Neuropeptide FF Analog Research Articles

Combinatorial lead optimization of a neuropeptide FF antagonist.

The tripeptide Pro-Gln-Arg-NH2, derivatized at the secondary amino group of the proline residue with 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl-PQR-NH2), antagonizes the central anti-opioid action of neuropeptide FF in animals after systemic administration and, therefore, is a therapeutic lead to treat opiate withdrawal. For a combinatorial optimization to improve potency, libraries focused on the possible replacement of the proline and glutamine residues of this lead compound were obtained by a solid-phase split-and-mix method using coded amino acids (excluding cysteine) as building blocks. After screening for competitive binding against a radioiodinated neuropeptide FF analogue, 5-(dimethylamino)-1-naphthalenesulfonyl-Gly-Ser-Arg-NH2 (dansyl-GSR-NH2) has emerged as one of the compounds in the library with high affinity to the NPFF receptor and even with a moderate increase compared to dansyl-PQR-NH2 in its predicted ability to penetrate the central nervous system.

Effect of 1DMe, a Neuropeptide FF Analog, on Acetylcholine Release From Myenteric Plexus of Guinea Pig Ileum

Since neuropeptide FF (NPFF) is a putative neurotransmitter to exert anti-opioid activity, we examined the effects of [D-Tyr', (NMe)Phe3]neuropeptide FF (IDMe), 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 microM 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.

Role of adenosine in the spinal antinociceptive and morphine modulatory actions of neuropeptide FF analogs

The neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2) 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-Tyr 1,(NMe)Phe 3]NPFF (1DMe, 0.86 nmol) and [ d-Tyr 1, d-leu 2, d-Phe 3]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.

The cardiovascular effects of PFRFamide and PFR(Tic)amide, a possible agonist and antagonist of neuropeptide FF (NPFF)☆

Neuropeptide FF (NPFF), an endogenous opioid-related neuromodulater, has been reported to show significant effects on the cardiovascular system, namely elevation of arterial blood pressure (BP) and heart rate (HR) in rats. In the present study, we synthesized two novel NPFF analogs, PFRFamide (putative NPFF agonist) and PFR(Tic)amide (putative NPFF antagonist), and examined their cardiovascular effect on BP and HR in anesthetized rats. The arterial mean BP and HR were measured by way of direct femoral artery catheterization. The data showed that PFRFamide increased BP in a dose-dependent manner, while PFR(Tic)amide decreased BP dose-dependently. These results revealed the possibility of PFRFamide and PFR(Tic)amide to be NPFF agonist and antagonist (or inverse agonist), respectively. These two NPFF analogs may possess potential in new drug design, and the NPFF system could be very important in mammalian cardiovascular function.

Neuropeptide FF selectively attenuates the effects of nociceptin on acutely dissociated neurons of the rat dorsal raphe nucleus

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.

Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs

Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists.

Effects of (1DMe)NPYF, a synthetic neuropeptide FF analogue, in different pain models

The antinociceptive effects of intrathecal (IT) (1DMe)NPYF were studied in adult Sprague–Dawley rats. (1DMe)NPYF produced dose-dependent antinociception that was reduced by subcutaneous injection of naloxone. (1DMe)NPYF (0.5 nmol) also potentiated the antinociceptive effects of intrathecal morphine 7.8 nmol. This suggests that the antinociceptive effects of (1DMe)NPYF are partially mediated by opioid receptor activation. In carrageenan inflammation, 5–10 nmol of (1DMe)NPYF was effective against both thermal hyperalgesia and mechanical allodynia. In the neuropathic pain model, the lowest dose tested (0.5 nmol) showed antiallodynic effects against cold allodynia. The results suggest a potential role for (1DMe)NPYF in the treatment of pain including neuropathic pain.

