Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding.

By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

Replacement of Gln280 by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids.

The ORL1 (Opioid Receptor-Like) receptor is the G protein-coupled receptor whose amino acid sequence is closest to those of opioid receptors. Residues that are conserved in ORL1 and the three types of opioid receptor, but also a residue, His in the sixth putative transmembrane (TM6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activation. Here we have sought to create an opioid binding pocket in the non-opioid ORL1 receptor by replacing residue Gln280 in its TM6 by the corresponding His residue of opioid receptors. The mutation affects neither the affinity of nociceptin - the natural ORL1 agonist - for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase via ORL1. In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor-BNI, bind the mutant Q280H receptor with substantially (5- to > 100-fold) higher apparent affinity than they do the wild-type receptor. Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor, but are endowed with clear antagonist properties at the mutant receptor. The mutation Q280H, which increases affinity while decreasing intrinsic activity of opioids at ORL1, emphasizes the importance of the His residue for opioid recognition and activation.

Different domains of the ORL1 and kappa-opioid receptors are involved in recognition of nociceptin and dynorphin A.

In order to gain further insight into the functional architecture of structurally related G protein-coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and mu-, delta- and kappa-opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1-kappa-opioid (O-K) hybrid construct has retained high affinity for non-type-selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and mu- and/or delta-opioid receptor-selective enkephalins analogs, thus behaving like a 'universal' opioid receptor. Most significantly however, whilst the ORL1 and kappa-opioid receptors display high binding preference (KD 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O-K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (KD < 1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and kappa-opioid receptors are involved in recognition of their endogenous peptide ligands.

Comparison of the structure-activity relationships of nociceptin and dynorphin A using chimeric peptides.

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.

ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization.

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the delta-opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane-spanning domains of a G protein-coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORL1 is 49-50% identical to the murine mu-, delta- and kappa-opioid receptors and, in CHO-K1 cells stably transfected with a pRc/CMV:hORL1 construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a 'universal' (nonselective) opiate agonist. Yet, hORL1 appears not to be a typical opioid receptor. Neither is it a somatostatin or sigma (N-allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula, ...) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

Photoactivatable opiate derivatives as irreversible probes of the mu-opioid receptor.

The synthesis of aryldiazonium and arylazido derivatives of carfentanil, etonitazene, and naltrexone and of a triazaspirodecane derivative is described. The chemical stability and the spectral characteristics of these compounds were verified, and their binding affinity constants for the different opioid receptor classes were determined, in the absence of light, from competition experiments. With the exception of the naltrexyl derivatives, which remained nonselective, all compounds tested displayed a pronounced mu-binding selectivity with mu/delta and mu/kappa ratios ranging from 12 to 1000. After irradiation, only the arylazido probes led to an irreversible mu-binding-site inactivation. This inactivation fulfilled the criteria for photoaffinity labeling such as protection against inactivation by other opiate ligands and absence of an effect of scavengers on the extent of the inactivation. Most of the photoactivatable probes formed long-lasting reversible complexes with the opioid binding sites: an efficient dissociation procedure was thus required to discriminate between pseudoirreversible and covalent complexes. The marked differences in labeling efficacy between aryldiazonium salts and their corresponding arylazido derivatives are discussed.

Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective radioligands.

The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively. By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS. The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord. The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.

Distribution of the nociceptin and nocistatin precursor transcript in the mouse central nervous system.

The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.

Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226.

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non-peptide or peptide NPFF-antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF(2) receptors. NPY Y(1) antagonist BIBP3226 and mixed Y(1) antagonist/Y(4) agonist GR231118 are able to displace with low affinity, 50 -- 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y(2), Y(4) or Y(5) receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin-stimulated cyclic AMP production mediated by NPFF(2) receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

Functional characterization of a human receptor for neuropeptide FF and related peptides.

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

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.).

[(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors.

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively). Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity. Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn. Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%. These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.

Structure-activity relationships of neuropeptide FF: role of C-terminal regions.

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine7 by a lysine or D. Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe8 and Arg7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.

[Phe1psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 is an agonist of the nociceptin (ORL1) receptor.

[Phe1psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2, a pseudopeptide analog of nociceptin, has been shown to be a selective 'antagonist' of the nociceptin receptor in the isolated guinea pig ileum and mouse vas deferens preparations (Guerrini et al., 1998. Br. J. Pharmacol. 123, 163-165). However, in recombinant chinese hamster ovary cells expressing the human nociceptin receptor, we find that the pseudopeptide is a potent (IC50 = 7.5 nM) and fully efficacious inhibitor of forskolin-induced accumulation of cAMP, thus behaving as a pure 'agonist' rather than an antagonist of the receptor. The contrary behaviour of the pseudopeptide in smooth muscle and transformed cells may suggest that different nociceptin receptor types are being addressed in the two systems.

