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A new radioiodinated photoaffinity compound, [ 125 I]YE(Bpa)WSLAAPQRFNH 2 , 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 NPFF 2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction ( K D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF 2 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 NPFF 2 receptor with apparent molecular weights of 140 and 95 kDa 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 NPFF 2 receptor is observed. [ 125 I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF 2 receptors in vitro.

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 NPFF 1 and NPFF 2 receptors in thermoregulation in mice. RF9 (10nmol) injected into the third ventricle did not modify the body temperature as compared to saline, but it completely antagonized the hypothermic effects of 10nmol NPVF, a NPFF 1 selective agonist, as well as the hyperthermic actions of dNPA (5nmol), a NPFF 2 selective agonist. The use of a specific antagonist demonstrates here that central NPFF 1 and NPFF 2 receptors control in an opposite manner the body temperature in mice.

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.

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.

NPFF receptors are expressed in several brain regions directly or indirectly involved in cognition and behavior. However, the cognitive effects of the NPFF system have been poorly studied. Therefore, the aim of our study was to analyze the effects of i.c.v. injections of 1 DMe, a stable agonist of NPFF receptors, on behavioral and cognitive performances in C57BL/6J mice. We measured locomotor activity, and an open field with objects was used to estimate the ability of mice to react to spatial changes and to measure short-term retention of information. The Morris navigation task was used to evaluate the acquisition, as well as long-term retention of a hippocampo-dependent spatial memory with a distributed training procedure. Finally, 1 DMe was tested in a contextual fear conditioning paradigm to study its effect on long-term memory of contextual information acquired in a single training session. Altogether, our results demonstrate a small but complex influence of the NPFF system on mouse behavior. 1 DMe injected i.c.v. induces a delayed hyperlocomotion and mildly impairs both short-term and long-term spatial memory processing without affecting contextual fear memory.

Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF2 and μ-opioid (MOP) receptors tagged with variants of the green fluorescent protein. Importantly, cyan fluorescent protein-tagged NPFF2 receptors retained their capacity to antagonize opioid receptors. Fluorescence resonance energy transfer (FRET) and coimmunoprecipitation studies indicate that NPFF and MOP receptors are close enough to generate a basal FRET signal. The opioid agonist Tyr-d-Ala-Gly-NMe-Phe-Gly-ol disrupts by 20-30% this FRET signal, mainly because it concomitantly induces 40% internalization of receptors. In contrast, the NPFF analog 1DMe significantly increases by 10-15% the basal FRET signal, suggesting an association between both receptors. In addition, 1DMe reduces, by half, MOP receptor internalization, indicating that, besides a functional blockade of opioid receptors, the NPFF analog also inhibits their internalization. Finally, as a first report showing the modulation of the mobility of a G-protein-coupled receptor by another one, fluorescence recovery after photobleaching analysis reveals that 1DMe modifies the lateral diffusion of MOP receptors in the cell membrane, changing them from a confined to a freely diffusing state. By promoting NPFF-MOP receptor heteromerization, 1DMe could disrupt the domain organization of MOP receptors in the membrane, resulting in a reduction of opioid response.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

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.

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFFA precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.

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.

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.

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 NPFF 2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF 2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine 8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine 7 by a lysine or D.Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe 8 and Arg 7 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 NPFF 2 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.

[ 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.

In rat dorsal raphe neurones, nociceptin (300 nM) reduced the peak [Ca(2+)](i) transient, triggered by depolarization, by 36.7+/-1.8% (n=46). This effect of nociceptin decreased to 16.7+/-2.9% (n=18) after pre-treatment of the neurones with pertussis toxin (5 microg/ml, 2-6 h) but was unchanged (37.4+/-2.1%, n=44) after pre-incubation with cholera toxin (5 microg/ml, 2-6 h). This suggests that, in dorsal raphe neurones, the ORL1 receptor couples to inhibitory (G(i/o)) G-proteins. The neuropeptide FF analogue, [D-Tyr1, (N-Me)Phe(3)]neuropeptide FF (10, 100, 1000 nM), acted as an anti-opioid and reduced the effect of nociceptin (300 nM, 30 s) by 62.0+/-3.3% (n=28). Following pre-incubation with cholera toxin (5 microg/ml, 2-6 h) [D-Tyr1, (N-Me)Phe3] neuropeptide FF was unable, at the three concentrations tested, to block nociceptin activity. We conclude that, in rat dorsal raphe neurones, neuropeptide FF receptors couple to stimulatory G-proteins (Gs).

In mice pretreated intracerebroventricularly (i.c.v.) with pertussis or cholera toxins, effects of neuropeptide FF (NPFF), on hypothermia and morphine-induced analgesia, were assessed. NPFF and a potent NPFF agonist, 1DMe (0.005–22 nmol) injected into the lateral ventricle decreased morphine analgesia and produced naloxone (2.5 mg kg −1 , s.c.)-resistant hypothermia after administration into the third ventricle. Cholera toxin (CTX 1 μg, i.c.v.) pretreatment (24 or 96 h before) inhibited the effect of 1DMe on body temperature, but failed to reverse its anti-opioid activity in the tail-flick test. CTX reduced hypothermia induced by a high dose of morphine (8 nmol, i.c.v.) but not the analgesic effect due to 3 nmol morphine. Pertussis toxin (PTX) pretreatment inhibited both morphine-hypothermia and -analgesia but did not modify hypothermia induced by 1DMe. The present results suggest that NPFF-induced hypothermia depends on the stimulation of Gs (but not Gi) proteins. In contrast, anti-opioid effects resulting from NPFF-receptor stimulation do not involve a cholera toxin-sensitive transducer protein.

