enkephalin--ala(2)-mephe(4)-gly(5)- and phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF1 and NPFF2 receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF2 receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF1 and NPFF2 receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF2 receptor and enhance the central antinociception of U69593 via both NPFF1 and NPFF2 receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.

Topics: Adamantane; Animals; Benzeneacetamides; Dipeptides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Naltrexone; Nociception; Oligopeptides; Pyrrolidines

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.

Topics: Calcium; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Neuroblastoma; Neuropeptide Y; Oligopeptides; Receptors, Neuropeptide; Receptors, Opioid; Staurosporine

Neurons that synthesize the morphine modulatory peptide neuropeptide FF (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) densely innervate the parabrachial nucleus (PBN), an area implicated in regulating food intake. We analyzed opioid-related actions of NPFF in feeding in adult male Sprague-Dawley rats. Unilateral infusion of 2 nmol/0.5 microl of the mu-opioid receptor agonist [d-Ala2,NMe-Phe4,glycinol5]enkephalin (DAMGO) into the lateral PBN increased 4-h food intake from 0.7 +/- 0.1 to 3.3 +/- 0.3 g. NPFF (1.25-5.0 nmol) prevented this hyperphagic mu-opioidergic action. In rats fed after 4-h deprivation (baseline = 12.3 +/- 0.3 g/2 h), 5 nmol of NPFF did not alter and larger doses (10 and 20 nmol) actually increased food intake (+36, 54%). Twenty nanomoles also elevated intake of freely feeding rats (from 0.7 +/- 0.1 to 5.1 +/- 1.0 g/4 h). The opioid receptor blocker naloxone (10 nmol) antagonized this increase. These data reveal both pro- and anti-opioid actions of NPFF in the PBN to modulate feeding. The mechanisms for the opposite actions of low and high concentrations of this neuropeptide in parabrachial regulation of food intake remain to be determined.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drinking; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Feeding Behavior; Hyperphagia; Immunohistochemistry; Male; Naloxone; Narcotic Antagonists; Oligopeptides; Pons; Rats; Rats, Sprague-Dawley

The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 microM) could not be blocked by the specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (1 microM), but application of delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (5 microM) almost completely prevented effects of NPFF. Moreover, the delta-opioid receptor agonist, Deltorphin (1 microM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta-opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta-opioid receptors.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Electric Conductivity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Ligands; Male; Naloxone; Narcotic Antagonists; Oligopeptides; Patch-Clamp Techniques; Peptides; Picrotoxin; Pons; Potassium; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Tetrahydroisoquinolines; Tetrodotoxin

Intracellular Ca2+ concentration ([Ca2+]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 [Ca2+]i but reduced the magnitude of the [Ca2+]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 microM) also reduced the magnitude of the [Ca2+]i transient. The selective 5-HT(1A) receptor antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-ben zamide hydrochloride (p-MPPI) (0.4 microM) strongly attenuated (by 72+/-7%, n=4) this effect. The responses to nociceptin and 5-HT were not affected by BaCl2 (100 microM). The neuropeptide FF analog [D-Tyr1, (N-Me)Phe3]NPFF (1DMe) altered neither the resting [Ca2+]i nor the [Ca2+]i transient triggered by depolarization but dose-dependently decreased the effect of nociceptin (EC50=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-Tyr1, D-Leu2, D-Phe3]NPFF (1 microM) 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 mu-opioid receptor agonist [D-Ala2, (N-Me)Phe4, Gly-ol5]-enkephalin (DAGO) with a decrease in the depolarization-induced [Ca2+]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.

Topics: Aminopyridines; Analgesics, Opioid; Animals; Barium Compounds; Biological Transport; Calcium; Chlorides; Dose-Response Relationship, Drug; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; In Vitro Techniques; Narcotic Antagonists; Neurons; Nociceptin; Oligopeptides; Opioid Peptides; Piperazines; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid, mu; Serotonin; Serotonin Antagonists; Vasodilator Agents

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.

Topics: Age Factors; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Mice, Inbred Strains; Morphine; Naltrexone; Narcotic Antagonists; Neuropeptides; Nociceptors; Oligopeptides; Receptors, Neuropeptide; Receptors, Opioid, delta; Receptors, Opioid, mu

The effect of continuous ICV infusion of NPFF (10 micrograms/microliter) and morphine (40 micrograms/microliter) on mu-opioid binding sites was examined in rats using the in vitro radiolabeled techniques of whole-brain homogenate receptor binding and quantitative autoradiography. Mu receptors were labeled with [3H][D-Ala2-MePhe4,Glyol5] enkephalin in the homogenate binding experiments and with [125I][D-Ala2-MePhe4,Gly-ol5]enkephalin in autoradiographic studies. In homogenate binding studies, chronic administration of NPFF or morphine significantly downregulated mu receptors by 20% and 44%, respectively. Quantitative autoradiographic experiments demonstrated downregulation of mu opioid receptors in specific brain nuclei for both NPFF- and morphine-treated animals. Within the striatum and several nuclei of the thalamus, the mu receptors of the NPFF- and morphine-treated animals were decreased by 20-30% and 38-73%, respectively. These results suggest that NPFF may modulate opioid-mediated responses in part by altering the density of mu-opioid receptors.