The neuropeptide FF analogue, 1DME, enhances in vivo met-enkephalin release from the rat spinal cord

Behavioural studies have suggested that endogenous opioids mediate the antinociceptive action of neuropeptide FF (FLFQPQRF–NH 2) at the spinal level in the rat. This hypothesis was directly assessed by investigating the effects of a NPFF analogue, 1DMe ([D-Tyr 1,(NMe)Phe 3]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 μM–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 μ-opioid receptor antagonist CTOP (1 μM) did not significantly affect the spinal MELM overflow due to 0.1 mM 1DMe. In contrast, both naltrindole and nor-binaltorphimine, at concentrations (10 μM) that allow the selective blockade of ∂- and κ-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.

Spinal effect of a neuropeptide FF analogue on hyperalgesia and morphine-induced analgesia in mononeuropathic and diabetic rats.

1DMe, a neuropeptide FF (NPFF) analogue, has been shown to produce antinociception and to enhance morphine analgesia in rats after intrathecal administration. To determine whether 1DMe could correct hyperalgesia and restore morphine efficacy in mononeuropathic (MN) and diabetic (D) rats we examined the spinal effect of 1DMe in MN and D rats without and after spinal blockade of mu- and delta-opioid receptors with CTOP and naltrindole, respectively. The influence of 1DMe on morphine-induced antinociception was assessed in the two models using isobolographic analysis. Whereas 1DMe intrathecally injected (0.1, 1, 7.5 microg rat(-1)) was ineffective in normal (N) rats, it suppressed mechanical hyperalgesia (decrease in paw pressure-induced vocalisation thresholds) in both MN and D rats. This effect was completely cancelled by CTOP (10 microg rat(-1)) and naltrindole (1 microg rat(-1)) suggesting that it requires the simultaneous availability of mu- and delta-opioid receptors. The combinations of morphine: 1DMe (80.6:19.4% and 99.8:0.2%, in MN and D rats, respectively) followed by isobolographic analysis, showed a superadditive interaction, relative to the antinociceptive effect of single doses, in D rats only. In N rats, the combination of morphine: 1DMe (0.5 mg kg(-1), i.v.: 1 microg rat(-1), i.t., ineffective doses) resulted in a weak short-lasting antinociceptive effect. These results show a different efficacy of 1DMe according to the pain model used, suggesting that the pro-opioid effects of the NPFF in neuropathic pain are only weak, which should contribute to hyperalgesia and to the impaired efficacy of morphine.

Biochemical, cellular and pharmacological activities of a human neuropeptide FF-related peptide

We report on the biochemical, cellular and pharmacological activities of SQA-neuropeptide FF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH 2), a peptide sequence contained in the human neuropeptide FF (neuropeptide FF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH 2) 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–NH 2) binding sites ( K i=0.33 nM) as did neuropeptide FF ( K i=0.38 nM). In acutely dissociated mouse dorsal root ganglion neurones, SQA-neuropeptide FF reduced by 40% the depolarisation-induced rise in intracellular Ca 2+ as measured with the Ca 2+ 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.

Affinity of Neuropeptide FF Analogs to Opioid Receptors in the Rat Spinal Cord

Several high-affinity analogs of neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2, NPFF) exhibiting both supraspinal anti-opioid and spinal analgesic activities were studied for their abilities to interact with specific μ, δ, and κ opioid binding in the rat spinal cord. Measurements by quantitative receptor autoradiography in the superficial layers of the spinal cord revealed that NPFF analogs tested have only a low affinity for opioid receptors since K i values ranged from 5 to 400 μ M. Taking into account the high efficacy of NPFF after intrathecal injection, these results indicate that analgesic effects of NPFF did not result from opioid receptor stimulation.

Neuropeptide FF, pain and analgesia.

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.

Hypothermic Effects of Neuropeptide FF Analogues in Mice

The effects of neuropeptide FF (NPFF) and its analogues on mouse body temperature were examined. In a thermoneutral environment, administration of NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2), 1DMe ([D.Tyr 1, (N.Me)Phe 3] NPFF), and 3D ([D.Tyr 1, D.Leu 2, D.Phe 3] NPFF) in the third ventricle produced marked hypothermia. The effect of 1DMe was dose-dependent, and 45 nmol decreased body temperature by 5.6°C. This effect was more pronounced when mice were placed at 4°C. Hypothermia was not reversed by naloxone, an opioid antagonist, and was not modified by morphine. After 5 days of chronic treatment with 1DMe, mice did not became tolerant to the hypothermic effect. These results indicate that central NPFF receptors may control body temperature independently from opioid functions.

