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.

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.

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.

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 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).

[Modulation of calcium conductance by opioid and anti-opioid peptides]. / Modulation de conductances calciques par les peptides opioïdes et anti-opioïdes.

In the central nervous system, opening of voltage-gated Ca2+ channels triggers the release of neurotransmitters. Numerous membrane receptors, particularly those belonging to the superfamily of G-protein coupled receptors modulate, in most cases inhibit the activity of these channels. In the present review, we describe the modulation of calcium channels by opioid and anti-opioid peptides. Following a brief presentation of the opioid system, we describe the characteristics of the modulation of calcium channels by opioids. Recent major advances concerning neuropeptide FF (NPFF), taken as an example of anti-opioid systems, are reviewed. Results from our laboratory demonstrating the anti-opioid activity of NPFF, in the modulation of Ca2+ channels in isolated neurones, are described.

Are neuropeptides FF and SF neurotransmitters in the rat?

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.

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.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.

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.

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-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 [125I]1DMe ([125I]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, 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.

Neuropeptide FF reverses the effect of mu-opioid on Ca2+ channels in rat spinal ganglion neurones.

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.

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.

Effects of metabolic inhibitors and hypoxia on the ATP, ADP and AMP content of the rabbit carotid body in vitro: the metabolic hypothesis in question.

The effects of metabolic inhibitors (cyanide, antimycin) and hypoxia on the nucleotide content of the carotid body were investigated in vitro. The mean ATP content of carotid bodies superfused for 1 h in normoxic conditions was around 200 pmol/organ. Whereas metabolic inhibitors induced a decrease in ATP and an increase in AMP, hypoxia (10% O2 in N2, either 4 or 30 min) did not induce any significant change in nucleotide content. The significance of these results is discussed with regard to the metabolic hypothesis.

Fate of the catecholamine stores in the rabbit carotid body superfused in vitro.

In rabbit carotid bodies (CBs) superfused during 1-5 h, with an air-equilibrated medium containing no tyrosine (TYR), the dopamine (DA) content decreased by approximately 60% after 1 h and remained constant afterwards. TYR and 3,4-dihydroxyphenylacetic acid (DOPAC) decreased with the same time course. Noradrenaline (NA) content exhibited a biphasic decrease of lesser magnitude than that of DA. Superfusions with a TYR-containing medium did not prevent the reduction in DA and TYR. Large amounts of DA and DOPAC were recovered in the effluent during the first hour of superfusion but after 90 min the two substances had declined below the detection limits (i.e. 0.5 and 1 pmol/5 min, respectively). The DA efflux decreased exponentially during the first hour and was not altered by changing the oxygen partial pressure (PO2) of the medium. The DOPAC efflux declined after 40 min of superfusion and was modulated by PO2. The DA and the DOPAC effluxes were not suppressed by omitting calcium ions from the superfusing medium. In 4 cat CBs equal amounts of DA and NA were recovered from the effluent during the first hour of superfusion.

Comparison of the monoamine and catabolite content in the cat and rabbit carotid bodies.

The monoamine and catabolite contents of a large number of rabbit (n = 95) and cat (n = 32) carotid bodies (CBs) have been measured by high performance liquid chromatography with electrochemical detection (HPLC-ED). The dopamine (DA) content as well as that of its precursors tyrosine (TYR), dihydroxyphenylalanine (DOPA) and catabolites dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA) were approximately equal in both species. The noradrenaline (NA) content was 10 times larger in the cat than in the rabbit CBs. Twenty-nine out of the 32 cat CBs contained more NA than DA while the reverse was true in 92 out of 95 rabbit CBs. In 11 cats the right CB was sympathectomized and its DA and NA contents were compared to those of intact contralateral organs.

Rate of dopamine metabolism in the rabbit carotid body in vivo.

In the rabbit carotid body (CB) in vivo, the rate of dopamine metabolism was estimated to 44.4 +/- 3.9 (SD) pmol/CB/h from the decrease in dihydroxyphenylacetic acid content after pargyline inhibition of monoamine oxidase.

Monoamines and their catabolites in the rabbit carotid body. Effects of reserpine, sympathectomy and carotid sinus nerve section.

Monoamines and their metabolites have been measured by high performance liquid chromatography with electrochemical detection, in control rabbit carotid bodies and under several experimental conditions: 1) at different times (3 h, 6 h, 24 h, 48 h) after intravenous injection of reserpine (5 mg/kg); 2) 14 days after sympathectomy; 3) 14 days after section of the carotid sinus nerve. The results were analyzed with probability plotting methods. Dopamine was the most important monoamine in the carotid body (CB) and its variations were very large. It was almost entirely depleted by reserpinization without simultaneous increase in 3,4-dihydroxyphenylacetic acid. Sympathectomy increased dopamine content but did not change noradrenaline content. However data analysis suggested that noradrenaline might be compartmented in two pools: one with a large variance, located in the type I cells was increased after sympathectomy, the other, more constant, located in the sympathetic nerve endings was entirely depleted after sympathectomy. Section of the carotid sinus nerve increased dopamine and noradrenaline and quadrupled the serotonin content of the CB. It is proposed that carotid sinus and sympathetic innervations regulate the monoamine metabolism of the CB.

Chemoreceptor response to hypoxia and hypercapnia in catecholamine depleted rabbit and cat carotid bodies in vitro.

The response of single chemoafferent fibres to hypoxic and hypercapnic stimulation was studied in vitro under different experimental conditions: 1. control, 2. 24 h after reserpinization (5 mg/kg iv) and 3. 18 h after iv injection of alpha-methyl-p-tyrosine (100 mg/kg in the rabbit, and 1. control and 2. 24 h after reserpinization (5 mg/kg ip) in the cat. The spontaneous activity was decreased by monoamine depletion. The amplitude of the response to hypoxia and to hypercapnia was decreased by reserpinization in the rabbit and in the cat, the change being less marked in the latter species. Similarly, treatment with alpha-methyl-p-tyrosine decreased the ability of chemoreceptors to respond to hypoxia and hypercapnia and, in a few instances, these receptors could only be excited by superfusion of nitrogen-equilibrated medium. These results emphasize to possible role of monoamine, and particularly dopamine, in modifying the sensitivity of arterial chemoreceptors to their natural stimuli.

A role of pulmonary rapidly adapting receptors in control of breathing.

We analysed the breathing pattern of anaesthetised rabbits during unloaded breathing when breathing was accelerated by inspired CO2 and when they breathed against positive or negative pressures before and during block of pulmonary stretch receptors by SO2, and after bilateral vagotomy. Before block moderate steps of inflation or deflation (0.5 kPa) produced relatively larger changes in duration of expiration than in duration of inspiration, indicating the relative sensitivities of the two phases. With stretch receptors, blocked inflation or deflation shortened expiration, demonstrating the influence of rapidly adapting receptors on that phase of breathing. If pulmonary stretch receptors were the major determinants of the duration of inspiration, we would have expected inspiratory duration in the stretch receptor blocked and vagotomised states to be almost identical. They were not, inspiratory duration being less in the blocked than in the vagotomised state. Possibly vagal afferent activity other than that of stretch receptors shortens inspiratory duration. However, we have found that rapidly adapting receptor activity (and any unmyelinated fibre activity provoked by rapid inflation or deflation of the lungs) never directly shortened inspiration. We therefore propose a mechanism whereby rapidly adapting receptors may indirectly affect duration of inspiration.