Since neuropeptide FF (NPFF) is a putative neurotransmitter to exert anti-opioid activity, we examined the effects of [D-Tyr1,(NMe)Phe3]neuropeptide FF (1DMe), a stable NPFF analog, on acetylcholine (ACh) release from a longitudinal muscle-myenteric plexus (LMMP) preparation of guinea pig ileum in which opioids were known to inhibit ACh release when muscarinic autoinhibition was not fully activated. In the presence of atropine, 1DMe increased spontaneous and electrical field stimulation (EFS)-evoked ACh release in a concentration-dependent manner. Naloxone also increased ACh release. The stimulatory effects of 1DMe and naloxone were not additive. In the absence of atropine, 1DMe did not affect ACh release. Morphine decreased spontaneous and EFS-evoked ACh release in the presence of 1 μM atropine. 1DMe as well as naloxone counteracted the inhibitory effects of morphine on EFS-evoked ACh release. The combination of 1DMe and naloxone was not more inhibitory than either drug alone. 1DMe had no appreciable effect on norepinephrine-induced inhibition of spontaneous and EFS-evoked ACh release. These results first demonstrated the effects of a NPFF analog on neurotransmitter release: 1DMe had a stimulatory effect on spontaneous and EFS-induced ACh release from the LMMP preparation of guinea pig ileum, probably by counteracting the inhibitory effect of endogenous opioids on ACh release.

The neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and its synthetic analogs bind to specific receptors in the spinal cord to produce antinociceptive effects that are partially attenuated by opioid antagonists, and at sub-effective doses neuropeptide FF receptor agonists augment spinal opioid antinociception. Since adenosine plays an intermediary role in the production of spinal opioid antinociception, this study investigated whether this purine has a similar role in the expression of spinal effects produced by neuropeptide FF receptor agonists. In rats bearing indwelling spinal catheters, injection of adenosine receptor agonists, N6-cyclohexyladenosine (CHA, 1.72 nmol) and N-ethylcarboxiamidoadenosine (NECA, 1.95 nmol), as well as morphine (13.2 nmol) elicited antinociception in the tail-flick and paw-pressure tests. Pretreatment with intrathecal 8-phenyltheophylline (5.9 and 11.7 nmol), an adenosine receptor antagonist, blocked the effect of all three agents without influencing baseline responses. Administration of two synthetic neuropeptide FF (NPFF) analogs, [ d -Tyr1,(NMe)Phe3]NPFF (1DMe, 0.86 nmol) and [ d -Tyr1, d -leu2, d -Phe3]NPFF (3D, 8.6 nmol) produced sustained thermal and mechanical antinociception. Pretreatment with doses of intrathecal 8-phenyltheophylline (5.9, 11.7 and 23.5 nmol), producing adenosine receptor blockade, significantly inhibited the antinociceptive effects of 1DMe or 3D. Injection of a sub-antinociceptive dose of 1DMe (0.009 nmol) significantly augmented the antinociceptive effect of intrathecal morphine (13.2 nmol) in the tail-flick and paw-pressure tests. Intrathecal 8-phenyltheophylline (11.7 nmol) reduced the effect of this combination. Administration of low dose of 1DMe (0.009 nmol) or 3D (0.009 nmol) very markedly potentiated the antinociceptive actions of the adenosine receptor agonist, N6-cyclohexyladenosine (0.43, 0.86 and 1.72 nmol) in the tail-flick and paw-pressure tests 50 min after injection. The results suggest that the antinociceptive and morphine modulatory effects resulting from activation of spinal NPFF receptors could be due to an increase in the actions or availability of adenosine.

This study examined the ability of the anti-opioid Neuropeptide FF (NPFF) to modify the endogenous activity of nitric oxide (NO). Antinociceptive and hypothermic effects of 1DMe (D.Tyr-Leu-(N.Me)Phe-Gln-Pro-Gln-Arg-Phe-NH2), an NPFF agonist, and of l -NAME (Nωnitro- l -arginine methyl ester), an inhibitor of nitric oxide synthase, were investigated in mice. Intraperitoneal (i.p.) injection of l -NAME induced, in the hot plate test, a dose-dependent antinociception not reversed by naloxone, an opioid antagonist, but inhibited by l -Arg, the NO synthesis precursor. Intracerebroventricular (i.c.v.) injections of 1DMe inhibit the antinociceptive activity of l -NAME in a dose-dependent manner. On the contrary, l -NAME markedly potentiated hypothermia induced by 1DMe injected in the third ventricle. These data show that Neuropeptide FF receptors exert a dual effect on endogenous NO functions and could modulate pain transmission independently of opioids.

Although neuropeptide FF (NPFF) is generally considered an anti-opioid, its intrathecal administration produces analgesia. In the present study, the stable analog 1DMe ([D.Tyr(1), (NMe)Phe(3)]neuropeptide FF) was used in quantitative autoradiographic experiments in combination with surgical and chemical lesions to precisely localize NPFF receptors in the rat spinal cord. Ligation of lumbar dorsal spinal roots revealed the presence of NPFF receptors in dorsal root fibers and it induced a significant accumulation of [(125)I]1DMe-specific binding on the side peripheral to the ligature, demonstrating that a population of NPFF receptors is synthesized in dorsal root ganglia and migrates anterogradely towards primary afferent nerve endings. Complete mid-thoracic spinal cord transection failed to modify the [(125)I]1DMe labeling density in the dorsal horn, indicating that NPFF receptors are not located on the descending fiber terminals. In contrast, unilateral microinjections of kainic acid into the dorsal horn dramatically reduced [(125)I]1DMe-specific binding in the superficial layers, revealing localization of a population of NPFF receptors on the spinal intrinsic neurons. NPFF receptor binding was not modified during the development of spinal opioid tolerance. The pre- and postsynaptic localization of spinal NPFF receptors provide further support for heterogeneity in the pain modulation by NPFF and related agonists.

