Neuropeptide FF Analog Research Articles

Peripheral administration of lipidized NPAF and NPFF analogs does not influence central food intake regulation but induces anxiety-like behavior

RF-amide peptides influence multiple physiological processes, including the regulation of appetite, stress responses, behavior, and reproductive and endocrine functions. In this study, we examined the roles of neuropeptide FF receptors (NPFFR1 and NPFFR2) by generating several lipidized analogs of neuropeptide AF (NPAF) and 1DMe, a stable analog of neuropeptide FF (NPFF). These analogs were administered peripherally for the first time to investigate their effects on food intake and other potential physiological outcomes. Lipidized NPAF and 1DMe analogs exhibited enhanced stability and increased pharmacokinetics. These analogs demonstrated preserved high affinity for NPFFR2 in the nanomolar range, while the binding affinity for NPFFR1 was tens of nanomoles. They activated the ERK and Akt signaling pathways in cells overexpressing the NPFFR1 and NPFFR2 receptors.Acute food intake in fasted mice decreased after the peripheral administration of oct-NPAF or oct-1DMe. However, this effect was not as pronounced as that observed after the injection of palm11-PrRP31, a potent anorexigenic compound used as a comparator that binds to GPR10 and the NPFFR2 receptor with high affinity. Neither oct-1DMe nor oct-NPAF decreased food intake or body weight in mice with diet-induced obesity during long-term treatment. In mice treated with oct-1DMe, we observed decreased activity in the central zone during the open field test and decreased activity in the open arms of the elevated plus maze. Furthermore, we observed a decrease in plasma noradrenaline levels and an increase in plasma corticosterone levels, as well as an increase in Crh expression in the hypothalamus. Moreover, neuronal activity in the hypothalamus was increased after treatment with oct-1DMe.In this study, we report that oct-1DMe did not have any long-term effects on the central regulation of food intake; however, it caused anxiety-like behavior.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Cholesterol-rich lipid rafts are involved in neuropeptide FF anti-nociceptin/orphanin FQeffect.

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

Neuropeptide FF receptors exhibit direct and anti-opioid effects on mice dorsal raphe nucleus neurons

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 1DMe (10µM) 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 1DMe. In contrast, cholera toxin treatment had no significant effect. Eighteen minute perfusion with 1DMe at a very low 10nM 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.

Neuropeptide FF analog RF9 is not an antagonist of NPFF receptor and decreases food intake in mice after its central and peripheral administration

Neuropeptide FF (NPFF) belongs to the RF-amide family of peptides bearing the identical C-terminal amino acid sequence (R-F-NH2). In addition to NPFF, prolactin-releasing peptide (PrRP), another RF-amide, binds to NPFF receptors with high affinity. A selective antagonist of PrRP has not yet been identified, but a selective antagonist of NPFF, 1-adamantanecarbonyl-RF-NH2 (RF9), was recently reported to antagonize the hyperalgesic effect of NPFF after central administration to mice. In the present study, RF9 competed with NPFF analog D-Y-L-(N-Me)-F-Q-P-Q-R-F-NH2 (1DMe) in binding to CHO-K1 cell membranes transfected with the human NPFF2 receptor. In rat pituitary RC-4B/C cells, where the expression of the NPFF2 receptor was proved by immunodetection, RF9 did not reverse the phosphorylation of MAPK/ERK1/2 induced by [Tyr1]NPFF. In vivo experiments with fasted mice confirmed that centrally injected [Tyr1]NPFF significantly lowered food intake. However, RF9, a putative NPFF2 antagonist, did not reverse the anorectic effect of [Tyr1]NPFF. Paradoxically, RF9 itself exhibited an anorectic effect in fasted mice not only after intracerebroventricular but also after subcutaneous administration. This finding casts doubt on claims that RF9 is an NPFF antagonist.

Neuropeptide FF (NPFF) reduces the expression of morphine- but not of ethanol-induced conditioned place preference in rats

Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr 1, (NMe)Phe 3]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.

Pharmacological effects of the dansylated neuropeptide FF analogues on body temperature and morphine analgesia

In our previous work, the two putative agonists (dansyl-GSRFamide and dansyl-PQRFamide) and the two putative antagonists (dansyl-GSRamide and dansyl-PQRamide) on neuropeptide FF (NPFF) receptors were synthesized to evaluate the importance of Phe(8) of NPFF. In the present study, these putative NPFF agonists/antagonists containing different N-terminal sequences were further examined for their pharmacological profiles in thermoregulatory and nociceptive tests. The results indicated that the two dansylated agonists potently possessed similar thermoregulation (rank order of potencies: dansyl-GSRFamide>>NPFF>dansyl-PQRFamide) and different modulation of opioid-induced analgesia; in contrast, both of the two putative antagonists exhibited marked hypothermia (rank order of potencies: dansyl-PQRamide>dansyl-GSRamide) and facilitation of morphine analgesia (rank order of potencies: dansyl-PQRamide > dansyl-GSRamide). These data reveal that the difference of the N-terminal residues of the two putative agonists causes their dissociation of pharmacological pro- and anti-opioid effects. In addition, their N-terminal part is important to determine the potency of the dansylated agonists/antagonists. Our work might be helpful to develop a highly potent and fluorescent NPFF ligand.

