Neuropeptide Y. Differential binding to rat intestinal laterobasal membranes

Neuropeptide Y (NPY), a neurotransmitter contained within intrinsic nerves of the small intestine, inhibits secretion when added to the serosal side of intestinal mucosa mounted in Ussing chambers. Using NPY radiolabeled with IODO-GEN, lactoperoxidase, or the Bolton-Hunter reagent, we have localized high affinity NPY receptors to the serosal laterobasal membranes of the rat intestinal epithelial cell, isolated according to a recently described protocol that minimizes contamination with endoplasmic reticulum, Golgi, and brush-border membranes. In addition, certain species of radiolabeled NPY, including those labeled with IODO-GEN at the tyrosine residue 36, also demonstrated an ability to bind to brush-border membranes. These receptors were specific for NPY since the homologous peptides, pancreatic polypeptide and peptide YY, were less efficient than NPY in inhibiting the membrane binding of radiolabeled NPY. By cross-linking NPY to its receptor with either disuccinimidyl suberate or dithiobis(succinimidyl propionate) and analyzing the resulting complexes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by radioautography, we identified two NPY receptor species with molecular sizes of 52-59 kDa and 37-39 kDa. The 37-39-kDa species further possesses a disulfide bond which may attach it to a separate approximately 5-kDa subunit, as evidenced by retarded migration in the absence of the reducing agent dithiothreitol. The intestinal NPY receptor is slightly smaller than the rat brain receptor previously characterized using similar techniques. The localization of NPY receptors on laterobasal membranes is consistent with previous anatomic and physiologic findings, and their identification by cross-linking techniques will constitute the basis for detailed characterization.     

Interaction between norepinephrine, NPY and VIP in the ovarian artery.

The in vitro effect and the interaction between norepinephrine (NE), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were studied in dissected segments of the rabbit ovarian artery. In addition, the structural requirement of the NPY receptor was investigated using NPY peptide analogs. NE induced a dose-dependent vasoconstriction with an Emax of 131.4 +/- 2.9% of K(+)-induced constriction. The vasoconstrictor effect of NPY was less than 5% of K(+)-induced vasoconstriction. Incubation of the artery with 10(-7) M NPY for 4 min induced a significant potentiation of NE-induced contractions. The selective NPY Y1 receptor agonist [Leu31, Pro34]NPY was also able to potentiate the NE response at the half-maximum contraction level, but not NPY(11-36), an NPY peptide fragment predominantly stimulating the NPY Y2 receptor. NPY exerted a dose-dependent vasoconstrictor effect on vessels contracted for 20 min with 10(-6) M NE. VIP induced a dose-dependent relaxation of vessels contracted with 10(-6) M NE. The VIP-induced relaxation could be reversed by NPY. In conclusion, receptors capable of interacting with NPY, presumably of the Y1 type, and VIP are present in the rabbit ovarian artery, and activation of these receptors may profoundly influence the response of the artery to norepinephrine.     

Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig

Summary The distributions within the coeliac ganglion of different chemically coded subgroups of noradrenaline neurons, and the relationships between these neurons and nerve fibres projecting to the ganglion from the intestine, have been assessed quantitatively by use of an immunohistochemical double-staining method. Noradrenaline (NA) neurons made up 99% of all cell bodies. Of these, 21% were also reactive for somatostatin (NA/SOM neurons), 53% were also reactive for NPY (NA/NPY neurons), and 26% were not reactive for either peptide. NA neurons without reactivity for any of the peptides whose localization was tested have been designated NA/-. A small percentage, about 1%, of neurons were reactive for both NPY and SOM. The three major types of NA neurons were arranged in clumps or ribbons throughout the ganglia, with a tendency for NA/SOM neurons to be medial and NA/NPY neurons to be lateral in the ganglia. A small group of neurons (<1%) encoded with dynorphin, NPY and vasoactive intestinal peptide (VIP) was encountered. VIP-immunoreactive nerve terminals, projecting to the ganglion from cell bodies in the intestine, ended around NA/SOM and NA/neurons but not around NA/NPY neurons. Thus, the VIP axons from the intestine end selectively around neurons that modify intestinal function (NA/SOM and NA/-neurons) but not around neurons, the terminals of which supply blood vessels (NA/NPY neurons).     

Ontogeny of peptide-containing neurons in human gut--an immunocytochemical study

Neurons containing enkephalin, substance P (SP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), galanin, calcitonin gene-related peptide (CGRP), peptide histidine isoleucine (PHI) or gastrin-releasing peptide (GRP) are known to occur in the human intestinal tract. The knowledge of the ontogeny of these neurones is, however, limited. Intestinal specimens from 24 human foetuses with gestational ages varying between 8 and 40 weeks were examined by immunocytochemistry. No peptide-containing neurones could be detected before the 14th week of gestation after which a rapid development was seen. Generally, peptide immunoreactivity was first noted in the myenteric ganglia and somewhat later in the other layers of the intestinal wall. There was no major difference between the peptides studied or between different parts of the intestinal tract with respect to time of appearance.     