Degradation of neuropeptide FF (NPFF) and SQA-neuropeptide FF (SQA-NPFF) by mouse brain sections was investigated by using capillary electrophoresis with UV detection for the separation and the identification of the degradation products. The half disappearance time of SQA-NPFF was 2-fold greater than that of NPFF. NPFF was cleaved preferentially into an inactive metabolite, Gln-Arg-Phe-NH2, in the cerebrum slices. SQA-NPFF was hydrolyzed by an unidentified degrading activity to generate NPFF, and NPFF accounted for a larger part of SQA-NPFF degradation in the hindbrain and cervical spinal cord than in the cerebrum slices. These findings suggest that, depending on the brain regions, NPFF produced from SQA-NPFF could prolong the biologic effects of SQA-NPFF.

Intracellular Ca 2+ concentration ([Ca 2+ ] i ) was measured in neurons, acutely dissociated from the rat dorsal raphe nucleus (DRN), with the fluorescent calcium probe Fluo3. Nociceptin (300 nM) had no effect on resting [Ca 2+ ] i but reduced the magnitude of the [Ca 2+ ] i transient triggered by depolarization in 90% of neurons having polygonal or fusiform perikarya. In 94% of neurons with the same morphology 5-HT (30 μM) also reduced the magnitude of the [Ca 2+ ] i transient. The selective 5-HT 1A receptor antagonist 4-iodo- N -[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]- N -2-pyridinyl-benzamide hydrochloride (p-MPPI) (0.4 μM) strongly attenuated (by 72±7%, n =4) this effect. The responses to nociceptin and 5-HT were not affected by BaCl 2 (100 μM). The neuropeptide FF analog [ d -Tyr 1 , ( N -Me)Phe 3 ]NPFF (1DMe) altered neither the resting [Ca 2+ ] i nor the [Ca 2+ ] i transient triggered by depolarization but dose-dependently decreased the effect of nociceptin (EC 50 =1.8 nM, maximal reduction: 68±5%). 1DMe had no effect on the response to 5-HT. Another neuropeptide FF analog, exhibiting a different pharmacological activity in mice and rats, [ d -Tyr 1 , d -Leu 2 , d -Phe 3 ]NPFF (1 μM) also reduced the effect of nociceptin by 74±11% ( n =4). Few neurons (5 out of 42), either with polygonal/fusiform or smaller ovoid cell bodies, responded to the μ-opioid receptor agonist [ d -Ala 2 , ( N -Me)Phe 4 , Gly-ol 5 ]-enkephalin (DAGO) with a decrease in the depolarization-induced [Ca 2+ ] i transient. 1DMe (100 nM) attenuated this response by 69±14%. These results suggest that, at the cellular level, neuropeptide FF selectively counteracts the effects of opioid receptor activation.

We report on the biochemical, cellular and pharmacological activities of SQA-neuropeptide FF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH2), a peptide sequence contained in the human neuropeptide FF (neuropeptide FF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH2) precursor. Quantitative autoradiography revealed that, in the superficial layers of the rat spinal cord, SQA-neuropeptide FF displayed the same high affinity for [ 125 I ]1DMe ([ 125 I ] d -Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe–NH2) binding sites (Ki=0.33 nM) as did neuropeptide FF (Ki=0.38 nM). In acutely dissociated mouse dorsal root ganglion neurones, SQA-neuropeptide FF reduced by 40% the depolarisation-induced rise in intracellular Ca2+ as measured with the Ca2+ indicator, Fluo-3. In mice, 1DMe and SQA-neuropeptide FF dose-dependently inhibited the antinociceptive effect of intracerebroventricular (i.c.v.) injections of morphine, but SQA-neuropeptide FF was less potent than 1DMe. Furthermore, SQA-neuropeptide FF, as well as 1DMe, produced marked hypothermia following third ventricle injections in mice. These data demonstrate that the human peptide, SQA-neuropeptide FF, exhibits biochemical and pharmacological properties similar to those of neuropeptide FF or neuropeptide FF analogues, and belongs to the neuropeptide FF family.

Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and the octadecapeptide neuropeptide AF (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2) were isolated from bovine brain, and were initially characterized as anti-opioid peptides. They can oppose the acute effects of opioids and an increase in their brain concentrations may be responsible for the development of tolerance and dependence to opioids. Numerous experiments suggest a possible neuromodulatory role for neuropeptide FF. A precursor protein has been identified, in particular in human brain. Neuropeptide FF immunoreactive neurons are present only in the medial hypothalamus, and the nucleus of the solitary tract, and in the spinal cord in the superficial layers of the dorsal horn and areas around the central canal. Depolarization induces a Ca2+-dependent release of neuropeptide FF immunoreactivity from the spinal cord. Neuropeptide FF acts through stimulation of its own receptors and high densities of specific binding sites are found in regions related either to sensory input and visceral functions or to the processing of nociceptive messages. In both isolated dorsal root ganglion neurons and CA1 pyramidal neurons of the hippocampus, neuropeptide FF has little effect of its own but reverses the effects of mu-opioid receptor agonists. In agreement with the hypothesized anti-opioid role of neuropeptide FF, supraspinal injection lowers the nociceptive threshold and reverses morphine-induced analgesia in rats. Furthermore, immunoneutralization of neuropeptide FF increases endogenous and exogenous opioid-induced analgesia. Similarly, microinfusion of neuropeptide FF or neuropeptide FF analogs into the nucleus raphe dorsalis, the parafascicular nucleus, or the ventral tegmental area has no effect on the nociceptive threshold but inhibits the analgesia induced by co-injected morphine. Furthermore, infusion of neuropeptide FF into the parafascicular nucleus or the nucleus raphe dorsalis reverses the analgesic effect of morphine infused into the nucleus raphe dorsalis or the parafascicular nucleus, respectively, demonstrating remote interactions between neuropeptide FF and opioid systems. By contrast, intrathecal administration of neuropeptide FF analogs induces a long lasting, opioid-dependent analgesia and potentiates the analgesic effect of morphine. Analgesic effects of neuropeptide FF after supraspinal injection could also be observed, for example during nighttime. In young mice, (1DMe)Y8Famide (D.Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2), a neuropeptide FF analog, increases delta-opioid receptor-mediated analgesia. These findings indicate that neuropeptide FF constitutes a neuromodulatory neuronal system interacting with opioid systems, and should be taken into account as a participant of the homeostatic process controlling the transmission of nociceptive information.