Physical Association between Neuropeptide FF and μ-Opioid Receptors as a Possible Molecular Basis for Anti-opioid Activity

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 micro-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.

In vitro and in vivo studies of dansylated compounds, the putative agonists and antagonists on neuropeptide FF receptors

To further evaluate the importance of C-terminal modification of neuropeptide FF (NPFF), in the present work, four dansylated NPFF analogues, including two putative agonists (dansyl-PQRFamide and dansyl-GSRFamide) and two putative antagonists (dansyl-PQRamide and dansyl-GSRamide), were synthesized and investigated to address their potencies and efficacies in a series of in vitro and in vivo assays. (1) In the isolated mouse colon bioassay, the four dansylated compounds showed agonistic profiles: both dansyl-GSRFamide (1–10 μM) and dansyl-GSRamide (1–10 μM) dose-dependently caused colonic contractions, which were attenuated by pretreatment with BIBP3226; dansyl-PQRFamide and dansyl-PQRamide evoked modest colonic contractions at a high dose of 50 μM. (2) In urethane-anaesthetized rats, both dansyl-PQRFamide (50–300 nmol/kg, i.v.) and dansyl-GSRFamide (15–50 nmol/kg, i.v.) dose-dependently increased the mean arterial pressure and heart rate in a manner similar to NPFF (50–300 nmol/kg, i.v.); on the contrary, the two putative antagonists (100–800 nmol/kg, i.v.) decreased blood pressure in a dose-dependent manner. All the results suggest that dansyl-PQRFamide and dansyl-GSRFamide are NPFF full agonists; in contrast, dansyl-GSRamide and dansyl-PQRamide behave as agonists in vitro and antagonists in vivo on NPFF receptors. The findings reveal that the C-terminal Phe might be a crucial residue to determine the efficacy. In addition, the novel analogue dansyl-GSRFamide may be developed as a highly potent agonist to investigate the NPFF system.

Comparison of pharmacological activities of Neuropeptide FF 1 and Neuropeptide FF 2 receptor agonists

The pharmacological effects of Neuropeptide FF (NPFF) analogs exhibiting different selectivities towards Neuropeptide FF 1 (NPFF 1) and Neuropeptide FF 2 (NPFF 2) receptors were investigated after supraspinal administration in mice. Injected into the third ventricle, VPNLPQRF-NH 2, which is selective for Neuropeptide FF 1 receptor induced a hypothermia while EFWSLAAPQRF-NH 2 and SPAFLFQPQRF-NH 2 which are selective for Neuropeptide FF 2 receptor, did not. Furthermore, EFWSLAAPQRF-NH 2 significantly increased the body temperature when compared to saline treated mice, indicating that Neuropeptide FF 1 receptor could be responsible for hypothermia while Neuropeptide FF 2 mediated an hyperthermic effect. After administration into the lateral ventricle, 1DMe ([D.Tyr 1,(N.Me)Phe 3]NPFF), a weakly selective Neuropeptide FF 2 receptor agonist, exerted a clear anti-opioid effect in the tail flick test. The selective Neuropeptide FF 1 receptor agonist VPNLPQRF-NH 2 did not induce significant anti-opioid actions but rather increased, dose-dependently, morphine analgesia while EFWSLAAPQRF-NH 2, the highest selective Neuropeptide FF 2 receptor analog, induced the same pharmacological effect as VPNLPQRF-NH 2 at comparable doses. These features indicate that the pro- and anti-opioid actions are not strictly related to the selectivity towards Neuropeptide FF 2 or Neuropeptide FF 1 receptor. Our data demonstrate that Neuropeptide FF 1 and Neuropeptide FF 2 receptors differently modulate nervous system functions.

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF2 Receptor-Transfected SH-SY5Y Neuroblastoma Cells

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.

Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay

We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [ 35S]guanosine-5′- O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH 2 exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.

Functional consequences of neuropeptide FF receptors stimulation in mouse: a cerebral glucose uptake study

