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1.
Peptides ; 134: 170406, 2020 12.
Article in English | MEDLINE | ID: mdl-32920044

ABSTRACT

The neuropeptide FF2 (NPFF2) receptor, predominantly expressed in the central nervous system, plays an important role in the modulation of sensory input and opioid analgesia, as well as in locomotion, feeding, intestinal motility, reward, and the control of obesity. The NPFF2 receptor belongs to the RFamide peptide receptor family and to the G protein coupled receptor (GPCR) super family, but contrary to many other class A GPCRs, no 3D structure has been solved. Thus, it is essential to perform mutagenesis to gain information on the fine functioning of the NPFF2 receptor. In this study, we examined the role of aspartic acid (D) from the "D/ERY/F" motif found in the second intracellular loop (ICL2) and the role of the C-terminal end of the receptor in ligand binding and signal transduction. We found that mutation D3.49A does not impair binding capacities but inhibits G protein activation as well as adenylyl cyclase regulation. Truncation of the C terminal part of the receptor has different effects depending on the position of truncation. When truncation was realized downstream of the putative acylation site, ligand binding and signal transduction capabilities were not lost, contrary to total deletion of the C terminus, which totally impairs the activity of the receptor.


Subject(s)
Analgesics, Opioid/pharmacology , Neuropeptides/pharmacology , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cricetinae , Humans , Mutagenesis , Receptors, Neuropeptide/genetics , Signal Transduction/drug effects , Structure-Activity Relationship
2.
Neurochem Int ; 132: 104588, 2020 01.
Article in English | MEDLINE | ID: mdl-31704091

ABSTRACT

This study compared pharmacological profiles between human mu opioid receptors (hMOR) overexpressed in the SH-SY5Y neuroblastoma cell line (SH-hMOR) and the methylotrophic yeast Pichia pastoris (Pp-hMOR). Affinity determinations were performed by direct binding with the tritiated agonist DAMGO and antagonist diprenorphine (DIP). Additionally, displacement of these drugs with agonists (morphine and DAMGO) and antagonists (ß-funaltrexamine, naloxone and diprenorphine) was examined. Tritiated DAMGO could bind to membranes prepared from Pp-hMOR, although the receptor was not coupled with G-proteins. The data obtained with this yeast strain suggested that only 7.5% of receptors were in a high-affinity-state conformation. This value was markedly less than that estimated in SH-hMOR membranes, which reached 50%. Finally, to understand the pharmacological discrepancies between Pp-hMOR and SH-hMOR, the role of sterols was evaluated. The major sterol in P. pastoris is ergosterol, while hMOR naturally functions in a cholesterol-containing membrane environment. Cell membranes were sterol-depleted or cholesterol-loaded with methyl-ß-cyclodextrine. The results indicated that cholesterol must be present to ensure Pp-hMOR function. The proportion of high-affinity-state conformation was reversibly increased by cholesterol complementation.


Subject(s)
Analgesics, Opioid/metabolism , Cholesterol/metabolism , Gene Expression Regulation, Fungal/physiology , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/metabolism , Saccharomycetales/metabolism , Analgesics, Opioid/pharmacology , Cell Line, Tumor , Cholesterol/genetics , Dose-Response Relationship, Drug , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Humans , Protein Binding/drug effects , Protein Binding/physiology , Receptors, Opioid, mu/genetics , Saccharomycetales/genetics
3.
PLoS One ; 14(3): e0213203, 2019.
Article in English | MEDLINE | ID: mdl-30845158

ABSTRACT

Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes.


Subject(s)
Antibodies, Monoclonal/immunology , Breast Neoplasms/metabolism , Fibroblasts/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Image Processing, Computer-Assisted/methods , Receptors, Lysosphingolipid/immunology , Receptors, Lysosphingolipid/metabolism , Animals , Breast Neoplasms/pathology , Cells, Cultured , Female , Fibroblasts/cytology , Humans , Immunization , Kidney/metabolism , Mice , Microscopy, Fluorescence , Sphingosine-1-Phosphate Receptors , Spinal Cord/metabolism
4.
J Neurochem ; 136(4): 778-790, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26617404