The ability of (1DMe)Y8Fa (D.Tyr–Leu–(NMe)Phe–Gln–Pro–Gln–Arg–Phe–NH2), a selective neuropeptide FF analog resistant to enzymatic degradation, to control morphine-induced analgesia was investigated in rat after microinfusion into the dorsal raphe nucleus and the nucleus parafascicularis of the thalamus. Infusion of (1DMe)Y8Fa (2.5 nmol) in the nucleus raphe dorsalis did not modify the animal response in the tail-immersion test but significantly reversed analgesia induced by coinjected morphine (27 nmol). Similarly, (1DMe)Y8Fa (5 nmol) inhibited morphine effects in the hot-plate test after co-injection into the parafascicular nucleus. Furthermore, (1DMe)Y8Fa injected into the parafascicular nucleus attenuated analgesia induced by morphine injected into the nucleus raphe dorsalis and similarly, the neuropeptide FF analog in the nucleus raphe dorsalis decreased the effects of 27 nmol morphine injected in the parafascicular nucleus. The density of neuropeptide FF receptors did not decrease in the nucleus raphe dorsalis after lesion of serotonergic neurons by 5,7-dihydroxytryptamine. However, after this lesion, (1DMe)Y8Fa injected in the nucleus raphe dorsalis was no longer able to modify analgesic effects of morphine in hot-plate and tail-immersion tests. Similarly, the serotonin (5-HT) depletion induced by a systemic administration of para-chlorophenylalanine did not modify morphine analgesia microinjected into the nucleus raphe dorsalis and the parafascicular nucleus but blocked the ability of (1DMe)Y8Fa to reverse morphine effects in both nuclei. These data show that neuropeptide FF exerts anti-opioid effects directly into both the nucleus raphe dorsalis and the parafascicular nucleus and acts also at distance on opioid functions. Furthermore, anti-opioid effects of neuropeptide FF require functional serotonergic neurons although neuropeptide FF receptors are not carried on these neurons.

The ability of neuropeptide FF (NPFF) to modulate mu- and delta-opioid-induced analgesia by intracerebroventricular administration was compared in adults and 14-day-old mice. In adults, opioid-induced analgesia was predominantly mediated by mu-receptors whereas mu- and delta-receptors were equally involved in pups. An NPFF analog, 1 DMe, reduced the analgesic effect of DAGO and [D.Ala2]deltorphin-I, mu and delta selective agonists respectively. However, a high dose of 1DMe (22 nmol) increased both morphine and [D.Ala2]deltorphin-I-induced analgesia. Dose-response curves for 1DMe in the presence of naltrindole or naltrexone, delta- and mu-opioid selective antagonists respectively, indicate that 1DMe preferentially reversed mu-receptor-mediated but increased delta-receptor-mediated analgesia. These findings demonstrate differences in control of mu- and delta-induced analgesia by NPFF receptors.

The binding properties of neuropeptide FF (NPFF) receptors were investigated in different laminae of the rat spinal cord by using quantitative autoradiography and [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF as radioligand. In the superficial layers, the specific binding of [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF was time-dependent, reversible, and saturable ( K D = 0.1 nM). Preincubation of spinal sections increased the maximal number of [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF binding sites. Bestatin, an inhibitor of aminopeptidases, increased significantly the apparent affinity of NPFF. Optimal binding of [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF was observed in the presence of 120 mM NaCl in all laminae of the spinal cord. No significant differences were noted in the salt dependence in laminae I–II, IV–V, and X, and the pharmacological profile of [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF binding was similar in each laminae. These results do not support the existence of NPFF receptors subtypes differentially localized in different area of the spinal cord. Our data reveal the effects of tissue treatments on binding characteristics of NPFF receptors and indicate that [ 125 I][D.Tyr 1 , (NMe)Phe 3 ]NPFF is a useful radioactive probe for the characterization of NPFF receptors in discrete brain areas.

Quantitative in vitro receptor autoradiography of [125I][ d -Tyr1,(NMe)Phe3]NPFF was used to study the regional distribution of neuropeptide FF receptors in rodent and lagomorph brain. In rat, mouse, rabbit, and Afghan pika [125I][ d -Tyr1,(NMe)Phe3]NPFF binding sites were enriched in the superficial layers of dorsal horn of the spinal cord and in parabrachial nucleus, central gray matter, hypothalamus, and reuniens thalamic nucleus. In other neuroanatomical regions, important species differences in NPFF receptor patterns are observed. In marked contrast, the brain and the spinal cord of the Octodon degus are devoid of NPFF receptors. The present study shows that in different species regional variations in brain NPFF receptor binding occur.

