Corticotropin-releasing factor-induced immunosuppression in human and invertebrate immunocytes

1. Corticotropin-releasing factor (CRF) appears to be a potentially important signal molecule in both vertebrate and invertebrate neuroimmune and autoimmunoregulatory processes. It appears to mimic the effects of alpha-melanocyte stimulating hormone (MSH) but has a longer duration of action. 2. alpha-Helical CRF, a specific inhibitor of CRF, antagonizes CRF-induced cellular immunosuppression but is ineffective in altering MSH-induced immunosuppression. 3. Both human and Mytilus immunocytes appear to have specific CRF receptors. 4. In another experiment, both CRF and MSH antagonize tumor necrosis factor stimulation of immunocytes. Again, alpha-helical CRF antagonizes only CRF activity, further suggesting the presence of a separate CRF receptor on these cells.     

大鼠NPY Y2受体基因的克隆及C端肽的表达和纯化

Y2受体亚型是早期发现的NPY的两种主要受体亚型之一,参与NPY所介导的多种生理和病理功能.为了制备Y2受体抗体和开展Y2受体的定位研究,用RT-PCR方法从大鼠海马的总RNA扩增NPY的Y2受体全长基因,接着PCR扩增Y2受体的C端片段,克隆入表达载体中,建立了重组NPY Y2受体C端肽的表达菌株,并对表达产物进行纯化.     

Neuropeptide Y and sympathetic vascular control in man

A parallel increase in systemic plasma levels of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) was found during thoracotomy and surgery involving cardiopulmonary bypass in man. Thus, plasma levels of NPY-LI increased from 29 +/- 4 pmol/l before anaesthesia to 59 +/- 10 after thoracotomy and to 87 +/- 8 pmol/l upon cardiopulmonary bypass. The corresponding NA levels increased from 1.3 +/- 0.1 nmol/l before anaesthesia to 3.0 +/- 0.6 and 4.2 +/- 5 nmol/l after thoracotomy and cardiopulmonary bypass, respectively. A significant correlation was found between plasma levels of NPY-LI and NA during the operation but not between NPY-LI and adrenaline. The NPY-LI in human plasma was found to be similar to synthetic porcine NPY on reversed phase high performance liquid chromatography. Human submandibular arteries contained high levels of NPY-LI (24 +/- 3 pmol/g). In in vitro experiments on isolated human submandibular arteries, NPY in low concentrations (1000 pmol/l) was found to potentiate the contractile effects of NA or transmural nerve stimulation and to exert vasoconstrictor activity per se in higher concentrations. The calcium-entry antagonist nifedipine abolished both the NPY-induced contractions and the enhancement of NA-evoked contractions. NPY depressed the nerve stimulation-evoked 3H-NA release from human submandibular arteries via a prejunctional mechanism which was resistant to nifedipine. NPY contracted human mesenteric veins and renal arteries, but not mesenteric arteries. In conclusion, NPY seems to be co-released with NA upon sympathetic activation in man. Furthermore, NPY exerts both pre- and postjunctional effects on sympathetic control of human blood vessels.     

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y₁, Y₂ and/or Y₅ receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 μM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y₂, Y₄ and Y₅ receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y₅ receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.     

Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real‐time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP⁺), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30–40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling. J. Cell. Biochem. 108: 621–630, 2009. © 2009 Wiley‐Liss, Inc.     

Sequence Variation within the Neuropeptide Y Gene and Obesity in Mexican Americans