Topics: Analgesics, Opioid; Animals; Autoradiography; Brain; Corpus Striatum; Down-Regulation; Drug Administration Routes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Morphine; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Subcellular Fractions; Thalamus; Tissue Distribution

Single neurones isolated from spinal ganglia of young rats were loaded with Fluo3, a fluorescent indicator of intracellular Ca2+ concentration. In 27 of 47 neurones the depolarization-evoked rise in fluorescence was reduced by 47.8 +/- 3.3% by prior perfusion with DAGO (1 microM, 30 s) a mu-opioid agonist. In 12 neurones an analogue of neuropeptide FF ((1DMe)Y8Fa, 10 nM, 30 min) did not affect the Ca2+ response to depolarization. (1DMe)Y8Fa reversed the effect of DAGO by 63 +/- 8% in seven of these 12 neurones. We conclude that stimulation of neuropeptide FF receptors antagonizes mu-opioid modulation of Ca2+ channels at the cellular level.

Topics: Analgesics; Aniline Compounds; Animals; Calcium Channels; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Ganglia, Spinal; Narcotic Antagonists; Narcotics; Neurons; Oligopeptides; Potassium Chloride; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Xanthenes

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.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Microinjections; Molecular Sequence Data; Morphine; Neuropeptides; Oligopeptides; Pain Measurement; Pain Threshold; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid

The central nervous system contains circuitry that inhibits pain sensitivity (analgesia), as well as circuitry that opposes pain inhibition (anti-analgesia). Activation of analgesia systems and anti-analgesia systems can each be brought under environmental control using classical conditioning procedures. Analgesia can be produced by cues present before and during aversive events such as electric shock, while active inhibition of analgesia comes to be produced by cues never present immediately before or during shock and therefore signal safety. We have recently reported that these analgesia and anti-analgesia systems interact at the level of the spinal cord. A series of 3 experiments were performed to examine how such interactions occur. First, potential opioid mediation of conditioned analgesia was investigated using systemic and intrathecal (i.t.) delivery of opiate antagonists. Conditioned analgesia was found to be mediated by activation of spinal mu and delta opiate receptors. Second, analgesia produced by each of these receptor subtypes was challenged by environmental signals for safety. Analgesias produced by mu and delta opiate agonists were each abolished by safety signals. Third, antagonists/antisera directed against several putative anti-opiate neurotransmitters were tested i.t. to identify which mediate conditioned anti-analgesia at the level of the spinal cord. A cholecystokinin antagonist abolished conditioned anti-analgesia. In contrast, neuropeptide FF antiserum and a kappa opiate antagonist were without effect.

Topics: Analgesia; Analgesics; Animals; Conditioning, Classical; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Immune Sera; Injections, Spinal; Male; Morphine; Naltrexone; Narcotic Antagonists; Oligopeptides; Proglumide; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Spinal Cord

Topics: Animals; Brain Chemistry; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Immunoglobulin G; Injections, Intraventricular; Ligands; Male; Morphine; Morphine Dependence; Oligopeptides; Rats; Rats, Inbred Strains; Receptors, Opioid; Up-Regulation

An in vivo preparation of the rat spinal cord was used to investigate the electrophysiological actions of two non-opioid peptides, cholecystokinin (CCK8) and FLFQPQRF-NH2 (FMRFamide-like peptide) applied intrathecally. These compounds were examined alone and as a pretreatment before DAGO, a mu opioid agonist, and DSTBULET, a delta opioid agonist, both which selectively reduce C-fibre evoked dorsal horn neurone activity elicited by transcutaneous electrical stimulation. Given alone, CCK8 (1 microgram) elicited a modest enhancement of C-fibre induced activity which returned to control levels after 20 min, while FLFQPQRF-NH2 (10 micrograms) had no significant effect on C-fibre evoked firing. As a pretreatment, however, both peptides selectively prevented the inhibition of C-fibre evoked activity normally resulting from intrathecal DAGO, while having no effect on that resulting from DSTBULET. Further, CCK8 enhanced the facilitation of C-fibre evoked firing normally observed with low doses of DAGO. These data indicate that the anti-opioid roles suggested for CCK8 and FLFQPQRF-NH2 may be specific for neural elements utilizing the mu opioid receptor.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Evoked Potentials; Injections, Spinal; Male; Nerve Fibers; Neurons; Oligopeptides; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sincalide; Spinal Cord