The effect of microinfusion into the nucleus raphe dorsalis (DR) of neuropeptide FF (NPFF) analogs on the antinociceptive effects of morphine was evaluated in rats, using the tail-immersion test. infusion of morphine into the DR induced a dose-dependent analgesia significantly reversed by co-infusion of 2.5 nmol opioid antagonist, naloxone. Similarly, 2.5 nmol NPFF and (1DMe)Y8Fa(D-Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2) or (3D)Y8Fa(D-Tyr-D-Leu-D-Phe-Gln-Pro-Gln-Arg-Phe-NH2), two neuropeptide FF analogs, inhibited morphine analgesia, although these peptides had no effect on nociceptive thresholds. This anti-opioid effect is indirect since NPFF analogs displayed no significant affinity towards mu and delta opioid binding sites in the DR. After intracerebroventricular infusion, morphine produced the same degree of analgesia as that measured after infusion into the nucleus raphe dorsalis and both NPFF analogs reversed morphine antinociception. This result is the first direct evidence that neuropeptide FF may act on opioid system at the DR and that several nuclei are involved in the suppression of morphine-induced antinociception.

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.

The distribution of β-opioid binding sites was studied by quantitative autoradiography in rat brain and spinal cord using the highly selective ligand [125I][D. Ala2]deltorphin-I. The binding properties of [125I][D. Ala2]deltorphin-I were investigated by microdensitometry of autoradiographic films with the aid of a computerassisted image-analysis system. [125I][D. Ala2]deltorphin-I appeared to interact with a single class of sites in all brain areas (KD = 0.9 nM). In 23 regions tested, whatever the δ site concentration, DTLET, a δ agonist, appears to be 2 orders of magnitude more effective than DAGO, a μ agonist, in inhibiting specific [125I][D. Ala2]deltorphin-I binding. The distribution of [125I][D. Ala2]deltorphin-I sites is globally consistent with that of other δ ligands and does not support the existence of a δ-receptor subtype recognized by [D. Ala2]deltorphin-I. [125I][D. Ala2]deltorphin-I binding sites were highly confined, exhibiting selective localization in the neocortex and a diffuse pattern in the striatum, accumbens nucleus, claustrum, layer of bulb, amygdaloid nucleus, pontine nuclei, and inferior colliculus. In several areas a rostro-caudal gradient of site concentration was indicated. [D. Ala2]deltorphin-I binding sites were also present in the substantia gelatinosa at all levels of the spinal cord and, unexpectedly, in deeper laminae and the ventral horn. These results demonstrate the ability of [125I][D. Ala2]deltorphin-I to characterize low concentrations of binding sites and to reveal new localizations of δ receptors. © 1993 Wiley-Liss, Inc.

This study presents the binding and functional properties of the mouse NPFF(2) (mNPFF(2)) receptor, in comparison with its human counterpart (hNPFF(2)). Binding experiments were performed by using the NPFF(2) selective radioligand [(3)H]-EYF in membranes from CHO cells transfected with mouse and human NPFF(2) receptors and compared to membranes from mouse olfactory bulb, the brain region expressing the highest density of NPFF(2) receptors in mouse. mNPFF(2) receptors exhibited a high affinity (Kd=0.2-0.4 nM) for [(3)H]-EYF, comparable to that of hNPFF(2) receptors. Also, the binding selectivity profile of mNPFF(2) receptors was comparable to that of hNPFF(2) receptors, except for three ligands (NPSF, NPVF, RF9) that were about tenfold more potent and active on mouse receptors than on human receptors. In particular, compared to hNPFF(2) receptors, mNPFF(2) receptors were less discriminative towards the proNPFF(B)-derived peptide. This suggests some species-related differences in the binding properties of NPFF(2) receptors that could have repercussion when evaluating the pharmacological properties of drugs in vivo.

The binding characteristics of [(3)H]-NPVF and [(3)H]-EYF, the two first tritiated probes for the respective labelling of NPFF(1) and NPFF(2) receptors, are presented. In membranes from CHO cells transfected with the human NPFF(1) receptor, [(3)H]-NPVF labelled one class of binding sites with a high affinity (Bmax=4pmol/mg protein, Kd=2.65nM). In membranes from CHO cells transfected with the human NPFF(2) receptor, [(3)H]-EYF labelled one class of binding sites with a high affinity (Bmax=16pmol/mg protein, Kd=0.54nM). Both radioligands exhibited time-dependent binding, low (10-20%) non-specific binding and poor cross-reactivity towards the related receptor subtype. The potency of different NPFF ligands to displace [(3)H]-NPVF and [(3)H]-EYF binding profiles was in good agreement with the profile previously measured by using (125)I-probes (NPFF(1) receptor: NPVF> or =1DMe=SPA-NPFF>NPFF=SQA-NPFF=QFW-NPSF>NPSF>RF9; NPFF(2) receptor: SPA-NPFF>>SQA-NPFF=QFW-NPSF=1DMe=NPFF>>NPSF=NPVF>RF9). Therefore, [(3)H]-NPVF and [(3)H]-EYF are new valuable tools for performing binding on NPFF receptors.

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.

Neuropeptide FF behaves as an opioid-modulating peptide that seems to be involved in morphine tolerance and physical dependence. Nevertheless, the effects of neuropeptide FF agonists on the rewarding properties of morphine remain unknown. C57BL6 mice were conditioned in an unbiased balanced paradigm of conditioned place preference to study the effect of i.c.v. injections of 1DMe (D-Tyr1(NMe)Phe3]NPFF), a stable agonist of the neuropeptide FF system, on the acquisition of place conditioning by morphine or alcohol (ethanol). Morphine (10 mg/kg, i.p.) or ethanol (2 g/kg, i.p.) induced a significant place preference. Injection of 1DMe (1-20 nmol), given 10 min before the i.p. injection of the reinforcing drug during conditioning, inhibited the rewarding effect of morphine but had no effect on the rewarding effect of ethanol. However, a single injection of 1DMe given just before place preference testing was unable to inhibit the rewarding effects of morphine. By itself, 1DMe was inactive but an aversive effect of this agonist could be evidenced if the experimental procedure was biased. These results suggest that neuropeptide FF, injected during conditioning, should influence the development of rewarding effects of morphine and reinforce the hypothesis of strong inhibitory interactions between neuropeptide FF and opioids.