Objective: Recently, we reported evidence for linkage between neuropeptide Y (NPY) and both obesity and several obesity‐related quantitative measures in a sample of Mexican Americans from Starr County, Texas. The purpose of this study was to investigate putative variation within the coding and promoter regions of NPY. Research Methods and Procedures: Five young, obese individuals (body mass index [BMI] 33 to 45 kg/m², age 14 to 30 years); five adult, lean individuals (BMI 20 to 26 kg/m², age 39 to 65 years); and five sibling pairs sharing no alleles that were identical by descent at a marker locus proximal to NPYwere selected for fluorescence‐based sequencing of approximately 1100 base pairs (bp) immediately 5′ from the start site and all four exons of NPY. We identified a total of eight variant sites, including a 2‐bp insertion/deletion (I/D) within a putative negative regulatory region (?880I/D) and a 17‐bp deletion at the exon 1/intron 1 junction (69I/D). The ?880I/D and 69I/D variants were typed in a separate random sample of Mexican Americans (N = 914) from Starr County, Texas. Results: Analyses of variance resulted in a significant association between ?880I/D and waist‐to‐hip ratio (p = 0.041) in the entire sample and between ?880I/D and BMI (p = 0.031), abdominal circumference (p = 0.044), and waist‐to‐hip ratio (p = 0.041) in a non‐obese subsample (BMI < 30 kg/m², n = 594). The 69I/D variant was observed in only one pedigree and does not appear to segregate with obesity within this pedigree. Discussion: This study reports newly identified common human sequence variation within the regulatory and coding sequence of NPY. Several variants were observed, and of those tested, the ?880I/D promoter region variant may influence body fat patterning in non‐obese individuals but does not appear to play a major role in the etiology of common forms of obesity in this population.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Effects of Fluoxetine Administration on Neuropeptide Y and Orexins in Obese Zucker Rat Hypothalamus

Objective: The aim of this work was to study the potential involvement of neuropeptide Y (NPY) and orexins in the anorexigenic mechanism of fluoxetine in obese Zucker rats, assessing the effects of chronic fluoxetine treatment on NPY and orexin immunostaining in several hypothalamic regions. Research Methods and Procedures: Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg intraperitoneally) daily for 2 weeks. The control group was administered 0.9% NaCl solution. Carcass composition was assessed using the official methods of the Association of Official Analytical Chemists. To test the potential thermogenic effect of fluoxetine administration, total body oxygen consumption was measured daily for 60 minutes before fluoxetine or saline injection and for 30 minutes after drug or saline injection. Hypothalamic arcuate and paraventricular nuclei, and the lateral hypothalamic area were immunostained for NPY, orexin A, and orexin B. Commercial kits were used for serum determinations. Results: Chronic fluoxetine administration in obese Zucker rats generated a reduction in body weight gain, food intake, adipocyte size, fat mass, and body protein. A decrease in NPY immunostaining in the paraventricular nucleus, without changes in the arcuate, was observed. However, no changes were observed in the number of neural cells immunostained for orexin A or orexin B in the lateral hypothalamic area. Discussion: Due to the hyperphagic effect of NPY in the paraventricular nucleus, these results suggest that NPY, but not orexins, could be involved in the anorexigenic effect of fluoxetine in obese Zucker rats.     

神经肽Y及其受体Y1、Y2对痛觉调制的研究进展

神经肽Y（neuropeptide Y,NPY）是一种由36个氨基酸残基组成的肽类激素,属胰多肽家族,广泛分布于中枢及外周神经组织的神经元中。NPY主要参与摄食行为、心血管活动、垂体分泌等生理功能的调节。NPY还参与了痛觉调制。NPY受体有Y1、Y2、Y3、Y4、Y5和Y6六种亚型。目前对Y1受体和Y2受体的研究较多,显示Y1受体和Y2受体参与痛觉调制。但现在对NPY在痛觉中的具体作用机制还不清楚。该文对NPY及其Y1受体、Y2受体在痛觉调制中的作用作一概述。     

Presynaptic Modulation Controlling Neuronal Excitability and Epileptogenesis: Role of Kainate, Adenosine and Neuropeptide Y Receptors

Based on the idea that seizures may arise from an overshoot of excitation over inhibition, all substances that may decrease glutamatergic function while having no effect or even increasing GABAergic neurotransmission are likely to be effective anticonvulsants. We now review the possible role of three such neuromodulators, kainate, adenosine, and neuropeptide Y receptors in controlling hyperexcitability and epileptogenesis. Particular emphasis is given on the robust neuromodulatory role of these three groups of receptors on the release of glutamate in the hippocampus, a main focus of epilepsy. Moreover, we also give special attention to the mechanisms of receptor activation and coupled signaling events that can be explored as attractive targets for the treatment of epilepsy and excitotoxicity. The present paper is a tribute to Arsélio Pato de Carvalho who has been the main driving force for the development of Neuroscience in Portugal, notably with a particular emphasis on the presynaptic mechanisms of modulation of neurotransmitter release.     