Effects of neuropeptide FF on intestinal motility and temperature changes induced by endotoxin and platelet-activating factor

Several effects of bacterial endotoxins involve an opioid pathway and neuropeptide FF is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. The aim of this study was to investigate in rats the role of central neuropeptide FF receptors in intestinal motor disturbances and body temperature changes induced by endotoxins and platelet-activating factor (PAF), a major endotoxin mediator. Rats were fitted with intestinal electrodes, an intraperitoneal thermistor probe and an intracerebroventricular (i.c.v.) cannula for long-term use. E. coli endotoxin (100 μg/kg, i.v.) disrupted the cyclic pattern of intestinal migrating myoelectric complexes and induced a biphasic increase in body temperature while PAF (25 μg/kg, i.p.) disrupted the migrating myoelectric complexes and induced hypothermia for about 2 h. The neuropeptide FF analog, (1 DME)Y8Fa ( d-Tyr- d-Leu[N–Me]–Phe–Gln–Pro–Gln–Arg–Phe–NH 2) administered i.c.v. 40 and 100 μg/kg reduced the duration of migrating myoelectric complex disruption induced by endotoxin and PAF and abolished the PAF-induced hypothermia. Only at the dose of 100 μg/kg did (1 DME)Y8Fa change the biphasic endotoxin-induced hyperthermia into a monophasic increase. Naloxone (1 mg/kg, s.c.) reduced only the duration of migrating myoelectric complex disruption induced by endotoxin. These results indicate that central neuropeptide FF modulates the intestinal motor disturbances and changes in body temperature induced by endotoxin and PAF. Its action against endotoxin may involve an anti-opioid pathway whereas its action against PAF does not.

Neuropeptide FF receptors control morphine-induced analgesia in the parafascicular nucleus and the dorsal raphe nucleus

The ability of (1DMe)Y8Fa (D.Tyr–Leu–(NMe)Phe–Gln–Pro–Gln–Arg–Phe–NH 2), 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.

Neuropeptide FF Receptors of Mouse Olfactory Bulb: Binding Properties and Stimulation of Adenylate Cyclase Activity

Gherardi, N. and J.-M. Zajac. Neuropeptide FF receptors of mouse olfactory bulb: Binding properties and stimulation of adenylate cyclase activity. Peptides 18(4) 577–583, 1997.—Neuropeptide FF (NPFF) receptors have been characterized in mouse olfactory bulb membranes by using [ 125I][1DMe]Y8Fa. The specific binding of this NPFF analogue was time and concentration dependent, reversible, saturable, and of high affinity ( K d = 0.022 n M, B max = 56.4 fmol/mg protein). In olfactory bulb membranes, NaCl increased the affinity of [ 125I][1DMe]Y8Fa by decreasing the dissociation rate constant ( k −1 ). In contrast, the nonhydrolyzable analogue of GTP, Gpp[NH]p, decreased the maximal number of binding sites suggesting a coupling of NPFF receptors to a G-protein. In mouse olfactory bulb and spinal cord membranes, NPFF analogues stimulated adenylate cyclase activity in a time- and dose-dependent manner, whereas in the cerebellum, which does not possess NPFF receptors, low cAMP production was stimulated by NPFF. Our data are consistent with guanine nucleotide binding protein regulation of NPFF receptors positively coupled to adenylate cyclase.