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses μ- and δ-opioid receptors, with the human NPFF 1 receptor. The SH 1 -C7 clone expresses high affinity NPFF 1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF 1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca 2+ currents and enhances the intracellular Ca 2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH 1 -C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.

Opioids are involved in the physiological control of numerous functions of the central nervous system, particularly nociception. It appears that some endogenous neuropeptides, called anti-opioids, participate in an homeostatic system tending to reduce the effects of opioids. Neuropeptide FF (NPFF) and cholecystokinin (CCK) possess these properties and, paradoxically, the opioid peptides nociceptin and dynorphin display some anti-opioid activity. All these peptides exhibit complex properties as they are able to both counteract and potentiate opioid activity, acting rather as modulators of opioid functions. The purpose of this review is to highlight that two different mechanisms are clearly involved in the control of opioid functions by opioid-modulating peptides: a circuitry-induced mechanism for nociceptin and dynorphin, and a cellular anti-opioid mechanism for NPFF and CCK. The knowledge of these mechanisms has potential therapeutic interest in the control of opioid functions, notably for alleviating pain and/or for the treatment of opioid abuse.

To elucidate the mechanism of the cellular antiopioid activity of neuropeptide FF (NPFF), we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu-and delta-opioid receptors, with the human NPFF2receptor. The selected clone, SH2-D9, expressed high-affinity NPFF2 receptors in the same range order as mu- and delta-opioid receptors (100-300 fmol/mg of protein). The NPFF analog [D-Tyr1, (NMe)Phe3]NPFF (1DMe) did not modify the binding parameters of the mu- and delta-specific agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin and deltorphin-I, respectively. 1DMe dose dependently inhibited 75 to 80% of the cAMP production stimulated by forskolin. Preincubation with 1DMe halved the maximal inhibition of N-type Ca2+ channels by opioid agonists. In the presence of carbachol, acting on muscarinic receptors to release Ca2+ from the intracellular stores, deltorphin-I and 1DMe enhanced this release. Preincubation with 1DMe reduced the maximal effect of deltorphin-I by 40%, demonstrating an antiopioid effect in this experimental model for the first time. By using peptides corresponding to the carboxyl terminus of the alphai1,2, alphai3, alphao, and alphas subunits of G proteins, which specifically uncouple receptors from G proteins, we demonstrated that mu-opioid and NPFF2 receptors couple to the four subunits assayed. The Ca2+ release from the intracellular stores by 1DMe resulted from the coupling of NPFF2 receptors with Galphao and Galphai1,2, whereas the coupling with Galphas reduced the antiopioid effect of 1DMe in the modulation of N-type channels. This SH2-D9 cell line now provides the opportunity to study the interaction between both receptors.

The pharmacological effects of Neuropeptide FF (NPFF) analogs exhibiting different selectivities towards Neuropeptide FF1 (NPFF1) and Neuropeptide FF2 (NPFF2) receptors were investigated after supraspinal administration in mice. Injected into the third ventricle, VPNLPQRF-NH2, which is selective for Neuropeptide FF1 receptor induced a hypothermia while EFWSLAAPQRF-NH2 and SPAFLFQPQRF-NH2 which are selective for Neuropeptide FF2 receptor, did not. Furthermore, EFWSLAAPQRF-NH2 significantly increased the body temperature when compared to saline treated mice, indicating that Neuropeptide FF1 receptor could be responsible for hypothermia while Neuropeptide FF2 mediated an hyperthermic effect. After administration into the lateral ventricle, 1DMe ([D.Tyr1,(N.Me)Phe3]NPFF), a weakly selective Neuropeptide FF2 receptor agonist, exerted a clear anti-opioid effect in the tail flick test. The selective Neuropeptide FF1 receptor agonist VPNLPQRF-NH2 did not induce significant anti-opioid actions but rather increased, dose-dependently, morphine analgesia while EFWSLAAPQRF-NH2, the highest selective Neuropeptide FF2 receptor analog, induced the same pharmacological effect as VPNLPQRF-NH2 at comparable doses. These features indicate that the pro- and anti-opioid actions are not strictly related to the selectivity towards Neuropeptide FF2 or Neuropeptide FF1 receptor. Our data demonstrate that Neuropeptide FF1 and Neuropeptide FF2 receptors differently modulate nervous system functions.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFFA cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms.

Neuropeptide FF (NPFF) and its analog 1DMe ([D-Tyr 1 ,(NMe)Phe 3 ]NPFF) have been shown to reverse or potentiate morphine analgesia in rat depending on the supraspinal or spinal site of injection. The properties, in the mouse tail-flick test, of 1DMe and its related compound Nic-1DMe (Nicotinoyl-Pro-1DMe) were investigated after their local (i.c.v. and i.t.) and systemic administration. Whereas Nic-1DMe and 1DMe exhibit the same affinity and selectivity towards NPFF 1 and NPFF 2 receptors, Nic-1DMe, in contrast to 1DMe, is unable to inhibit morphine-induced analgesia after i.c.v. and i.p. administration. Conversely, after i.t. and i.p. administration, both neuropeptide FF analogs could potentiate morphine analgesia. Differences in disposition parameters between 1DMe and Nic-1DMe are evidenced, suggesting that the two neuropeptide FF analogs could stimulate differentially supraspinal neuropeptide FF receptors. The predominant activation of spinal neuronal pathways by Nic-1DMe could explain the selective pro-opioid action of this compound after i.t., i.c.v. and i.p. administration.