ABSTRACT

The participation of a signaling platform to the anti-nociceptin/orphanin FQ (N/OFQ) effect of neuropeptide FF (NPFF) receptors was investigated in both acutely dissociated neurons and SH-SY5Y human neuroblastoma cells. The NPFF anti-N/OFQ, not anti-µ opioid effect, on the Ca2+ transient triggered by depolarization was reversed by methyl-ß-cyclodextrin which depletes cholesterol from cell membranes. While the inactive α-cyclodextrin had no effect. By using [35 S]GTPγS binding assay, a significant 20% decrease in the activity of nociceptin/orphanin FQ peptide receptors induced by the NPFF analog 1DMe was observed in detergent-resistant membranes, but not in total membranes of SH-SY5Y cells. Moreover, siRNA knock-down of G-protein-coupled receptor kinase 2 indicated that G-protein-coupled receptor kinase 2, but not protein kinase C, acted as the mediator in the NPFF anti-N/OFQ process. These data indicate that cholesterol-rich lipid rafts play an important role in the anti-N/OFQ effect of NPFF receptors. We proposed the participation of a signaling platform to the anti-Nociceptin/Orphanin FQ (N/OFQ) effect of Neuropeptide FF (NPFF) receptors both in mouse neurons and SH-SY5Y cells, with GRK2 protein acting as the mediator in this process. These findings should provide a more precise way to understand the anti-opioid effect of NPFF. NOP, Nociceptin/Orphanin FQ peptide.

5.
J Proteome Res ; 14(8): 3162-73, 2015 Aug 07.
Article in English | MEDLINE | ID: mdl-26090583

ABSTRACT

Human µ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.


Subject(s)
Chromatography, Liquid/methods , Receptors, Opioid, mu/chemistry , Recombinant Fusion Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Blotting, Western , Chymotrypsin/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Peptides/metabolism , Pichia/genetics , Protein Structure, Secondary , Proteomics/instrumentation , Proteomics/methods , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Recombinant Fusion Proteins/metabolism , Trypsin/metabolism
6.
J Biol Chem ; 289(49): 33754-66, 2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25326382

ABSTRACT

The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.


Subject(s)
Neurons/metabolism , Oligopeptides/metabolism , Protein Processing, Post-Translational , Receptors, Neuropeptide/chemistry , Amino Acid Sequence , Cell Line, Tumor , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Neurons/cytology , Oligopeptides/chemistry , Peptide Mapping , Phosphorylation , Protein Transport , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/metabolism , Sequence Alignment , Signal Transduction
7.
Eur J Pharmacol ; 740: 271-6, 2014 Oct 05.
Article in English | MEDLINE | ID: mdl-25046840

ABSTRACT

By using acutely dissociated dorsal raphe nucleus neurons (DRN) from young mice, direct and anti-opioid effects of Neuropeptide FF (NPFF) receptors were measured. The NPFF analog 1 DMe (10 µM) had no effect on resting Ca2+ channels but reduced the magnitude of Ca2+ transients induced by depolarization in 83.3% neurons tested, of which the inhibition rate is 45.4±2.9%. Pertussis toxin treatment reduced to 18.9% the number of responding neurons and attenuated by 47% the response of 1 DMe. In contrast, cholera toxin treatment had no significant effect. Eighteen minute perfusion with 1 DMe at a very low 10 nM concentration, that did not directly inhibit Ca2+ transients triggered by depolarization in every neuron, attenuated by 78% the inhibitory effect of Nociceptin/orphanin FQ (N/OFQ) on Ca2+ transients, but not that of by serotonin. These results demonstrated for the first time that NPFF receptors on mice DRN inhibit Ca2+ transients induced by depolarization via Gi/o protein and also exhibit a specific anti-opioid activity on nociceptin receptors, and that their specific anti-opioid activity is not a direct consequence of their activity on Ca2+ transients.


Subject(s)
Dorsal Raphe Nucleus/cytology , Neurons/physiology , Receptors, Neuropeptide/physiology , Receptors, Opioid/physiology , Animals , Calcium/physiology , Cholera Toxin/pharmacology , Mice, Inbred C57BL , Neurons/drug effects , Oligopeptides/pharmacology , Opioid Peptides/pharmacology , Pertussis Toxin/pharmacology , Receptors, Neuropeptide/agonists , Serotonin/pharmacology , Nociceptin Receptor , Nociceptin
8.
Peptides ; 55: 79-84, 2014 May.
Article in English | MEDLINE | ID: mdl-24582609

ABSTRACT

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.


Subject(s)
Receptors, Opioid, mu/chemistry , Animals , CHO Cells , Cell Line, Tumor , Cholic Acids/chemistry , Cricetulus , Detergents/chemistry , Diprenorphine/chemistry , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Humans , Ligands , Narcotic Antagonists/chemistry , Polyethylene Glycols/chemistry , Protein Binding , Protein Refolding , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/biosynthesis , Solubility
9.
Anal Biochem ; 453: 50-4, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24613258

ABSTRACT

A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.


