Michel Roumy

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.

Opioid-modulating Peptides: Mechanisms of Action

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.

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

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

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

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.

Anti-opioid activities of NPFF1 receptors in a SH-SY5Y model

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 mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ 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. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.

Neuropeptide FF receptors 1 and 2 exert an anti-opioid activity in acutely dissociated rat dorsal raphe and periventricular hypothalamic neurones.

We measured the reduction by nociceptin of the [Ca(2+)](i) transient triggered by depolarization in acutely dissociated neurones of the rat dorsal raphe and periventricular hypothalamic nuclei that express NPFF(2) and NPFF(1) receptors, respectively, in the absence and presence of 10 nM of NPA-NPFF or NPVF, two peptides selective for NPFF(2) and NPFF(1) receptors, respectively. In dorsal raphe neurones, NPA-NPFF reduces the inhibition of Ca(2+) conductances by nociceptin while NPVF is inactive. In periventricular hypothalamic neurones, both peptides reduce the inhibition of Ca(2+) transients by nociceptin, NPVF having a significantly larger effect than NPA-NPFF. These results demonstrate that activation of both NPFF(1) and NPFF(2) receptors has the same cellular anti-opioid effect.

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 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.

Dual localization of neuropeptide FF receptors in the rat dorsal horn.

Although neuropeptide FF (NPFF) is generally considered an anti‐opioid, its intrathecal administration produces analgesia. In the present study, the stable analog 1DMe ([D.Tyr1,(NMe)Phe3]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 [125I]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 [125I]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 [125I]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. Synapse 35:45–52, 2000.

Effects of neuropeptide FF on intracellular Ca2+ in mouse spinal ganglion neurons

Intracellular Ca2+ was measured in freshly dissociated mouse dorsal root ganglion neurons by using Fluo3 as fluorescent Ca2+ probe. Short perifusions (5-10 s) with 30 mM K+ induced a sharp rise in fluorescence due to the entry of Ca2+ ions, in particular through L and N voltage sensitive Ca2+ 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 Ca2+ rise during depolarization. These features indicate that neuropeptide FF and its analogs exert their pharmacological effects by reducing the [Ca2+]i transient induced by short depolarizations.