Pharmacological studies have implicated the anti-opioid neuropeptide FF (NPFF) in the modulation of pain transmission. Since its physiological role has not yet been fully elucidated, the present study examined whether antisense peptide nucleic acid (PNA) complementary to the NPFF precursor (proNPFFA) modified pain sensitivity. Mice received three intraperitoneal (i.p.) injections (10 mg/kg) of antisense PNA (As-proNPFFA) over a period of 24 h. As-proNPFFA treatment significantly increased the basal tail withdrawal latency in the tail-flick test. This analgesia persisted during 2 days and was completely reversed by naloxone. Thus, antisense PNAs, by decreasing anti-opioid effects, revealed a basal endogenous opioid activity. Our results evidence a physiological interplay between NPFF and opioid systems and further support the use of PNA as effective antisense agents, for studying gene function in vivo.

Intrathecal infusion of the neuropeptide FF analogue, [D-Tyr1, (NMe)Phe3]neuropeptide FF (1DMe; 0.1 microm-0.1 mm) in anaesthetized rats produced a concentration-dependent decrease in the spinal outflow of dynorphin A (1-8)-like material, which persisted for at least 90 min after treatment with 10 microm-0.1 mm of the compound. Co-administration of d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 1 microm) to block spinal micro-opioid receptors did not modify this effect, whereas naltrindole (10 microm) totally prevented it and nor-binaltorphimine (10 microm) reduced the post-effect. These data suggest that 1DMe triggers the release of endogenous opioids that stimulate mainly delta-opioid receptors, and secondarily kappa-opioid receptors, thereby exerting a negative influence on dynorphin A (1-8)-like material outflow. Because dynorphin has pronociceptive properties, such a decrease in spinal dynorphin A (1-8)-like material release might underlie the long-lasting antinociceptive effects of intrathecally administered neuropeptide FF and analogues.

We assessed the possible influence of a neuropeptide FF analogue, 1DMe ([D-Tyr(1),(NMe)Phe(3)]neuropeptide FF), on the inhibitory action of endogenous and exogenous partial differential-opioid receptor agonists on K(+)-evoked [Met(5)]-enkephalin release from superfused rat spinal cord slices. 1DMe (0.1-10 microM) dose-dependently enhanced the increase in superfusate [Met(5)]-enkephalin content due to the peptidase inhibitors thiorphan (1 microM) and bestatin (20 microM), and prevented the reduction in [Met(5)]-enkephalin release due to stimulation of partial differential receptors by 1 microM deltorphin I. Because it had the same effects as partial differential-opioid receptor antagonists, 1DMe might act through the functional blockade of presynaptically located partial differential-opioid autoreceptors.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

Experimental data suggest that neuropeptide FF (NPFF), a peptide isolated from brain, is part of a neurotransmitter system that acts as an anti-opioid or at the very least as a modulator of endogenous opioid function. Our understanding of the cellular basis of this neurotransmission in the CNS is limited. However, following the molecular screening of orphan G-protein-coupled receptors (GPCRs) [aimed at the identification of novel neuropeptide Y (NPY) receptors and putative pharmaceutical targets], two NPFF receptors and a second precursor that contains novel NPFF-related peptides have been cloned. Consequently, some confusion with respect to the nomenclature used for NPY and NPFF receptors has emerged.Elshourbagy et al.1xReceptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. Elshourbagy, N.A et al. J. Biol. Chem. 2000; 275: 25965–25971Crossref | PubMed | Scopus (198)See all References1 have described a cDNA encoding a novel NPY-like human orphan GPCR referred to as HLWAR77 for which NPFF and NPFF-related peptides display high affinity, strongly suggesting that this receptor is a NPFF receptor. An apparent splice variant of this cDNA is referred to, by Cikos et al.2xSequence and tissue distribution of a novel G-protein-coupled receptor expressed prominently in human placenta. Cikos, S et al. Biochem. Biophys. Res. Commun. 1999; 256: 352–356Crossref | PubMed | Scopus (23)See all References2, as NPGPR, and Parker et al.3xMolecular cloning and characterisation of GPR74 a novel G-protein coupled receptor closest related to the Y-receptor family. Parker, R.M et al. Mol. Brain Res. 2000; 77: 199–208Crossref | PubMed | Scopus (25)See all References3 have cloned the same receptor as GPR74, ‘a novel G-protein-coupled receptor’ that is related to the NPY receptor family. Furthermore, Bonini et al.4xIdentification and characterization of two G protein-coupled receptors for neuropeptide FF. Bonini, E.A et al. J. Biol. Chem. 2000; 275: 39324–39331Crossref | PubMed | Scopus (253)See all References4 have identified two GPCRs for NPFF, NPFF1 and NPFF2 receptors, which exhibit different localization in human and rat brain. The NPPF2 receptor is identical to HLWAR77 described by Elshourbagy et al.1xReceptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. Elshourbagy, N.A et al. J. Biol. Chem. 2000; 275: 25965–25971Crossref | PubMed | Scopus (198)See all References1 and does not have the extra 102 amino acids that are present in NPGPR, which suggests that NPGPR is an aberrant form of this receptor. NPFF1 and NPFF2 receptors exhibit high affinities for NPFF, neuropeptide AF (NPAF) and 1DMe, a synthetic agonist of NPFF. Finally, a recent study by Hinuma et al.5xNew neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Hinuma, S et al. Nat. Cell Biol. 2000; 2: 703–708Crossref | PubMed | Scopus (380)See all References5 describes the cloning of a rat and human gene encoding a seven-transmembrane-domain receptor, OT7TO22, which responds to NPFF and possesses exactly the same amino acid sequence as the NPFF1 receptor characterized by Bonini et al.4xIdentification and characterization of two G protein-coupled receptors for neuropeptide FF. Bonini, E.A et al. J. Biol. Chem. 2000; 275: 39324–39331Crossref | PubMed | Scopus (253)See all References4With respect to the NPFF precursor, Hinuma et al.5xNew neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Hinuma, S et al. Nat. Cell Biol. 2000; 2: 703–708Crossref | PubMed | Scopus (380)See all References5 have described the cloning of a human gene that encodes at least two RF-amide-related peptides (RFRPs), one of which (VPNLPQRF-NH2) acts specifically via the NPFF1 receptor (OT7TO22) with a higher potency than NPFF.Thus, it is possible that two different NPFF-mediated systems exist, with two different proproteins releasing several NPFF-related peptides, which in turn act at two different receptors. I would like to propose a nomenclature to name the recently identified NPFF receptors and precursors. Two preproproteins, a prepro-NPFF (which could be named A) that contains FLFQPQRF-NH2 and SLAAPQRF-NH2 sequences, and a prepro-NPFF (which could be named B) that contains LPLRF-NH2 and LPQRF-NH2 sequences, exist in mammals. ProNPFF-A should produce peptides related to NPFF that do not discriminate between two receptors that could be named NPFF1 and NPFF2 receptors. ProNPFF-B-derived peptides exhibit a high affinity towards NPFF1 receptors but, to date, no data are available concerning their affinity for NPFF2 receptors.