Neuropeptides,Including Neuropeptide Y and Melanocortins,Mediate Lipolysis in Murine Adipocytes

Objective: To determine whether key appetite‐regulating neuropeptides such as melanin‐concentrating hormone (MCH), neuropeptide Y (NPY), and α‐melanocyte—stimulating hormone (α‐MSH), which are known to mediate energy balance through centrally mediated pathways, also have direct acute effects on the lipolytic activity of murine adipocytes. Research Methods and Procedures: Fully differentiated 3T3‐L1 adipocytes serum starved overnight in Dulbecco's modified Eagle medium containing 2% bovine serum albumin or freshly isolated mouse adipocytes were incubated for up to 2 hours in the absence and presence of 100 nM each of NPY, MCH, α‐MSH, the melanocortin receptor agonist MTII, or isoproterenol as a control. Free fatty acids secreted into the incubation medium were measured using a commercially available nonesterified fatty acid C test kit. Results: Treatment of 3T3‐L1 cells with 100 nM NPY decreased basal free fatty acid secretion (basal, 0.006 ± 0.001 vs. NPY, 0.001 ± 0.0003 nM at 90 minutes; p < 0.05), whereas both α‐MSH and MTII stimulated up to a 7‐fold increase in free fatty acid release (MTII, 0.238 ± 0.004 vs. basal, 0.024 ± 0.002 nM at 2 hours; p < 0.05; and α‐MSH, 0.22 ± 0.005 vs. basal, 0.04 ± 0.003 nM at 2 hours; p < 0.05). Treatment with 100 nM MCH had no effect on basal free fatty acid release or on α‐MSH—induced lipolysis during concurrent treatment. Conversely, concurrent treatment with 100 nM NPY dramatically inhibited (by ～90%) α‐MSH—induced lipolysis. Similar treatment of freshly isolated mouse adipocytes showed virtually identical results. Discussion: In addition to their centrally mediated actions, appetite‐regulating neuropeptides modulate adipose tissue mass through direct peripheral effects. Systemic administration of pharmacological agents altering the effects of these neuropeptides may form the basis of future obesity therapies. Thus, some of these agents will likely have direct effects on adipocytes that may serve to alter their therapeutic effectiveness.     

Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers

Neuropeptide Y (NPY)-immunoreactive nerve fibers were numerous around arteries and few around veins. NPY probably co-exists with noradrenaline in such fibers since chemical or surgical sympathectomy eliminated both NPY and noradrenaline from perivascular nerve fibers and since double staining demonstrated dopamine-beta-hydroxylase, the enzyme that catalyzes the conversion of dopamine to noradrenaline, and NPY in the same perivascular nerve fibers. Studies on isolated blood vessels indicated that NPY is not a particularly potent contractile agent in vitro. NPY greatly enhanced the adrenergically mediate contractile response to electrical stimulation and to application of adrenaline, noradrenaline or histamine, as studied in the isolated rabbit gastro-epiploic and femoral arteries. The potentiating effect of NPY on the response to electrical stimulation is probably not presynaptic since NPY affected neither the spontaneous nor the electrically evoked release of [3H]noradrenaline from perivascular sympathetic nerve fibers.     

The Solution Structure of a Cyclic Analog of Neuropeptide Y with High Y<Subscript>1</Subscript> Receptor Affinity by NMR,CD and MD Simulations

The conformation of a cyclic analog of neuropeptide Y [Tyr¹--Lys--Gly--Arg--cyclo^5/8-(Glu⁵--Tyr--Ile--Lys⁸)--Leu--Ile¹⁰--Thr--Arg--Pro--Arg--Tyr¹⁵--NH₂; cEK-NPY] with high Y₁ receptor affinity was studied using ¹H, ¹³C and ¹⁵N 2D-NMR and CD in three diverse media-viz. DMSO-d₆, water (pH 4.0) and 50% hexafluoroacetone (HFA). The conformation of cEK-NPY was interpreted based on chemical shift (¹H, ¹³C and ¹⁵N), temperature coefficients of the NH chemical shifts, ³J_NHα coupling constants and the pattern of intra and inter-residue NOE’s and the CD spectrum. In both DMSO and water, there is a preponderance of a β-strand structure, while HFA promotes an α-helical structure, which is discontinuous in the mid-region of the peptide, due to the constraints of the lactam ring. The solution structures were generated using Restrained Molecular Dynamics simulations and further refined by Mardigras to R factors between 0.55 and 0.65. The role of its conformations in its biological activity is discussed.     