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.     

Physiological role of neuropeptide Y in the regulation of the ascending phase of the peristaltic reflex

The physiological role of neuropeptide Y (NPY) and of specific NPY receptors in regulating the intestinal peristaltic reflex was examined in three-compartment flat-sheet preparations of rat colon. Graded muscle stretch or mucosal stimulation applied to the central compartment inhibited NPY release in the orad compartment where ascending contraction was measured. NPY and the Y1-receptor agonist [Leu31, Pro34]NPY inhibited, whereas the selective Y1-receptor antagonist BIBP 3226 augmented ascending contraction and substance P (SP) release in the orad compartment induced by muscle stretch or mucosal stimulation. Neither agonist nor antagonist had any effect on descending relaxation or VIP release in the caudad compartment. The Y2-receptor agonist NPY13-36 and antagonist BIIE 0246 had no effect on peptide release or mechanical response. The results indicate that suppression of a tonic inhibitory influence of NPY neurons on excitatory neurotransmitter release contributes substantially to the orad contractile phase of the peristaltic reflex. The effect of NPY on neurotransmitter release is mediated by Y1 receptors.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Neuropeptide Y Inhibits β-Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.     

Distribution and characterisation of neuropeptide Y immunoreactivity in the brain and gastrointestinal tract of the rainbow trout,Oncorhynchus mykiss

1. Neuropeptide Y (NPY) immunoreactivity has been localised cytochemically in neuronal somata and fibres in rainbow trout brain, nerve fibres and mucosal epithelial endocrine cells within the gastrointestinal tract and in endocrine cells within pancreatic islets.2. Using a C-terminal specific NPY radioimmunoassay, immunoreactivity was detected in extracts of brain (519 pmol/g), cardiac stomach (37.9 pmol/g), pyloric stomach plus pancreas (37.9 ol/g) and intestine (29.2 pmol/g).3. Gel permeation and reverse-phase HPLC analysis of brain and intestinal extracts resolved a single NPY immunoreactive peptide.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.     

Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry

The immunocytochemical distribution of substance P (SP), gastrin releasing peptide (GRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and neuropeptide Y (NPY) was studied in the ovary and the Fallopian tube (oviduct) of rats, guinea-pigs, cows, pigs and humans. Generally, the nerve supply was better developed in the oviduct than in the ovary. GRP fibers were most scarce in all tissues. Nerves containing SP were particularly numerous in the oviduct of rat and guinea-pig, supplying the muscular wall and blood vessels. VIP and PHI coexisted in dense plexuses of nerves, not only around blood vessels but also in the follicular wall and the interstitial gland of the ovary, as well as within the smooth muscle layers and subepithelially in the oviduct. The general distribution of NPY was similar, but these immunoreactive nerves were even more numerous. Sequential staining for dopamine-beta-hydroxylase and NPY together with results of chemical sympathectomy with 6-hydroxydopamine suggested that NPY was stored in the noradrenergic sympathetic nerves.     

Helical structure and self-association in a 13 residue neuropeptide Y Y2 receptor agonist: relationship to biological activity

The solution structure and self-association behaviour of a 13 residue peptide analogue of the C-terminal region of human neuropeptide Y (NPY) have been investigated. NMR analysis of Ac[Leu(28,31)]NPY(24-36), a potent Y2 receptor agonist, shows that it is unstructured in aqueous solution at 5-20 degrees C, but forms a well-defined helix (encompassing residues 25-35) in 40% trifluoroethanol/water at 20 degrees C. Sedimentation experiments show that, in contrast to many peptides in aqueous trifluoroethanol, Ac[Leu(28,31)]NPY(24-36) associates to form a trimer or, more likely, a tetramer in 40% trifluoroethanol, even though it is monomeric in water. This is consistent with the observation of inter-molecular nuclear Overhauser enhancements in trifluoroethanol. Possible models of the associated form that are consistent with the NMR data are described. The relevance of the helical structure observed in trifluoroethanol to the structure of this peptide bound to the NPY Y2 receptor is discussed.     

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.     

The neuropeptide Y monomer in solution is not folded in the pancreatic-polypeptide fold

Fluorescence-labelled analogs of NPY, a 36-amino acid peptide amide, were synthesized by solid-phase peptide synthesis and used for fluorescence-resonance energy transfer studies to investigate the conformation. Energy-transfer efficiency measurements in different media at the concentration of 10 microM are in agreement with a model of the NPY structure proposed by NMR studies (performed at millimolar concentration) in which the C-terminal part of the molecule adopts an alpha-helical conformation while the N-terminal part is flexible. According to this model, the alpha-helix is stabilized by intermolecular hydrophobic interactions because of the formation of dimers. The decrease of the peptide concentration causes a shift of the dimerization equilibrium toward the monomeric form. Energy-transfer efficiency measurements performed at lower concentrations do not support the hypothesis of the folding back of the N-terminal tail onto the C-terminal alpha-helix to yield the so-called "PP-fold" conformation. This structure is observed in the crystal structure of avian pancreatic polypeptide, a member of the NPY peptide hormone family, and it has been considered to be the bioactive one. Our results complete the structural characterization of NPY in solution at concentration ranges in which NMR experiments are not feasible. Furthermore, these results open the way to study the conformation of the receptor-bound ligand.     