Subject(s)
Affinity Labels/chemistry , Oligopeptides/chemistry , Phenylalanine/analogs & derivatives , Radioligand Assay/methods , Receptors, Neuropeptide/analysis , Affinity Labels/chemical synthesis , Amino Acid Sequence , Animals , Humans , Iodine Radioisotopes , Ligands , Mice , Mice, Inbred C57BL , Molecular Weight , Neuroblastoma/metabolism , Olfactory Bulb/metabolism , Oligopeptides/analysis , Phenylalanine/chemistry , Photolysis , Rats , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/metabolism , Tumor Cells, Cultured
10.
Neuropsychopharmacology ; 38(4): 596-604, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23169349

ABSTRACT

Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction.


Subject(s)
Behavior, Addictive/metabolism , Morphine/administration & dosage , Opioid-Related Disorders/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin/metabolism , Animals , Behavior, Addictive/psychology , Male , Mice , Mice, Inbred C57BL , Opioid-Related Disorders/psychology , Proteolysis/drug effects , Rats , Rats, Sprague-Dawley , Self Administration
11.
Peptides ; 37(1): 157-60, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22813580

ABSTRACT

Neuropeptide FF (NPFF) has been shown to act as an endogenous anti-analgesic peptide. In this paper, several peptide analogs of the selective ligand dNP(NMe)AFLFQPQRF-NH(2) modified in the putative address segment, were designed to be selective NPFF(2) receptor probes, synthesized and assayed. One peptide dA(NMe)AAFLFQPQRF-NH(2) displays a very high affinity for NPFF(2) receptors transfected in CHO cells, and a high selectivity versus NPFF(1) receptors. The exact residues carried in the N-terminal part of the ligands are not decisive to obtain a high affinity only the length of the peptide in itself seems important to create selectivity.


Subject(s)
Peptide Fragments/pharmacology , Receptors, Neuropeptide/agonists , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Humans , Ligands , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Neuropeptide/metabolism
12.
J Biol Chem ; 287(16): 12736-49, 2012 Apr 13.
Article in English | MEDLINE | ID: mdl-22375000

ABSTRACT

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of µ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit ß-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.


Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , Receptors, Neuropeptide/metabolism , Receptors, Opioid, mu/metabolism , Amino Acid Sequence , Analgesics, Opioid/pharmacology , Arrestins/metabolism , Cell Line, Tumor , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , G-Protein-Coupled Receptor Kinase 2/genetics , Gene Knockdown Techniques , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Neuroblastoma , Phosphorylation/physiology , Receptors, G-Protein-Coupled/metabolism , Receptors, Opioid, mu/agonists , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Serine/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , beta-Arrestins
13.
Anal Biochem ; 420(1): 99-100, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-21951781

ABSTRACT

Functional and structural studies on G-protein-coupled receptors (GPCRs) at molecular levels require producing and purifying high levels of receptors, and recombinant mammalian cell expression systems constitute the best systems to obtain receptors resembling those expressed in natural environments. In the course of increasing GPCR expression in Chinese hamster ovary (CHO) cells, we have expressed mu (µ)- and kappa (κ)-opioid receptors and neuropeptide FF(1) and FF(2) receptors (NPFF(1) and NPFF(2), respectively) in dimethyl sulfoxide. This treatment did not modify the affinity (K(d)) for any receptor, but a significant increase in functional expression levels was observed for all receptors with the noticeable exception of NPFF(1).


Subject(s)
Dimethyl Sulfoxide/pharmacology , Receptors, Neuropeptide/metabolism , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/metabolism , Recombinant Proteins/metabolism , Animals , CHO Cells/drug effects , Cricetinae , Cricetulus , Female , Protein Engineering/methods , Receptors, Neuropeptide/genetics , Receptors, Opioid, kappa/genetics , Receptors, Opioid, mu/genetics , Recombinant Proteins/genetics
14.
Pharmacol Rep ; 63(4): 1061-5, 2011.
Article in English | MEDLINE | ID: mdl-22001995

ABSTRACT

This study investigated the presence of neuropeptide FF (NPFF) receptors on F-11 cells, a hybridoma derived from rat dorsal root ganglia (DRG) and mouse neuroblastoma. Binding experiments revealed a low density (4 fmol/mg) of high affinity (0.5 nM) [(3)H]-EYF binding sites in these cells. The whole-cell planar patch-clamp technique showed that dNPA, a selective NPFF(2) agonist, increased the voltage-dependent potassium outward currents (about 30 pA/pF) by 21%; this reversible effect on sustained delayed potassium currents is blocked by tetraethylammonium. The similar effects of NPFF and opioid agonists on K(+) currents in this cell line may explain their similar antinociceptive actions at the spinal level.