We have compared the affinities and anti-opioid activities of the different peptides putatively produced by the rat NPFF precursor, NPAFLFQPQRF-NH(2) (NPA-NPFF) and EFWSLAAPQRF-NH(2) (EFW-NPSF), with those already identified in nervous tissue, FLFQPQRF-NH(2) (NPFF) and SLAAPQRF-NH(2) (NPSF). NPFF and NPA-NPFF exhibit a high affinity (0.34 and 0.14 nM, respectively) for [(125)I]1DMe binding sites of the rat spinal cord. In contrast, EFW-NPSF displays an affinity 13 times higher than NPSF (1.99 and 9.5 nM, respectively). In rat dorsal raphe neurones, EFW-NPSF, NPFF, and NPA-NPFF maximally reduce the inhibitory effect of nociceptin on the [Ca(2+)](i) transients triggered by depolarization by 39, 31, and 58%, respectively. NPSF is inactive in the same test. We conclude that NPA-NPFF and EFW-NPSF are likely to be the physiologically active neurotransmitters in rat brain.

Behavioural studies have suggested that endogenous opioids mediate the antinociceptive action of neuropeptide FF (FLFQPQRF-NH2) at the spinal level in the rat. This hypothesis was directly assessed by investigating the effects of a NPFF analogue, 1DMe ([D-Tyr1,(NMe)Phe3]NPFF), on the spinal outflow of met-enkephalin-like material (MELM) in halothane-anaesthetised rats. Intrathecal infusion (0.1 ml/min) of 1DMe (0.1 microM-0.1 mM, for 45 min) produced a concentration-dependent increase in spinal MELM outflow which persisted for at least 90 min at the highest concentration tested. Intrathecal coadministration of the micro-opioid receptor antagonist CTOP (1 microM) did not significantly affect the spinal MELM overflow due to 0.1 mM 1DMe. In contrast, both naltrindole and nor-binaltorphimine, at concentrations (10 microM) that allow the selective blockade of alpha- and kappa-opioid receptors, respectively, significantly reduced the stimulatory effect of 1DMe on spinal MELM outflow. These data provide the first direct demonstration that met-enkephalin (among other opioid peptides) can mediate the antinociceptive action of NPFF at the spinal level in rats. In addition, they suggest that reciprocal excitatory interactions between opioids and opioid-modulatory factors (such as NPFF) participate in the physiological control of nociception.

Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) is able to modulate opioid analgesia. Intracerebroventricular treatment for 5 days with antisense-oligodeoxynucleotides complementary to the sequence of human SQA-neuropeptide FF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) precursor gene or by mismatch-oligodeoxynucleotides did not change the antinociceptive activity of morphine in the mouse tail flick test. In contrast, antisense- but not mismatch-oligodeoxynucleotides attenuated significantly the tolerance to the analgesic activity of morphine and the withdrawal syndrome precipitated by naloxone in morphine-treated mice. These treatments with oligodeoxynucleotides did not modify neuropeptide FF-immunoreactivity content in whole brain but repeated injections of an agonist of neuropeptide FF receptors increased the intensity of morphine tolerance. These results demonstrate the important role of neuropeptide FF in opioid pharmacodependence.

The modulatory effects of 1DMe (d-Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2), an agonist of Neuropeptide FF (NPFF) receptors, on opioid antinociceptive activity have been compared in naive and tolerant mice in the tail-flick and the hot-plate tests. In naive mice, 1DMe alone had no effect on pain threshold but decreased dose-dependently (3-22 nmol) the analgesic activity of morphine in both tests. In tolerant mice, injections of 60-fold lower doses of 1DMe (0.05-0.5 nmol) reverse morphine-induced analgesia in the tail-flick test but this anti-opioid effect was no longer observed with the highest doses of 1DMe tested (3-22 nmol). In the hot-plate test, the anti-opioid action of 1DMe was not detected, whatever doses tested. Neither the NPFF-like immunoreactivity content of spinal cord and of olfactory bulbs, nor the density of NPFF receptors in olfactory bulbs, were altered. These results indicate that a chronic morphine treatment modifies the pharmacological properties of NPFF but the type of pain test is crucial in determining NPFF effects.