Lionel Moulédous

Tissue distribution of the opioid receptor-like (ORL1) receptor

The ORL1 receptor is a G protein-coupled receptor structurally related to the opioid receptors, whose endogenous ligand is the heptadecapeptide nociceptin/orphanin FQ. In this review, data which have contributed to the mapping of the anatomic distribution of the ORL1 receptor have been collated with an emphasis on their relation to physiological functions. The ORL1 receptor is widely expressed in the central nervous system, in particular in the forebrain (cortical areas, olfactory regions, limbic structures, thalamus), throughout the brainstem (central periaqueductal gray, substantia nigra, several sensory and motor nuclei), and in both the dorsal and ventral horns of the spinal cord. Regions almost devoid of ORL1 receptors are the caudate-putamen and the cerebellum. ORL1 mRNA and binding sites exhibit approximately the same distribution pattern, indicating that the ORL1 receptor is located on local neuronal circuits. The ORL1 receptor is also expressed at the periphery in smooth muscles, peripheral ganglia, and the immune system. The anatomic distribution of ORL1 receptor suggests a broad spectrum of action for the nociceptin/orphanin FQ system (sensory perception, memory process, emotional behavior, etc.).

Central apelin controls glucose homeostasis via a nitric oxide-dependent pathway in mice

AIMS Apelin and its receptor have emerged as promising targets for the treatment of insulin resistance. Indeed, peripheral administration of apelin stimulates glucose utilization and insulin sensitivity via a nitric oxide (NO) pathway. In addition to being expressed on peripheral metabolically active adipose tissues, apelin is also found in the brain. However, no data are available on the role of central effects of apelin on metabolic control. We studied glucose metabolism in response to acute and chronic intracerebroventricular (i.c.v.) injection of apelin performed in normal and obese/diabetic mice. RESULTS We demonstrate that i.c.v. injection of apelin into fed mice improves glucose control via NO-dependent mechanisms. These results have been strengthened by transgenic (eNOS-KO mice), pharmacological (L-NMMA i.c.v. treated mice), and real-time measurement of NO release with amperometric probes detection. High-fat diet-fed mice displayed a severely blunted response to i.c.v. apelin associated with a lack of NO response by the hypothalamus. Moreover, central administration of high dose apelin in fasted normal mice provoked hyperinsulinemia, hyperglycemia, glucose intolerance, and insulin resistance. CONCLUSION These data provide compelling evidence that central apelin participates in the regulation of glucose homeostasis and suggest a novel pathophysiological mechanism involved in the transition from normal to diabetic state.

Opioid-modulating properties of the neuropeptide FF system

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.

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

Background: Neuropeptide FF2 receptors interact with μ-opioid receptors and decrease their activity. Results: The phosphorylation pattern of MOP receptor is similar after homologous (DAMGO) and heterologous (neuropeptide FF) stimulation. Conclusion: GPCR kinase 2 (GRK2) contributes to the NPFF-induced phosphorylation and loss of function of MOP receptor. Significance: GRK2-mediated transphosphorylation within receptor heteromers could play a role in heterologous desensitization of GPCRs. 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 NPFF2 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 NPFF2 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 Gi/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, NPFF2 receptor activation was sufficient to recruit β-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.

Modulation of basal and morphine-induced neuronal activity by a NPFF2 selective agonist measured by c-Fos mapping of the mouse brain

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 NPFF2‐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 NPFF2 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. Synapse 64:672–681, 2010.

Direct identification of a peptide binding region in the ORL1 receptor by photo-affinity labelling with [Bpa10, Tyr14]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe10,Tyr14]nociceptin ([Bpa10,Tyr14]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa10,Tyr14]noc binds the receptor with high affinity (K i ∼0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC50 ∼0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa10,Tyr14]noc results in the irreversible labeling of a glycoprotein of ∼65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (∼6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of ∼3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu295 → Asp mutant receptor also yields a single labeled fragment of ∼6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296–302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa10]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile300 within the experimentally identified photoreactive region.

Opposite control of body temperature by NPFF1 and NPFF2 receptors in mice

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.

Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

BACKGROUND The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. METHODS In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography-tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. RESULTS Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. CONCLUSIONS These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells.

Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF(2)) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF(2) receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

Heterologous regulation of Mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells.

Background: MOP receptor function is presumably linked to a specific dynamic organization in the membrane. Results: Inhibition of MOP receptor signaling by NPFF2 and α2 receptors is accompanied by diffusion changes, with a particular behavior for heterodimers. Conclusion: MOP receptor function, diffusion, and confinement are subject to specific heterologous regulation by other GPCRs. Significance: Specific GPCR regulation is associated with particular dynamic organization in the membrane. The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.