Regulation by Na+ ions and GppNHp of the interaction between a G protein and the amphibian type of opioid receptor.

Digitonin treatment of frog brain membranes in 50 mM Tris-HCl yields a soluble extract that contains nearly equal amounts of free and G protein-bound opioid receptor molecules (Mollereau et al., 1988, J. Biol. Chem. 263, 18003). We report here that the balance of the two forms of the opioid receptor in digitonin solution is dependent on the environment of the membrane suspension at the time of solubilization with the detergent. Preincubating the membrane suspension with 50 microM GppNHp or with 120 mM NaCl results, in the two cases, in a digitonin extract that no longer displays the G protein-bound form of the receptor, i.e., the form of the receptor which exhibits high affinity for the opiate agonist etorphine in binding studies, as well as large apparent molecular size in sucrose gradients. Assuming that the situation in soluble extracts faithfully reflects the one in the membrane, these results would exclude the possibility that in a physiological environment the opioid receptor is in part precoupled with a G protein in the absence of an opioid agonist.

Evidence for a new type of opioid binding site in the brain of the frog Rana ridibunda.

The crude membrane fraction from the brain of the frog Rana ridibunda was shown to contain 0.7-0.8 pmol/mg protein for a site with high (KD = 0.1 nM) and about 3.2 pmol/mg protein for a site with lower (KD = 10-15 nM) affinity for the opiate agonist [3H]etorphine and for the opiate antagonist [3H]diprenorphine. In addition to its very high affinity for the two tritiated oripavine derivatives, the high affinity site displayed (i) a considerably reduced ability to bind the agonist but not the antagonist in the presence of Na+ ions and (ii) pronounced stereospecificity. These properties are all typical of an opioid receptor site. The lower affinity site, which was about four times as abundant as the other exhibited none of the aforementioned characteristics and is therefore probably not opioid in nature. Detailed testing of the potency of various unlabelled opioid ligands to inhibit the binding of [3H]etorphine at the high affinity site showed that the latter consists of a mixture of several types of opioid sites, including a major type with an apparent binding profile clearly different from those of mammalian brain mu, delta- and kappa-opioid sites. In particular, this major type of site, which accounted for about 70% of the opioid binding in frog brain membranes, bound mu ([D-Ala2,MePhe4,Glyol5]enkephalin), delta ([D-Thr2,Leu5]enkephalyl-Thr) and kappa (U50,488) selective ligands with much lower affinity than did mu-, delta- and kappa-opioid receptor sites, respectively.

Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids.

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.

Involvement of neuropeptide FF receptors in neuroadaptive responses to acute and chronic opiate treatments.

BACKGROUND AND PURPOSE Opiates remain the most effective compounds for alleviating severe pain across a wide range of conditions. However, their use is associated with significant side effects. Neuropeptide FF (NPFF) receptors have been implicated in several opiate-induced neuroadaptive changes including the development of tolerance. In this study, we investigated the consequences of NPFF receptor blockade on acute and chronic stimulation of opioid receptors in mice by using RF9, a potent and selective antagonist of NPFF receptors that can be administered systemically. EXPERIMENTAL APPROACH The effects of RF9 were investigated on opioid pharmacological responses including locomotor activity, antinociception, opioid-induced hyperalgesia, rewarding properties and physical dependence. KEY RESULTS RF9 had no effect on morphine-induced horizontal hyperlocomotion and slightly attenuated the decrease induced in vertical activity. Furthermore, RF9 dose-dependently blocked the long-lasting hyperalgesia produced by either acute fentanyl or chronic morphine administration. RF9 also potentiated opiate early analgesic effects and prevented the development of morphine tolerance. Finally, RF9 increased morphine-induced conditioned place preference without producing any rewarding effect by itself and decreased naltrexone-precipitated withdrawal syndrome following chronic morphine treatment. CONCLUSION AND IMPLICATIONS The NPFF system is involved in the development of two major undesirable effects: tolerance and dependence, which are clinically associated with prolonged exposure to opiates. Our findings suggest that NPFF receptors are interesting therapeutic targets to improve the analgesic efficacy of opiates by limiting the development of tolerance, and for the treatment of opioid dependence.