Effects of Electroconvulsive Stimuli and MK-801 on Neuropeptide Y,Neurokinin A,and Calcitonin Gene-Related Peptide in Rat Brain

Rats were pretreated with 0.9% NaCl, or 0.1 or 1.0 mg/kg MK-801, an anticonvulsant and a psychotomimetic drug, and 60 minutes later given ECS or sham ECS. After six sessions the animals were sacrificed and neuropeptide Y (NPY-), neurokinin A (NKA-), and calcitonin gene-related peptide (CGRP-) like immunoreactivity (-LI) measured with radioimmunoassays. ECS increased NPY-LI in frontal cortex, striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex and hippocampus. MK-801 increased CGRP in a dose-response manner in frontal cortex, and NKA-LI in occipital cortex. Although the higher MK-801 dose reduced seizure duration by 50%, the ECS induced NPY-LI increase in striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex was not diminished. In contrast, there was a parallel decrease in seizures and NPY-LI and NKA-LI changes in frontal cortex and hippocampus, respectively. Investigation of neuropeptides in brain may contribute to understanding of the mechanisms of action of antide-pressive and antipsychotic treatments and of psychotomimetic drugs.     

Neuropeptide Y regulates the hematopoietic stem cell microenvironment and prevents nerve injury in the bone marrow

Many reports have revealed the importance of the sympathetic nervous system (SNS) in the control of the bone marrow environment. However, the specific role of neuropeptide Y (NPY) in this process has not been systematically studied. Here we show that NPY‐deficient mice have significantly reduced hematopoietic stem cell (HSC) numbers and impaired regeneration in bone marrow due to apoptotic destruction of SNS fibers and/or endothelial cells. Furthermore, pharmacological elevation of NPY prevented bone marrow impairments in a mouse model of chemotherapy‐induced SNS injury, while NPY injection into conditional knockout mice lacking the Y1 receptor in macrophages did not relieve bone marrow dysfunction. These results indicate that NPY promotes neuroprotection and restores bone marrow dysfunction from chemotherapy‐induced SNS injury through the Y1 receptor in macrophages. They also reveal a new role of NPY as a regulator of the bone marrow microenvironment and highlight the potential therapeutic value of this neuropeptide.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis

Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.     

Biosynthesis, Development, and Regulation of Neuropeptide Y in Superior Cervical Ganglion Culture

The biosynthesis of neuropeptide Y (NPY) and norepinephrine (NE) has been examined in dissociated neuronal cultures from newborn rat superior cervical ganglion (SCG). NPY synthetic rate was measured by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after incubation in medium containing a labeled amino acid. The authenticity of the NPY was confirmed by reverse-phase HPLC analyses of tryptic peptides. The NPY synthetic rate in cultures grown in complete serum free medium increased 30-fold after plating, in parallel to catecholamine synthesis; both NPY and the catecholamines reached the rate for adult SCG neurons. This development in culture is seen without spinal cord input, target organs, or significant numbers of glial cells. NPY synthesis was maintained in the face of a major decrease in the rate of NE production after cholinergic induction.     

Neuropeptide Y (NPY) and depression: from animal studies to the human condition

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions. In addition, both pre-clinical and clinical evidence have suggested that NPY, together with its receptors, may have a direct implication in several psychiatric disorders, including depression and related illnesses. NPY-like immunoreactivity and NPY receptors are expressed throughout the brain, with varying concentrations being found throughout the limbic system. Such brain structures have been repeatedly implicated in the modulation of emotional processing, as well as in the pathogenesis of depressive disorders. This review will concentrate on the distribution of NPY, its receptors, and the putative role played by this peptide in depressive illness based on both pre-clinical and clinical evidence.