calyx as well. Immunoreactive NPY is colocalized in most of these nerves, but some intrarenal noradrenergic nerves do not contain NPY but do contain VIP immunoreactivity. The distribution of NPYI nerves resembles that of DBHI nerves, whereas most perivascular noradrenergic nerves immunoreactive for VIP innervate selected arcuate and interlobular arteries. A small population of nonadrenergic, VIPI nerves innervates the renal calyx.     

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.     

Innervation of human omental arteries and veins and vasomotor response to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide

Human omental arteries and veins are supplied with nerve fibers containing noradrenaline (NA) and neuropeptide Y (NPY); these two agents probably co-exist in perivascular sympathetic nerve fibers. Substance P (SP)- or vasoactive intestinal peptide (VIP)-containing fibers could not be detected. In studies on isolated omental vessels NA produced constriction. The results of blockade experiments suggest that human omental arteries are equipped predominantly with alpha 1-adrenoceptors and omental veins with a mixture of alpha 1- and alpha 2-adrenoceptors. NPY at a concentration of 10(-7) M or higher had a weak contractile effect on veins and virtually no effect on arteries. NPY at a concentration of 3 X 10(-8) M shifted the NA concentration response curve to the left in arteries (pD2 = 5.8 for NA versus 6.6. for NA in the presence of NPY; P less than 0.001) but not in veins. Both SP and VIP relaxed arteries precontracted with NA or prostaglandin F2 alpha (PGF2 alpha). The potency of SP as a relaxant agent was similar in arteries and veins; the effect of VIP was elicited at lower concentrations in veins than in arteries.     

Distribution of peptidergic nerves in the choroid plexus, focusing on coexistence of neuropeptide Y, vasoactive intestinal polypeptide and peptide histidine isoleucine

Choroid plexus from rat, guinea-pig, rabbit and pig was investigated by light-microscopic immunohistochemistry and by radioimmunoassay for the presence of neuropeptides. A moderately dense supply of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), respectively, was found around blood vessels and in close relation to the secretory epithelium in both pig and rabbit, while lower densities of nerve fibers were found in rat and guinea-pig. Peptide concentrations ranged from 10-40 pmolequivalents/g (pmoleqv/g) for NPY and 0.5-6 pmoleqv/g for VIP in all four species. Peptide histidine isoleucine (PHI) immunoreactive nerve fibers were present in pig choroid plexus at a lower density than NPY and VIP but with a similar distribution. Low concentrations of substance P (0.3-3 pmoleqv/g) and calcitonin gene-related peptide (0.1-3 pmoleqv/g) were found to a varying degree in choroid plexus tissue from the different species, while immunohistochemical investigation was unable to detect any immunoreactive nerve fibers. NPY was often found to coexist with VIP and PHI in pig choroid plexus, while a lesser amount of nerve fibers showed coexistence of NPY and the noradrenaline synthetizing enzyme, dopamine-beta-hydroxylase. Surgical sympathetic denervation by excision of the superior cervical ganglion in the rabbit abolished NPY-containing nerve fibers, as revealed by immunohistochemistry, but only decreased NPY levels by one third, which may be due to different identity of the peptide being detected by the two techniques. It is concluded that NPY-containing nerve fibers have a dual origin in the choroid plexus and coexist with either noradrenaline or VIP/PHI.     

Neuropeptide (neuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin) immunohistochemistry and ultrastructure of renal nerves

With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.     

Neuropeptide (neuropeptide Y,neurotensin, vasoactive intestinal polypeptide,substance P,calcitonin gene-related peptide,somatostatin) immunohistochemistry and ultrastructure of renal nerves

Summary With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.Dedicated to Prof. Dr. T.H. Schiebler on the occasion of his 65th birthday     

Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries.