Neuropeptide control of thyroid blood flow and hormone secretion in the rat

The effects of peptide HI (PHI), neuropeptide Y (NPY), and substance P (SP) on thyroid blood flow and hormone levels were studied in anesthetized rats. Regional blood flows were determined using radioactive microspheres. No change in heart rate or mean left ventricular pressure occurred during these neuropeptide infusions (0.625 micrograms iv over 2 min). PHI treatment resulted in a four-fold increase in thyroid blood flow. Blood flows to the pancreas and salivary gland also increased during PHI treatment. Infusions of NPY or SP did not significantly alter thyroid blood flow. However, SP decreased blood flow to the spleen and small intestine. These neuropeptides had no effect on blood flows to the adrenal, kidney, brain, heart, and adipose tissues. Following PHI, NPY, and SP infusions, plasma triiodothyronine and thyroxine levels were not different from values in saline-treated rats. This study demonstrates that PHI, like vasoactive intestinal peptide, is a potent thyroidal vasodilator at a dose that does not affect circulating thyroid hormone secretion.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

VIP and PHI coexist with an NPY-like peptide in intramural neurones of the small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and neuropeptide Y (NPY) are neuropeptides present in all layers of the small intestine. NPY-immunoreactive fibres in the gut seem to derive from two sources. One population is of extramural (sympathetic) origin and contains noradrenaline, another is of intramural origin and does not contain noradrenaline. In the present study of mouse, rat and pig, immunocytochemistry showed immunoreactive PHI to coexist completely with immunoreactive VIP. This was predictable, since VIP and PHI derive from the same precursor. In addition, however, VIP and PHI were found to coexist with immunoreactive NPY in non-adrenergic (but not in adrenergic) nerve fibres and nerve cell bodies. This coexistence was unexpected, since the VIP precursor does not contain NPY-like sequences.     

Immunohistochemical characterisation of pelvic autonomic ganglia in male mice

Pelvic ganglia are mixed sympathetic-parasympathetic ganglia and provide the majority of the autonomic innervation to the urogenital organs. Here we describe the structural and histochemical features of the major pelvic ganglion in the male mouse and compare two different mouse strains. The basic structural features of the ganglion are similar to those in the male rat. Almost all pelvic ganglion cells are monopolar and most are cholinergic. All contain either neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP), or both peptides together. The peptide coexistence varies between strains, with C57BL/6 mice having similar proportions of neurons with NPY alone, VIP alone or both peptides. In contrast, virtually all pelvic neurons in the Quackenbush-Swiss (QS) strain express NPY, i.e. the level of VIP/NPY coexistence is much higher. Cholinergic axons provide the major nerve supply to epithelia of reproductive organs, bladder smooth muscle and, as described previously, penile erectile tissue. They also provide a minor component of the smooth muscle innervation of the prostate gland, seminal vesicles and vas deferens. Virtually all non-cholinergic pelvic ganglion cells are noradrenergic and contain NPY. Their major target is smooth muscle of reproductive organs. This study shows that the male mouse pelvic ganglion bears many similarities to that in the rat, but that VIP/NPY colocalisation is much more common in the mouse. We also show that there are differences in peptide expression in parasympathetic pelvic neurons between strains of mice. These studies provide the framework for future investigations on neural regulation of urogenital function, particularly in transgenic and knockout models.     

Hibernation-related changes in the immunoreactivity of neuropeptide systems in the suprachiasmatic nucleus of the ground squirrel,Spermophilus richardsonii

Summary The pattern of distribution and reactivity of the neuropeptides vasopressin (AVP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), and thyrotropin-releasing hormone (TRH) were studied in the suprachiasmatic nucleus (NSC) of 20 Richardson's ground squirrels (and 7 European hedgehogs) of both sexes during hibernation and euthermia. The total area of immunostained structures revealed by application of the individual immunocytochemical techniques was measured by means of computer-aided image analysis. In both species, elements of all peptide systems examined were related to particular subdivisions of the NSC. The pattern of immunoreactivity was strongly correlated with the physiological stage of hibernation or euthermia both in ground squirrels and hedgehogs. The immunoreactivities to AVP and SP increased in area during hibernation (AVP: 25%; SP: 25%), whereas the respective area immunoreactive to NPY and VIP decreased (NPY: 45%; VIP: 100%) in comparison to nonhibernating controls. The TRH-immunoreactive nerve fibers were rare and rather scattered; thus, the quantitative procedure was not applicable for this immunoreaction.Abbreviations AVP argnine vasopressin - NPY neuropeptide Y - NSC suprachiasmatic nucleus - SP substance P - TRH thyrotropinreleasing hormone - VIP vasoactive intestinal peptide The results have been partly presented at the 10^th International Symposium on Neurosecretion held in Bristol, UK, September 1987     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.