The brain substrates involved in the pharmacological effects of neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2) including interactions with opioid systems, were investigated with the [ 14C]-2-deoxyglucose ([ 14C]-2-DG) autoradiography technique in mouse. The changes in cerebral activity were mapped after i.p. administration of 1DMe ([ d-Tyr 1,(NMe)Phe 3]NPFF; 70 mg/kg), a neuropeptide FF analogue partially resistant to peptidases, alone or in combination with morphine (15 mg/kg). 1DMe induced a rapid decrease in the cerebral activity in the thalamus, the pontine reticular nuclei and the cerebellar cortex, brain regions involved in the control of motor activity and/or the processing of sensory data. This decrease, observed when 1DMe was administered 5 min before [ 14C]-2-DG, was reversed by morphine, which was devoid of significant effect at this time. When administered 30 min before the radioisotope, 1DMe was without effect, whereas morphine induced a significant increase in cerebral glucose utilization in the caudate putamen, the primary somatosensory cortex, the thalamus, the superior colliculus, the pontine reticular nuclei and the spinal cord. The association of morphine and 1DMe significantly increased cerebral glucose utilization in the same regions as morphine alone and also in three additional regions: the auditory cortex, the inferior colliculus and the dorsomedial periaqueductal gray. Following systemic administration, 1DMe and morphine modulated cerebral activity in brain regions involved in pain transmission and motor control, but their effects were temporally shifted, as were their effects on horizontal locomotor activity. However, neuropeptide FF-induced changes in brain activity were modulated in part by opioid receptors activation.

Functional properties of PFR(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [ 35 S ]guanosine-5′- O-(3-thio)triphosphate ([ 35 S ]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [ 35 S ]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.

Dissociation of pharmacological pro- and anti-opioid effects by neuropeptide FF analogs

Neuropeptide FF (NPFF) and its analog 1DMe ([D-Tyr1,(NMe)Phe3]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 NPFF1 and NPFF2 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.

The neuropeptide FF analogue, 1DMe, reduces in vivo dynorphin release from the rat spinal cord.

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.

The neuropeptide FF analogue, 1DMe, acts as a functional opioid autoreceptor antagonist in the rat spinal cord

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 ∂-opioid receptor agonists on K +-evoked [Met 5]-enkephalin release from superfused rat spinal cord slices. 1DMe (0.1–10 μM) dose-dependently enhanced the increase in superfusate [Met 5]-enkephalin content due to the peptidase inhibitors thiorphan (1 μM) and bestatin (20 μM), and prevented the reduction in [Met 5]-enkephalin release due to stimulation of ∂ receptors by 1 μM deltorphin I. Because it had the same effects as ∂-opioid receptor antagonists, 1DMe might act through the functional blockade of presynaptically located ∂-opioid autoreceptors.

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 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.

Response properties of neurons in the rostroventromedial medulla of neuropathic rats: attempted modulation of responses by [1DMe]NPYF, a neuropeptide FF analogue

We determined whether chronic neuropathy changes response properties of neurons in the rostroventromedial medulla of rats, and whether (d-Tyr)L(Me-Phe)QPQRF-amide, a neuropeptide FF analogue, in the periaqueductal gray produces changes in responses of rostroventromedial medullary neurons that might underlie its antiallodynic effect described earlier. Single unit recordings of medullary neurons were performed in lightly anesthetized neuropathic and control animals. Spontaneous activity and the responses to noxious thermal and mechanical stimulation of the hind paw were determined with and without administration of (d-Tyr)L(Me-Phe)QPQRF-amide. The neurons were classified into three groups: ON-neurons increased, OFF-neurons decreased, and NEUTRAL-neurons did not change their discharge rate prior to a limb withdrawal induced by noxious stimulation of the skin. Spontaneous activity and heat-evoked responses of ON-neurons were not different between neuropathic and control animals, whereas their mechanically evoked responses were reduced in neuropathy. Response properties of OFF-neurons were not different between neuropathic and control animals. Spontaneous activity of NEUTRAL-neurons was not different between neuropathic and control animals. (d-Tyr)L(Me-Phe)QPQRF-amide in the periaqueductal gray had no significant effect on evoked responses or spontaneous activity of ON- or OFF-neurons, independent of the experimental group. However, (d-Tyr)L(Me-Phe)QPQRF-amide produced a significant attenuation of spontaneous activity of NEUTRAL-neurons in neuropathic animals. In a behavioral study performed in unanesthetized animals it was found that intrathecal administration of methysergide, a serotonin antagonist, selectively attenuated neuropathic symptoms. Also, light pentobarbitone anesthesia markedly attenuated, but did not abolish, behaviorally determined neuropathic symptoms. From these results we suggest that NEUTRAL-neurons of the rostroventromedial medulla may have a role in neuropathy and they may be involved in attenuation of mechanical hypersensitivity by (d-Tyr)L(Me-Phe)QPQRF-amide in the periaqueductal gray. It is proposed that in neuropathy the synaptic effects of descending impulses from medullary NEUTRAL-neurons on their axonal targets in the spinal cord are changed so that this contributes to mechanical hypersensitivity, due to mechanisms that are at least partly serotoninergic.

Neuropeptide FF receptors couple to a cholera toxin-sensitive G-protein in rat dorsal raphe neurones

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 μg/ml, 2–6 h) but was unchanged (37.4±2.1%, n=44) after pre-incubation with cholera toxin (5 μg/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-Tyr 1, (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 μg/ml, 2–6 h) [ d-Tyr 1, (N-Me)Phe 3] 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 (G s).