Nicotine abstinence syndrome precipitated by an analog of neuropeptide FF

In a recently introduced rodent model of nicotine abstinence syndrome the observed behavioral signs closely resembled those typical of rat opiate abstinence syndrome. Nicotine-induced release of endogenous opioids may contribute to nicotine dependence; morphine potently reverses nicotine abstinence signs, while naloxone precipitates abstinence signs and prevents nicotine from alleviating them. Considerable evidence suggests that neuropeptide FF, an endogenous antiopiate peptide, contributes to opiate dependence. Third ventricle injection of neuropeptide FF precipitates abstinence syndrome in morphine-dependent rats, as does SC injection of its lipophilic analogs, dansyl-PQRFamide and dansyl-RFamide. Might NPFF also play a role in nicotine dependence? In the present study, SC injection of 15 or 25 mg/kg dansyl-RFamide or vehicle alone dose dependently precipitated an abstinence syndrome in nicotine-dependent rats. There was a significant, p < 0.01, positive linear trend of abstinence signs as a function of dose. Categories of abstinence signs had the same rank ordering by frequency as observed in spontaneous nicotine abstinence. Injection of 25 mg/kg dansyl-RFamide SC had no significant effect in nondependent rats.

Effects of neuropeptide FF on intracellular Ca 2+ in mouse spinal ganglion neurons

Intracellular Ca 2+ was measured in freshly dissociated mouse dorsal root ganglion neurons by using Fluo3 as fluorescent Ca 2+ probe. Short perifusions (5–10 s) with 30 mM K + induced a sharp rise in fluorescence due to the entry of Ca 2+ ions, in particular through L and N voltage sensitive Ca 2+ channels opened by the action potentials that were triggered by depolarization. Perifusions with 1 or 10 nM (1DMe)Y8Fa (DYL(NMe)FQPQRFamide), a neuropeptide FF analog, suppressed the rise in fluorescence induced by short (5–10 s) K + perifusions within 30 min. However, when K + perifusions of longer duration were applied, Fluo3 fluorescence rose after an increased latency. Two other analogs, (2DMe)Y8Fa (DYDL(NMe)FQPQRFamide) and (3D)Y8Fa (DYDLDFQPQRFamide), had the same effect; similarly neuropeptide FF (FLFQPQRFamide, 1 nM, 30 min) reduced intracellular Ca 2+ rise during depolarization. These features indicate that neuropeptide FF and its analogs exert their pharmacological effects by reducing the [Ca 2+] i transient induced by short depolarizations.

Role of opioid receptors in the spinal antinociceptive effects of neuropeptide FF analogues.

1. Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphine-induced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFF analogues, -D-Tyr1,(NMe)Phe3-NPFF (1DMe) and [D-Tyr1,D-Leu2,D-Phe3]NPFF (3D), which are resistant to degradation and exhibit a high affinity for NPFF binding sites, were examined in tests of thermal and mechanical nociception. 2. 1DMe and 3D produced potent dose-dependent spinal antinociception in the tail-flick test. On a molar basis, 1DMe was 20 and 50 times more potent than 3D and morphine, respectively, and high doses of 1DMe and 3D produced a sustained antinociceptive effect without visible signs of motor impairment. 3. Spinal antinociceptive effects produced by 1DMe (0.86 nmol) or 3D (8.6 nmol) were significantly reduced by i.t. co-administration of naloxone (11 nmol) or i.t. pre-administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 9.25 nmol) or beta-funaltrexamine (beta-FNA, 2 nmol) or naltrindole (2.2 nmol). The doses of the mu-antagonists (CTOP and beta-FNA) or the delta-antagonist (naltrindole) used in 1DMe and 3D experiments blocked the antinociceptive effects of mu- or delta-receptor-selective agonists. 4. When administered in combination with antinociceptive doses of the mu-receptor agonist, morphine (13.2 nmol) or the delta-receptor agonist, [D-Ala2]deltorphin I (20 nmol), sub-effective dose of 1DMe or 3D (0.009 nmol) enhanced and prolonged the spinal effects of these opioid agonists. 5. The results of this study show that spinal mu- and delta-opioid receptors play a role in antinociception produced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptive signals at the spinal level.

Effects of neuropeptide FF analogs on morphine analgesia in the nucleus raphe dorsalis

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-NH 2) or (3D)Y8Fa ( d-Tyr- d-Leu- d-Phe-Gln-Pro-Gln-Arg-Phe-NH 2), 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 μ and δ 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.