Subject(s)
Hybridomas , Oligopeptides/pharmacology , Potassium Channels/metabolism , Receptors, Neuropeptide/metabolism , Analgesics, Opioid/pharmacology , Animals , Binding Sites , Cell Line , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Mice , Neuroblastoma/metabolism , Patch-Clamp Techniques , Potassium Channels/drug effects , Rats , Receptors, Neuropeptide/drug effects , Tetraethylammonium/pharmacology
15.
Pharmacol Rep ; 63(1): 102-11, 2011.
Article in English | MEDLINE | ID: mdl-21441617

ABSTRACT

We studied whether noradrenaline release is affected by interleukin-1ß and the neuropeptides urotensin II, melanin-concentrating hormone (MCH), neuropeptide W (NPW) and neuropeptide FF (NPFF). Rodent tissues preincubated with [3H]noradrenaline were superfused, and the effect of peptides on the electrically-evoked tritium overflow ("noradrenaline release") was studied. In mouse brain cortex, interleukin-1ß at 0.3 nM and the prostaglandin E2 analogue sulprostone at 3 nM inhibited noradrenaline release by about 40% the effect of interleukin-1ß developed gradually, whereas the effect of sulprostone occurred promptly. Urotensin II at 0.001-1 µM did not affect noradrenaline release in rat kidney cortex, whereas 0.01 µM angiotensin II increased it (positive control). MCH at 0.01-1 µM did not alter noradrenaline release in the rat brain cortex, and NPW 1 µM did not affect noradrenaline release in the mouse hypothalamus or hippocampus. In each model, 0.1 µM sulprostone inhibited noradrenaline release (positive control). NPFF and the NPFF2 receptor agonist dNPA (1 µM) did not affect noradrenaline release in the mouse atria; the inhibitory effect of the δ opioid receptor agonist 1 µM DPDPE on noradrenaline release in this tissue was not altered by NPFF or dNPA at 0.32 µM but was counteracted by the δ opioid antagonist naltrindole at 0.001 µM. In conclusion, interleukin-1ß inhibits noradrenaline release in the mouse cortex; the effect develops gradually, suggesting that it affects protein biosynthesis. Noradrenergic neurons in various tissues from rodents are devoid of presynaptic receptors for urotensin II, MCH, NPW and NPFF. Finally, an interaction between a δ opioid agonist and NPFF could not be detected.


Subject(s)
Interleukin-1beta/pharmacology , Neuropeptides/pharmacology , Norepinephrine/metabolism , Animals , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Dose-Response Relationship, Drug , Heart Atria/drug effects , Heart Atria/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Hypothalamus/drug effects , Hypothalamus/metabolism , Interleukin-1beta/administration & dosage , Kidney Cortex/drug effects , Kidney Cortex/metabolism , Male , Mice , Neuropeptides/administration & dosage , Protein Biosynthesis/drug effects , Rats , Rats, Wistar , Time Factors
16.
Neurosci Lett ; 488(3): 305-9, 2011 Jan 25.
Article in English | MEDLINE | ID: mdl-21111027

ABSTRACT

The present study investigates the modulatory effects of neuropeptide FF (NPFF) receptors on the mesolimbic dopaminergic pathway controlled by opioid receptors. A stable NPFF(2) receptor agonist, dNPA, was injected into the ventral tegmental area (VTA) and the release of dopamine and serotonin within the nucleus accumbens (NAc), induced by intraperitoneal injection of morphine, was monitored using the brain microdialysis, in non-constrained rat. dNPA decreased systemic morphine-induced elevation of dopamine and serotonin metabolites within the NAc. Furthermore, co-injected with morphine into the VTA, NPFF inhibited morphine-induced stereotypy 60-120min after the injection. This neurochemical and behavioural anti-opioid effect mediated by NPFF(2) receptors at the level of VTA suggests the involvement of NPFF in the rewarding effects of opiates on the mesolimbic dopamine system.


Subject(s)
Morphine Dependence/metabolism , Morphine/pharmacology , Narcotics/pharmacology , Receptors, Neuropeptide/metabolism , Ventral Tegmental Area/metabolism , Animals , Chromatography, High Pressure Liquid , Dopamine/metabolism , Male , Microdialysis , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Serotonin/metabolism , Ventral Tegmental Area/drug effects
17.
Biofactors ; 36(6): 423-9, 2010.
Article in English | MEDLINE | ID: mdl-20803521

ABSTRACT

Opioid receptors are involved in the control of pain perception in the central nervous system together with endogenous neuropeptides, termed opioid-modulating peptides, participating in a homeostatic system. Neuropeptide FF (NPFF) and related peptides possess anti-opioid properties, the cellular mechanisms of which are still unclear. The purpose of this review is to detail the phenomenon of cross-talk taking place between opioid and NPFF systems at the in vivo pharmacological level and to propose cellular and molecular models of functioning. A better knowledge of the mechanisms underlying opioid-modulating properties of NPFF has potential therapeutic interest for the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.