A sparse to moderate supply of nerve fibers containing neuropeptide Y-like immunoreactivity (NPY-LI), vasoactive intestinal polypeptide (VIP-LI), substance P (SP-LI), and calcitonin gene-related peptide (CGRP-LI) was demonstrated in the walls of human middle meningeal arteries. Comparison with similar studies on human cerebral and temporal arteries indicated a similar distribution and density. The immunoreactive material in all three arterial regions was characterized by reversed-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA). The major peak of NPY-LI, VIP-LI, SP-LI, and CGRP-LI in each extract eluted approximately with the same elution volume as that of the corresponding synthetic analogues. The concentration of NPY in the middle meningeal arteries was lower as compared to the temporal arteries. Low concentrations of SP-LI and CGRP-LI were found in the middle meningeal arteries as compared to the cerebral arteries. In isolated ring segments of human middle meningeal and cerebral arteries, NPY caused vasoconstriction but did not potentiate the contractile response of noradrenaline. In the temporal artery, NPY did not induce contraction but potentiated the vasoconstrictor response to noradrenaline. Vasoactive intestinal polypeptide, peptide histidine methionine-27, SP, neurokinin A, and CGRP relaxed all three types of cephalic arteries. The peptide effects were not antagonized by propranolol, atropine, or cimetidine. Comparison of the responses to VIP and SP of vessels from the different regions showed a similar pattern of reactivity. The response to SP was slightly (p less than 0.05) more potent, whereas the responses to CGRP were less potent in the middle meningeal as compared to that in cerebral (p less than 0.005) vessels.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Studies on colocalization of neuropeptide Y,vasoactive intestinal polypeptide,catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat

Summary In the present immunohistochemical study, the distribution of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the larynx was examined and compared with that of fibers containing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (BDH), and with that of acetylcholinesterase (AChE)-positive nerve fibers, in intact and vagotomized rats and in rats subjected to removal of the superior cervical ganglion (SCG). Fibers showing TH/DBH-like immunoreactivity (LI) were only found in the walls of arteries and arterioles, whereas AChE-positive nerve fibers were located close to the acini and ducts of the glands, in blood vessel walls, in the perichondrium and in the lamina propria. NPY-LI and VIP-LI coexisted in local AChE-positive ganglionic cells and in a subpopulation of the AChE-positive fibers, NPY-LI also being present in some periarterial fibers showing TH/DBH-LI. Unilateral removal of the SCG eliminated the TH/DBH-innervation in the upper but not the lower parts of the larynx ipsilaterally, whereas the NPY-innervation of the arteries in the upper parts only partly disappeared and the NPY-innervation of the other structures remained unchanged. The distribution of VIP-innervation was unchanged after vagotomy and removal of the SCG. The results suggest that VIP is present in the postganglionic parasympathetic innervation, whereas NPY is present in both the postganglionic parasympathetic and sympathetic innervation of the rat larynx.     

Localization of neuropeptide Y and its C-terminal flanking peptide in human renal tissue

We produced and characterized three anti-C-flanking peptides of neuropeptide Y (CPON) monoclonal antibodies. The K_a for these antibodies ranged from 0.4 to 0.8 × 10⁸ l/mol with an IC₅₀ for CPON(1–30) at about 20 nM as determined by ELISA. All these antibodies are IgG1 and recognize the 16–30 part of CPON. These antibodies and a specific anti-NPY monoclonal antibody were used to study the localization of CPON and NPY in the human kidney. The avidin-biotin technique was employed. NPY and CPON immunoreactivities were present in large amount in the renal tubules of the human kidney but not in the glomeruli. No labeling was found within the renal arterioles and veins, but some immunoreactivity was evidenced in the perivascular area. Because no specific receptor for CPON has been described to date, the presence of this peptide in the tubules may be due to a tubular reabsorption or perhaps to a local synthesis of pro-NPY.     

Lipid-induced conformational changes in glucagon,secretin, and vasoactive intestinal peptide

The CD of glucagon, secretin, and vasoactive intestinal peptide has been studied as a function of temperature in water and in aqueous solutions of dodecyl sulfate, phosphatidyl glycerol, and L -α-phosphatidic acid (dipalmitoyl). The anionic detergent and lipids induce helix formation in all three peptides, with the amount of induced helical content increasing in the order glucagon < secretin < vasoactive intestinal peptide. These observations are subject to quantitative rationalization using a matrix formulation for the configuration partition function. In this formulation the major conformational consequences of the interaction with anionic lipids or detergents is an increase in the probability for helix formation by arginyl, histidyl, and lysyl residues. The region in which helix formation is maximal is found to be at amino acid residues 13–20 in all three peptides. Other studies have implicated this portion of the polypeptide chain in receptor binding. Thus, the helical segment induced by interaction with anionic lipids may play an important physiological role.     

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.     

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.     

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.     

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.