Subject(s)
Analgesics, Opioid/pharmacology , Morphine/pharmacology , Narcotic Antagonists , Neurons/drug effects , Oligopeptides , Opioid Peptides/antagonists & inhibitors , Receptor Cross-Talk/drug effects , Receptors, Opioid/metabolism , Amino Acid Sequence , Animals , Cattle , Humans , Mice , Models, Molecular , Molecular Sequence Data , Neurons/metabolism , Oligopeptides/pharmacology , Opioid Peptides/metabolism , Rats
18.
Neuropeptides ; 44(5): 453-6, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20554321

ABSTRACT

Neuropeptide FF (NPFF) is a neurotransmitter known to modulate opioid functions. This study investigates the effects of RF9, a new antagonist of NPFF receptors, on the roles of NPFF1 and NPFF2 receptors in thermoregulation in mice. RF9 (10 nmol) injected into the third ventricle did not modify the body temperature as compared to saline, but it completely antagonized the hypothermic effects of 10 nmol NPVF, a NPFF1 selective agonist, as well as the hyperthermic actions of dNPA (5 nmol), a NPFF2 selective agonist. The use of a specific antagonist demonstrates here that central NPFF1 and NPFF2 receptors control in an opposite manner the body temperature in mice.


Subject(s)
Body Temperature Regulation/drug effects , Body Temperature/physiology , Receptors, Neuropeptide/metabolism , Adamantane/analogs & derivatives , Adamantane/pharmacology , Analysis of Variance , Animals , Body Temperature/drug effects , Dipeptides/pharmacology , Male , Mice , Oligopeptides/pharmacology
19.
Neurochem Int ; 56(6-7): 768-73, 2010.
Article in English | MEDLINE | ID: mdl-20211672

ABSTRACT

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF(2) receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [(35)S]GTPgammaS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [(35)S]GTPgammaS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with alpha(s), alpha(i2) and beta subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.


Subject(s)
GTP-Binding Proteins/metabolism , Narcotic Antagonists/pharmacology , Oligopeptides/pharmacology , Receptors, Opioid, mu/drug effects , Analgesics, Opioid/pharmacology , Cell Line , Cell Line, Tumor , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Gene Expression , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Immunosorbent Techniques , Neuroblastoma , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/physiology , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Sulfur Radioisotopes , Transfection
20.
Synapse ; 64(9): 672-81, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20336629

ABSTRACT

Neuropeptide FF (NPFF) is a neurotransmitter known to modulate opioid-induced analgesia, sensitization, and reward. The expression of the immediate early gene c-Fos was analyzed to map the distribution of neurons whose activity is regulated by central administration of the NPFF(2)-selective agonist dNPA in naive mice and in animals who had received a systemic injection of morphine. The number of c-Fos positive nuclei was quantified in 28 brain regions. Intracerebro-ventricular injection of 1 nmol dNPA alone produced an overall inhibition of basal c-Fos expression in the brain with a statistically significant decrease in the lateral ventral part of the bed nucleus of the stria terminalis, the medial preoptic area, and the medial parvicellular part of the paraventricular nucleus of the hypothalamus. In contrast, intraperitoneal injection of morphine 5 mg.kg(-1) induced a statistically significant increase in c-Fos expression in the prelimbic cortex, the nucleus accumbens core and shell, the ventral pallidum, the lateral hypothalamus, and the nucleus of the tractus solitarius. dNPA counteracted morphine effect only in the nucleus accumbens shell and the ventral pallidum. The inhibitory effects of a low dose of dNPA in the hypothalamus and its afferents suggest that NPFF(2) receptors negatively regulate the hypothalamic-pituitary-adrenal axis in mouse. Moreover, our study identified the nucleus accumbens shell and ventral pallidum as putative sites of interaction between NPFF and opioid systems in relation with the modulation of acute morphine rewarding and locomotor effects.


Subject(s)
Analgesics, Opioid/pharmacology , Brain Chemistry/drug effects , Morphine/pharmacology , Neurons/drug effects , Proto-Oncogene Proteins c-fos/metabolism , Receptors, Neuropeptide/agonists , Animals , Brain/anatomy & histology , Female , Immunohistochemistry , Injections, Intraventricular , Mice , Mice, Inbred C57BL
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